Prof Peter J McHugh

Research Area: Cell and Molecular Biology
Technology Exchange: Drug discovery and Ex vivo models
Scientific Themes: Cancer and Molecular, Cell & Systems Biology
Web Links:

During every division cycle, cells must maintain their genomic integrity to retain normal functionality. To achieve this, complex pathways to repair genomic damage and abnormalities encountered during DNA replication have evolved. A DNA lesion that is particularly associated with spontaneous and chemically-induced genome attrition is the interstrand cross-links (ICL). ICLs, which tether the two strand of the double helix together, are extremely toxic since they block replication and transcription. Endogenously generated ICLs are a major threat to genome integrity and therefore the normal development and ageing programme of mammals. This is exemplified by the genetic disorder Fanconi anemia (FA), where affected individuals are unable to repair ICLs. This leads to very serious health problems including a range of developmental defects, haematopoietic failure and highly elevated cancer predisposition. Multiple DNA repair pathways co-operate to combat ICLs and related damage, but how they do this remain poorly understood.

 

One the other hand, ICLs are the active cytotoxic lesion produced by a wide range of clinically important anticancer drugs. For example, platinum DNA cross-linking drugs are a mainstay of chemotherapy for many common cancers, but resistance to therapy remains a major problem, all to often limiting their effective use. In contrast, such chemotherapy has dramatic and frequently curative effects in testicular (germ cell) tumours, highlighting the enormous benefit that could result from understanding mechanisms of resistance to drugs of this class. Since DNA repair pathways directly combat ICL-inducing therapies, by removing the toxic DNA damage they induce, targeting of DNA repair response is one of the most compelling strategies for sensitising or re-sensitising tumours ICL-inducing chemotherapy. The goal of my lab is to understand the mechanism of repair of DNA replication-blocking DNA damage such as ICLs in order that we can move towards rational therapeutic targeting of the ICL repair-response in tumours, and also use this knowledge in cancer prevention.

 

My group utilises a combination of genetic, cell biological and biochemical techniques to dissect the mechanisms of ICL repair in human cells. Here we have made recent progress in showing how endonucleases in the XPF-family interface with factors mutated in FA to initiate ICL repair. Moreover, we have identified a novel exonuclease activity present in the SNM1A protein that is required for the initiation of ICL repair in human cells. Future work will focus on cell biology studies to determine how the key components of the ICL repair apparatus (XPF family nucleases, SNM1A and FA factors) are recruited to ICLs during DNA replication, coupled to efforts to biochemically reconstitute core ICL repair reactions. This will be integrated into studies that employ structure-guided approaches to rationally develop small molecule inhibitors of ICL repair factors, with a focus on the SNM1A-family, in collaboration with the groups of Chris Schofield (Oxford, Chemistry) and Opher Gileadi (Oxford, Structural Genomics Consortium). These inhibitors will be powerful experimental tools, and also allow to test proof-of-principle that inhibition ICL repair will improve response to a number of important anticancer drugs, paving the way for new combined therapies to overcome resistance.

Name Department Institution Country
Prof Christopher Schofield Chemistry University of Oxford United Kingdom
Dr Opher Gileadi Structural Genomics Consortium Oxford University, Old Road Campus Research Building United Kingdom
Abdullah UB, McGouran JF, Brolih S, Ptchelkine D, El-Sagheer AH, Brown T, McHugh PJ. 2017. RPA activates the XPF-ERCC1 endonuclease to initiate processing of DNA interstrand crosslinks. EMBO J, 36 (14), pp. 2047-2060. | Show Abstract | Read more

During replication-coupled DNA interstrand crosslink (ICL) repair, the XPF-ERCC1 endonuclease is required for the incisions that release, or "unhook", ICLs, but the mechanism of ICL unhooking remains largely unknown. Incisions are triggered when the nascent leading strand of a replication fork strikes the ICL Here, we report that while purified XPF-ERCC1 incises simple ICL-containing model replication fork structures, the presence of a nascent leading strand, modelling the effects of replication arrest, inhibits this activity. Strikingly, the addition of the single-stranded DNA (ssDNA)-binding replication protein A (RPA) selectively restores XPF-ERCC1 endonuclease activity on this structure. The 5'-3' exonuclease SNM1A can load from the XPF-ERCC1-RPA-induced incisions and digest past the crosslink to quantitatively complete the unhooking reaction. We postulate that these collaborative activities of XPF-ERCC1, RPA and SNM1A might explain how ICL unhooking is achieved in vivo.

Ward TA, McHugh PJ, Durant ST. 2017. Small molecule inhibitors uncover synthetic genetic interactions of human flap endonuclease 1 (FEN1) with DNA damage response genes. PLoS One, 12 (6), pp. e0179278. | Show Abstract | Read more

Flap endonuclease 1 (FEN1) is a structure selective endonuclease required for proficient DNA replication and the repair of DNA damage. Cellularly active inhibitors of this enzyme have previously been shown to induce a DNA damage response and, ultimately, cell death. High-throughput screens of human cancer cell-lines identify colorectal and gastric cell-lines with microsatellite instability (MSI) as enriched for cellular sensitivity to N-hydroxyurea series inhibitors of FEN1, but not the PARP inhibitor olaparib or other inhibitors of the DNA damage response. This sensitivity is due to a synthetic lethal interaction between FEN1 and MRE11A, which is often mutated in MSI cancers through instabilities at a poly(T) microsatellite repeat. Disruption of ATM is similarly synthetic lethal with FEN1 inhibition, suggesting that disruption of FEN1 function leads to the accumulation of DNA double-strand breaks. These are likely a result of the accumulation of aberrant replication forks, that accumulate as a consequence of a failure in Okazaki fragment maturation, as inhibition of FEN1 is toxic in cells disrupted for the Fanconi anemia pathway and post-replication repair. Furthermore, RAD51 foci accumulate as a consequence of FEN1 inhibition and the toxicity of FEN1 inhibitors increases in cells disrupted for the homologous recombination pathway, suggesting a role for homologous recombination in the resolution of damage induced by FEN1 inhibition. Finally, FEN1 appears to be required for the repair of damage induced by olaparib and cisplatin within the Fanconi anemia pathway, and may play a role in the repair of damage associated with its own disruption.

McHugh PJ, Kisker C, Chovanec M, Van Houten B. 2017. Third EU-US workshop on “Nucleotide excision repair and crosslink repair—From molecules to mankind”, Smolenice Castle, Slovak Republic, May 7th–11th 2017 DNA Repair, 58 pp. 62-66. | Show Abstract | Read more

© 2017. The purpose of this workshop was to bring together scientists from the EU and US to discuss our current understanding of how protein machines assemble and sort through genomic DNA to identify specific damaged sites which are repaired through the two critical repair pathways: nucleotide excision repair or interstrand crosslink repair. Several of the enzymes perform dual functions in both pathways, thus combining these two topics in one meeting provided tremendous synergy and was a unique feature of this workshop. Many endogenous agents, environmental toxicants and chemotherapeutic agents cause a spectrum of DNA lesions that are repaired through these two mechanisms. The importance of the repair pathways acting on these forms of DNA damage is underscored by the fact that their loss or dysregulation is associated with a variety of devastating heritable human conditions including cancer, neurodegeneration, premature aging and severe developmental abnormalities. The workshop covered research conducted on molecules to mankind, and included discussions of disease syndromes associated with poor repair of these forms of damage. The meeting also provided an unparalleled opportunity to interact with scientists from the EU, US, and throughout the world, who are driving the field.

Rendeková J, Ward TA, Šimoničová L, Thomas PH, Nosek J, Tomáška Ľ, McHugh PJ, Chovanec M. 2016. Mgm101: A double-duty Rad52-like protein. Cell Cycle, 15 (23), pp. 3169-3176. | Show Abstract | Read more

Mgm101 has well-characterized activity for the repair and replication of the mitochondrial genome. Recent work has demonstrated a further role for Mgm101 in nuclear DNA metabolism, contributing to an S-phase specific DNA interstrand cross-link repair pathway that acts redundantly with a pathway controlled by Pso2 exonuclease. Due to involvement of FANCM, FANCJ and FANCP homologues (Mph1, Chl1 and Slx4), this pathway has been described as a Fanconi anemia-like pathway. In this pathway, Mgm101 physically interacts with the DNA helicase Mph1 and the MutSα (Msh2/Msh6) heterodimer, but its precise role is yet to be elucidated. Data presented here suggests that Mgm101 functionally overlaps with Rad52, supporting previous suggestions that, based on protein structure and biochemical properties, Mgm101 and Rad52 belong to a family of proteins with similar function. In addition, our data shows that this overlap extends to the function of both proteins at telomeres, where Mgm101 is required for telomere elongation during chromosome replication in rad52 defective cells. We hypothesize that Mgm101 could, in Rad52-like manner, preferentially bind single-stranded DNAs (such as at stalled replication forks, broken chromosomes and natural chromosome ends), stabilize them and mediate single-strand annealing-like homologous recombination event to prevent them from converting into toxic structures.

Lee SY, Brem J, Pettinati I, Claridge TDW, Gileadi O, Schofield CJ, McHugh PJ. 2016. Cephalosporins inhibit human metallo β-lactamase fold DNA repair nucleases SNM1A and SNM1B/apollo. Chem Commun (Camb), 52 (40), pp. 6727-6730. | Show Abstract | Read more

Bacterial metallo-β-lactamases (MBLs) are involved in resistance to β-lactam antibiotics including cephalosporins. Human SNM1A and SNM1B are MBL superfamily exonucleases that play a key role in the repair of DNA interstrand cross-links, which are induced by antitumour chemotherapeutics, and are therefore targets for cancer chemosensitization. We report that cephalosporins are competitive inhibitors of SNM1A and SNM1B exonuclease activity; both the intact β-lactam and their hydrolysed products are active. This discovery provides a lead for the development of potent and selective SNM1A and SNM1B inhibitors.

Broderick R, Nieminuszczy J, Baddock HT, Deshpande R, Gileadi O, Paull TT, McHugh PJ, Niedzwiedz W. 2016. EXD2 promotes homologous recombination by facilitating DNA end resection. Nat Cell Biol, 18 (3), pp. 271-280. | Show Abstract | Read more

Repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is critical for survival and genome stability of individual cells and organisms, but also contributes to the genetic diversity of species. A vital step in HR is MRN-CtIP-dependent end resection, which generates the 3' single-stranded DNA overhangs required for the subsequent strand exchange reaction. Here, we identify EXD2 (also known as EXDL2) as an exonuclease essential for DSB resection and efficient HR. EXD2 is recruited to chromatin in a damage-dependent manner and confers resistance to DSB-inducing agents. EXD2 functionally interacts with the MRN complex to accelerate resection through its 3'-5' exonuclease activity, which efficiently processes double-stranded DNA substrates containing nicks. Finally, we establish that EXD2 stimulates both short- and long-range DSB resection, and thus, together with MRE11, is required for efficient HR. This establishes a key role for EXD2 in controlling the initial steps of chromosomal break repair.

Pettinati I, Brem J, Lee SY, McHugh PJ, Schofield CJ. 2016. The Chemical Biology of Human Metallo-β-Lactamase Fold Proteins. Trends Biochem Sci, 41 (4), pp. 338-355. | Show Abstract | Read more

The αββα metallo β-lactamase (MBL) fold (MBLf) was first observed in bacterial enzymes that catalyze the hydrolysis of almost all β-lactam antibiotics, but is now known to be widely distributed. The MBL core protein fold is present in human enzymes with diverse biological roles, including cell detoxification pathways and enabling resistance to clinically important anticancer medicines. Human (h)MBLf enzymes can bind metals, including zinc and iron ions, and catalyze a range of chemically interesting reactions, including both redox (e.g., ETHE1) and hydrolytic processes (e.g., Glyoxalase II, SNM1 nucleases, and CPSF73). With a view to promoting basic research on MBLf enzymes and their medicinal targeting, here we summarize current knowledge of the mechanisms and roles of these important molecules.

Allerston CK, Lee SY, Newman JA, Schofield CJ, McHugh PJ, Gileadi O. 2015. The structures of the SNM1A and SNM1B/Apollo nuclease domains reveal a potential basis for their distinct DNA processing activities. Nucleic Acids Res, 43 (22), pp. 11047-11060. | Show Abstract | Read more

The human SNM1A and SNM1B/Apollo proteins are members of an extended family of eukaryotic nuclease containing a motif related to the prokaryotic metallo-β-lactamase (MBL) fold. SNM1A is a key exonuclease during replication-dependent and transcription-coupled interstrand crosslink repair, while SNM1B/Apollo is required for maintaining telomeric overhangs. Here, we report the crystal structures of SNM1A and SNM1B at 2.16 Å. While both proteins contain a typical MBL-β-CASP domain, a region of positive charge surrounds the active site of SNM1A, which is absent in SNM1B and explains the greater apparent processivity of SNM1A. The structures of both proteins also reveal a putative, wide DNA-binding groove. Extensive mutagenesis of this groove, coupled with detailed biochemical analysis, identified residues that did not impact on SNM1A catalytic activity, but drastically reduced its processivity. Moreover, we identified a key role for this groove for efficient digestion past DNA interstrand crosslinks, facilitating the key DNA repair reaction catalysed by SNM1A. Together, the architecture and dimensions of this groove, coupled to the surrounding region of high positive charge, explain the remarkable ability of SNM1A to accommodate and efficiently digest highly distorted DNA substrates, such as those containing DNA lesions.

Brem J, van Berkel SS, Zollman D, Lee SY, Gileadi O, McHugh PJ, Walsh TR, McDonough MA, Schofield CJ. 2015. Structural Basis of Metallo-β-Lactamase Inhibition by Captopril Stereoisomers. Antimicrob Agents Chemother, 60 (1), pp. 142-150. | Show Abstract | Read more

β-Lactams are the most successful antibacterials, but their effectiveness is threatened by resistance, most importantly by production of serine- and metallo-β-lactamases (MBLs). MBLs are of increasing concern because they catalyze the hydrolysis of almost all β-lactam antibiotics, including recent-generation carbapenems. Clinically useful serine-β-lactamase inhibitors have been developed, but such inhibitors are not available for MBLs. l-Captopril, which is used to treat hypertension via angiotensin-converting enzyme inhibition, has been reported to inhibit MBLs by chelating the active site zinc ions via its thiol(ate). We report systematic studies on B1 MBL inhibition by all four captopril stereoisomers. High-resolution crystal structures of three MBLs (IMP-1, BcII, and VIM-2) in complex with either the l- or d-captopril stereoisomer reveal correlations between the binding mode and inhibition potency. The results will be useful in the design of MBL inhibitors with the breadth of selectivity required for clinical application against carbapenem-resistant Enterobacteriaceae and other organisms causing MBL-mediated resistant infections.

Iyama T, Lee SY, Berquist BR, Gileadi O, Bohr VA, Seidman MM, McHugh PJ, Wilson DM. 2015. CSB interacts with SNM1A and promotes DNA interstrand crosslink processing Nucleic Acids Research, 43 (1), pp. 247-258. | Show Abstract | Read more

© 2015 Published by Oxford University Press on behalf of Nucleic Acids Research. Cockayne syndrome (CS) is a premature aging disorder characterized by photosensitivity, impaired development and multisystem progressive degeneration, and consists of two strict complementation groups, A and B. Using a yeast two-hybrid approach, we identified the 5′-3′ exonuclease SNM1A as one of four strong interacting partners of CSB. This direct interaction was confirmed using purified recombinant proteins-with CSB able to modulate the exonuclease activity of SNM1A on oligonucleotide substrates in vitro-and the two proteins were shown to exist in a common complex in human cell extracts. CSB and SNM1A were also found, using fluorescently tagged proteins in combination with confocal microscopy and laser microirradiation, to be recruited to localized trioxsalen-induced ICL damage in human cells, with accumulation being suppressed by transcription inhibition. Moreover, SNM1A recruitment was significantly reduced in CSB-deficient cells, suggesting coordination between the two proteins in vivo. CSB-deficient neural cells exhibited increased sensitivity to DNA crosslinking agents, particularly, in a non-cycling, differentiated state, as well as delayed ICL processing as revealed by a modified Comet assay and γ-H2AX foci persistence. The results indicate that CSB coordinates the resolution of ICLs, possibly in a transcription-associated repair mechanism involving SNM1A, and that defects in the process could contribute to the post-mitotic degenerative pathologies associated with CS.

Iyama T, Lee SY, Berquist BR, Gileadi O, Bohr VA, Seidman MM, McHugh PJ, Wilson DM. 2015. CSB interacts with SNM1A and promotes DNA interstrand crosslink processing. Nucleic Acids Res, 43 (1), pp. 247-258. | Show Abstract | Read more

Cockayne syndrome (CS) is a premature aging disorder characterized by photosensitivity, impaired development and multisystem progressive degeneration, and consists of two strict complementation groups, A and B. Using a yeast two-hybrid approach, we identified the 5'-3' exonuclease SNM1A as one of four strong interacting partners of CSB. This direct interaction was confirmed using purified recombinant proteins-with CSB able to modulate the exonuclease activity of SNM1A on oligonucleotide substrates in vitro-and the two proteins were shown to exist in a common complex in human cell extracts. CSB and SNM1A were also found, using fluorescently tagged proteins in combination with confocal microscopy and laser microirradiation, to be recruited to localized trioxsalen-induced ICL damage in human cells, with accumulation being suppressed by transcription inhibition. Moreover, SNM1A recruitment was significantly reduced in CSB-deficient cells, suggesting coordination between the two proteins in vivo. CSB-deficient neural cells exhibited increased sensitivity to DNA crosslinking agents, particularly, in a non-cycling, differentiated state, as well as delayed ICL processing as revealed by a modified Comet assay and γ-H2AX foci persistence. The results indicate that CSB coordinates the resolution of ICLs, possibly in a transcription-associated repair mechanism involving SNM1A, and that defects in the process could contribute to the post-mitotic degenerative pathologies associated with CS.

McHugh P. 2014. The role of exonucleases in replication-coupled DNA interstrand crosslink repair MUTAGENESIS, 29 (6), pp. 532-532.

Hatch SB, Swift LP, Caporali S, Carter R, Hill EJ, Macgregor TP, D'Atri S, Middleton MR, McHugh PJ, Sharma RA. 2014. XPF protein levels determine sensitivity of malignant melanoma cells to oxaliplatin chemotherapy: Suitability as a biomarker for patient selection International Journal of Cancer, 134 (6), pp. 1495-1503. | Show Abstract | Read more

As the options for systemic treatment of malignant melanoma (MM) increase, the need to develop biomarkers to identify patients who might benefit from cytotoxic chemotherapy becomes more apparent. In preclinical models, oxaliplatin has activity in cisplatin-resistant cells. In this study, we have shown that oxaliplatin forms interstrand crosslinks (ICLs) in cellular DNA and that loss of the heterodimeric structure-specific endonuclease XPF-ERCC1 causes hypersensitivity to oxaliplatin in mammalian cells. XPF deficiency resulted in late S-phase arrest and persistence of double-strand breaks following oxaliplatin treatment. In a panel of 12 MM cell lines, oxaliplatin sensitivity correlated with XPF and ERCC1 protein levels. The knockdown of ERCC1 and XPF protein levels by RNA interference increased sensitivity of cancer cells to oxaliplatin; overexpression of exogenous ERCC1 significantly decreased drug sensitivity. Following immunohistochemical optimization, XPF protein levels were quantified in MM tissue samples from 183 patients, showing variation in expression and no correlation with prognosis. In 57 patients with MM treated with cisplatin or carboplatin, XPF protein levels did not predict the likelihood of clinical r esponse. We propose that oxaliplatin should not be discarded as a potential treatment for MM on the basis of the limited activity of cisplatin in unselected patients. Moreover, we show that XPF-ERCC1 protein levels are a key determinant of the sensitivity of melanoma cells to oxaliplatin in vitro. Immunohistochemical detection of XPF appears suitable for development as a tissue biomarker for potentially selecting patients for oxaliplatin treatment in a prospective clinical trial. What's new? With options for systemic treatment of malignant melanoma (MM) on the rise, there is an increasing need to develop biomarkers for patient selection. To that end, this study explored the possibility of a biomarker to improve objective response rates to the drug oxaliplatin. The study reveals a mechanism by which mammalian cells are rendered hypersensitive to oxaliplatin that centers around the loss of endonuclease XPF-ERCC1. Sensitivity to oxaliplatin was directly related to XPF and ERCC1 protein levels. The findings indicate that XPF may be a suitable biomarker for MM patient selection for oxaliplatin therapy. © 2013 UICC.

Srivas R, Costelloe T, Carvunis A-R, Sarkar S, Malta E, Sun SM, Pool M, Licon K, van Welsem T, van Leeuwen F et al. 2013. A UV-induced genetic network links the RSC complex to nucleotide excision repair and shows dose-dependent rewiring. Cell Rep, 5 (6), pp. 1714-1724. | Show Abstract | Read more

Efficient repair of UV-induced DNA damage requires the precise coordination of nucleotide excision repair (NER) with numerous other biological processes. To map this crosstalk, we generated a differential genetic interaction map centered on quantitative growth measurements of >45,000 double mutants before and after different doses of UV radiation. Integration of genetic data with physical interaction networks identified a global map of 89 UV-induced functional interactions among 62 protein complexes, including a number of links between the RSC complex and several NER factors. We show that RSC is recruited to both silenced and transcribed loci following UV damage where it facilitates efficient repair by promoting nucleosome remodeling. Finally, a comparison of the response to high versus low levels of UV shows that the degree of genetic rewiring correlates with dose of UV and reveals a network of dose-specific interactions. This study makes available a large resource of UV-induced interactions, and it illustrates a methodology for identifying dose-dependent interactions based on quantitative shifts in genetic networks.

Hatch SB, Swift LP, Caporali S, Carter R, Hill EJ, MacGregor TP, D'Atri S, Middleton MR, McHugh PJ, Sharma RA. 2014. XPF protein levels determine sensitivity of malignant melanoma cells to oxaliplatin chemotherapy: suitability as a biomarker for patient selection. Int J Cancer, 134 (6), pp. 1495-1503. | Show Abstract | Read more

As the options for systemic treatment of malignant melanoma (MM) increase, the need to develop biomarkers to identify patients who might benefit from cytotoxic chemotherapy becomes more apparent. In preclinical models, oxaliplatin has activity in cisplatin-resistant cells. In this study, we have shown that oxaliplatin forms interstrand crosslinks (ICLs) in cellular DNA and that loss of the heterodimeric structure-specific endonuclease XPF-ERCC1 causes hypersensitivity to oxaliplatin in mammalian cells. XPF deficiency resulted in late S-phase arrest and persistence of double-strand breaks following oxaliplatin treatment. In a panel of 12 MM cell lines, oxaliplatin sensitivity correlated with XPF and ERCC1 protein levels. The knockdown of ERCC1 and XPF protein levels by RNA interference increased sensitivity of cancer cells to oxaliplatin; overexpression of exogenous ERCC1 significantly decreased drug sensitivity. Following immunohistochemical optimization, XPF protein levels were quantified in MM tissue samples from 183 patients, showing variation in expression and no correlation with prognosis. In 57 patients with MM treated with cisplatin or carboplatin, XPF protein levels did not predict the likelihood of clinical response. We propose that oxaliplatin should not be discarded as a potential treatment for MM on the basis of the limited activity of cisplatin in unselected patients. Moreover, we show that XPF-ERCC1 protein levels are a key determinant of the sensitivity of melanoma cells to oxaliplatin in vitro. Immunohistochemical detection of XPF appears suitable for development as a tissue biomarker for potentially selecting patients for oxaliplatin treatment in a prospective clinical trial.

Nguyen GH, Dexheimer TS, Rosenthal AS, Chu WK, Singh DK, Mosedale G, Bachrati CZ, Schultz L, Sakurai M, Savitsky P et al. 2013. A small molecule inhibitor of the BLM helicase modulates chromosome stability in human cells. Chem Biol, 20 (1), pp. 55-62. | Show Abstract | Read more

The Bloom's syndrome protein, BLM, is a member of the conserved RecQ helicase family. Although cell lines lacking BLM exist, these exhibit progressive genomic instability that makes distinguishing primary from secondary effects of BLM loss problematic. In order to be able to acutely disable BLM function in cells, we undertook a high throughput screen of a chemical compound library for small molecule inhibitors of BLM. We present ML216, a potent inhibitor of the DNA unwinding activity of BLM. ML216 shows cell-based activity and can induce sister chromatid exchanges, enhance the toxicity of aphidicolin, and exert antiproliferative activity in cells expressing BLM, but not those lacking BLM. These data indicate that ML216 shows strong selectivity for BLM in cultured cells. We discuss the potential utility of such a BLM-targeting compound as an anticancer agent.

McHugh PJ, Ward TA, Chovanec M. 2012. A prototypical Fanconi anemia pathway in lower eukaryotes? Cell Cycle, 11 (20), pp. 3739-3744. | Show Abstract | Read more

DNA interstrand cross-links (ICLs) present a major challenge to cells, preventing separation of the two strands of duplex DNA and blocking major chromosome transactions, including transcription and replication. Due to the complexity of removing this form of DNA damage, no single DNA repair pathway has been shown to be capable of eradicating ICLs. In eukaryotes, ICL repair is a complex process, principally because several repair pathways compete for ICL repair intermediates in a strictly cell cycle-dependent manner. Yeast cells require a combination of nucleotide excision repair, homologous recombination repair and postreplication repair/translesion DNA synthesis to remove ICLs. There are also a number of additional ICL repair factors originally identified in the budding yeast Saccharomyces cerevisiae, called Pso1 though 10, of which Pso2 has an apparently dedicated role in ICL repair. Mammalian cells respond to ICLs by a complex network guided by factors mutated in the inherited cancer-prone disorder Fanconi anemia (FA). Although enormous progress has been made over recent years in identifying and characterizing FA factors as well as in elucidating certain aspects of the biology of FA, the mechanistic details of the ICL repair defects in FA patients remain unknown. Dissection of the FA DNA damage response pathway has, in part, been limited by the absence of FA-like pathways in highly tractable model organisms, such as yeast. Although S. cerevisiae possesses putative homologs of the FA factors FANCM, FANCJ and FANCP (Mph1, Chl1 and Slx4, respectively) as well as of the FANCM-associated proteins MHF1 and MHF2 (Mhf1 and Mhf2), the corresponding mutants display no significant increase in sensitivity to ICLs. Nevertheless, we and others have recently shown that these FA homologs, along with several other factors, control an ICL repair pathway, which has an overlapping or redundant role with a Pso2-controlled pathway. This pathway acts in S-phase and serves to prevent ICL-stalled replication forks from collapsing into DNA double-strand breaks.

Ward TA, Dudášová Z, Sarkar S, Bhide MR, Vlasáková D, Chovanec M, McHugh PJ. 2012. Components of a Fanconi-like pathway control Pso2-independent DNA interstrand crosslink repair in yeast. PLoS Genet, 8 (8), pp. e1002884. | Show Abstract | Read more

Fanconi anemia (FA) is a devastating genetic disease, associated with genomic instability and defects in DNA interstrand cross-link (ICL) repair. The FA repair pathway is not thought to be conserved in budding yeast, and although the yeast Mph1 helicase is a putative homolog of human FANCM, yeast cells disrupted for MPH1 are not sensitive to ICLs. Here, we reveal a key role for Mph1 in ICL repair when the Pso2 exonuclease is inactivated. We find that the yeast FANCM ortholog Mph1 physically and functionally interacts with Mgm101, a protein previously implicated in mitochondrial DNA repair, and the MutSα mismatch repair factor (Msh2-Msh6). Co-disruption of MPH1, MGM101, MSH6, or MSH2 with PSO2 produces a lesion-specific increase in ICL sensitivity, the elevation of ICL-induced chromosomal rearrangements, and persistence of ICL-associated DNA double-strand breaks. We find that Mph1-Mgm101-MutSα directs the ICL-induced recruitment of Exo1 to chromatin, and we propose that Exo1 is an alternative 5'-3' exonuclease utilised for ICL repair in the absence of Pso2. Moreover, ICL-induced Rad51 chromatin loading is delayed when both Pso2 and components of the Mph1-Mgm101-MutSα and Exo1 pathway are inactivated, demonstrating that the homologous recombination stages of ICL repair are inhibited. Finally, the FANCJ- and FANCP-related factors Chl1 and Slx4, respectively, are also components of the genetic pathway controlled by Mph1-Mgm101-MutSα. Together this suggests that a prototypical FA-related ICL repair pathway operates in budding yeast, which acts redundantly with the pathway controlled by Pso2, and is required for the targeting of Exo1 to chromatin to execute ICL repair.

Sengerová B, Allerston CK, Abu M, Lee SY, Hartley J, Kiakos K, Schofield CJ, Hartley JA, Gileadi O, McHugh PJ. 2012. Characterization of the human SNM1A and SNM1B/Apollo DNA repair exonucleases. J Biol Chem, 287 (31), pp. 26254-26267. | Show Abstract | Read more

Human SNM1A and SNM1B/Apollo have both been implicated in the repair of DNA interstrand cross-links (ICLs) by cellular studies, and SNM1B is also required for telomere protection. Here, we describe studies on the biochemical characterization of the SNM1A and SNM1B proteins. The results reveal some fundamental differences in the mechanisms of the two proteins. Both SNM1A and SNM1B digest double-stranded and single-stranded DNA with a 5'-to-3' directionality in a reaction that is stimulated by divalent cations, and both nucleases are inhibited by the zinc chelator o-phenanthroline. We find that SNM1A has greater affinity for single-stranded DNA over double-stranded DNA that is not observed with SNM1B. Although both proteins demonstrate a low level of processivity on low molecular weight DNA oligonucleotide substrates, when presented with high molecular weight DNA, SNM1A alone is rendered much more active, being capable of digesting kilobase-long stretches of DNA. Both proteins can digest past ICLs induced by the non-distorting minor groove cross-linking agent SJG-136, albeit with SNM1A showing a greater capacity to achieve this. This is consistent with the proposal that SNM1A and SNM1B might exhibit some redundancy in ICL repair. Together, our work establishes differences in the substrate selectivities of SNM1A and SNM1B that are likely to be relevant to their in vivo roles and which might be exploited in the development of selective inhibitors.

Olsen AL, Davies JM, Medley L, Breen D, Talbot DC, McHugh PJ. 2012. Quantitative analysis of survivin protein expression and its therapeutic depletion by an antisense oligonucleotide in human lung tumors. Mol Ther Nucleic Acids, 1 pp. e30. | Show Abstract | Read more

RNA-directed antisense and interference therapeutics are a promising treatment option for cancer. The demonstration of depletion of target proteins within human tumors in vivo using validated methodology will be a key to the application of this technology. Here, we present a flow cytometric-based approach to quantitatively determine protein levels in solid tumor material derived by fiber optic brushing (FOB) of non-small cell lung cancer (NSCLC) patients. Focusing upon the survivin protein, and its depletion by an antisense oligonucleotide (ASO) (LY2181308), we show that we can robustly identify a subpopulation of survivin positive tumor cells in FOB samples, and, moreover, detect survivin depletion in tumor samples from a patient treated with LY2181308. Survivin depletion appears to be a result of treatment with this ASO, because a tumor treated with conventional cytotoxic chemotherapy did not exhibit a decreased percentage of survivin positive cells. Our approach is likely to be broadly applicable to, and useful for, the quantification of protein levels in tumor samples obtained as part of clinical trials and studies, facilitating the proof-of-principle testing of novel targeted therapies.

Sengerová B, Wang AT, McHugh PJ. 2011. Orchestrating the nucleases involved in DNA interstrand cross-link (ICL) repair. Cell Cycle, 10 (23), pp. 3999-4008. | Show Abstract | Read more

DNA interstrand cross-links (ICLs) pose a significant threat to genomic and cellular integrity by blocking essential cellular processes, including replication and transcription. In mammalian cells, much ICL repair occurs in association with DNA replication during S phase, following the stalling of a replication fork at the block caused by an ICL lesion. Here, we review recent work showing that the XPF-ERCC1 endonuclease and the hSNM1A exonuclease act in the same pathway, together with SLX4, to initiate ICL repair, with the MUS81-EME1 fork incision activity becoming important in the absence of the XPF-SNM1A-SLX4-dependent pathway. Another nuclease, the Fanconi anemia-associated nuclease (FAN1), has recently been implicated in the repair of ICLs, and we discuss the possible ways in which the activities of different nucleases at the ICL-stalled replication fork may be coordinated. In relation to this, we briefly speculate on the possible role of SLX4, which contains XPF and MUS81- interacting domains, in the coordination of ICL repair nucleases.

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Wang AT, Sengerová B, Cattell E, Inagawa T, Hartley JM, Kiakos K, Burgess-Brown NA, Swift LP, Enzlin JH, Schofield CJ et al. 2011. Human SNM1a and XPF-ERCC1 collaborate to initiate DNA interstrand cross-link repair Genes and Development, 25 (17), pp. 1859-1870. | Show Abstract | Read more

One of the major DNA interstrand cross-link (ICL) repair pathways in mammalian cells is coupled to replication, but the mechanistic roles of the critical factors involved remain largely elusive. Here, we show that purified human SNM1A (hSNM1A), which exhibits a 59-39 exonuclease activity, can load from a single DNA nick and digest past an ICL on its substrate strand. hSNM1A-depleted cells are ICL-sensitive and accumulate replicationassociated DNA double-strand breaks (DSBs), akin to ERCC1-depleted cells. These DSBs are Mus81-induced, indicating that replication fork cleavage by Mus81 results from the failure of the hSNM1A- and XPF-ERCC1-dependent ICL repair pathway. Our results reveal how collaboration between hSNM1A and XPF-ERCC1 is necessary to initiate ICL repair in replicating human cells. © 2011 by Cold Spring Harbor Laboratory Press.

Ashton TM, Mankouri HW, Heidenblut A, McHugh PJ, Hickson ID. 2011. Pathways for Holliday junction processing during homologous recombination in Saccharomyces cerevisiae. Mol Cell Biol, 31 (9), pp. 1921-1933. | Show Abstract | Read more

The Saccharomyces cerevisiae Rmi1 protein is a component of the highly conserved Sgs1-Top3-Rmi1 complex. Deletion of SGS1, TOP3, or RMI1 is synthetically lethal when combined with the loss of the Mus81-Mms4 or Slx1-Slx4 endonucleases, which have been implicated in Holliday junction (HJ) resolution. To investigate the causes of this synthetic lethality, we isolated a temperature-sensitive mutant of the RMI1 strain, referred to as the rmi1-1 mutant. At the restrictive temperature, this mutant phenocopies an rmi1Δ strain but behaves like the wild type at the permissive temperature. Following a transient exposure to methyl methanesulfonate, rmi1-1 mutants accumulate unprocessed homologous recombination repair (HRR) intermediates. These intermediates are slowly resolved at the restrictive temperature, revealing a redundant resolution activity when Rmi1 is impaired. This resolution depends on Mus81-Mms4 but not on either Slx1-Slx4 or another HJ resolvase, Yen1. Similar results were also observed when Top3 function was impaired. We propose that the Sgs1-Top3-Rmi1 complex constitutes the main pathway for the processing of HJ-containing HRR intermediates but that Mus81-Mms4 can also resolve these intermediates.

Tafel AA, Wu L, McHugh PJ. 2011. Human HEL308 localizes to damaged replication forks and unwinds lagging strand structures. J Biol Chem, 286 (18), pp. 15832-15840. | Show Abstract | Read more

HEL308 is a superfamily II DNA helicase, conserved from archaea through to humans. HEL308 family members were originally isolated by their similarity to the Drosophila melanogaster Mus308 protein, which contributes to the repair of replication-blocking lesions such as DNA interstrand cross-links. Biochemical studies have established that human HEL308 is an ATP-dependent enzyme that unwinds DNA with a 3' to 5' polarity, but little else is know about its mechanism. Here, we show that GFP-tagged HEL308 localizes to replication forks following camptothecin treatment. Moreover, HEL308 colocalizes with two factors involved in the repair of damaged forks by homologous recombination, Rad51 and FANCD2. Purified HEL308 requires a 3' single-stranded DNA region to load and unwind duplex DNA structures. When incubated with substrates that model stalled replication forks, HEL308 preferentially unwinds the parental strands of a structure that models a fork with a nascent lagging strand, and the unwinding action of HEL308 is specifically stimulated by human replication protein A. Finally, we show that HEL308 appears to target and unwind from the junction between single-stranded to double-stranded DNA on model fork structures. Together, our results suggest that one role for HEL308 at sites of blocked replication might be to open up the parental strands to facilitate the loading of subsequent factors required for replication restart.

Sarkar S, Kiely R, McHugh PJ. 2010. The Ino80 chromatin-remodeling complex restores chromatin structure during UV DNA damage repair. J Cell Biol, 191 (6), pp. 1061-1068. | Show Abstract | Read more

Chromatin structure is modulated during deoxyribonucleic acid excision repair, but how this is achieved is unclear. Loss of the yeast Ino80 chromatin-remodeling complex (Ino80-C) moderately sensitizes cells to ultraviolet (UV) light. In this paper, we show that INO80 acts in the same genetic pathway as nucleotide excision repair (NER) and that the Ino80-C contributes to efficient UV photoproduct removal in a region of high nucleosome occupancy. Moreover, Ino80 interacts with the early NER damage recognition complex Rad4-Rad23 and is recruited to chromatin by Rad4 in a UV damage-dependent manner. Using a modified chromatin immunoprecipitation assay, we find that chromatin disruption during UV lesion repair is normal, whereas the restoration of nucleosome structure is defective in ino80 mutant cells. Collectively, our work suggests that Ino80 is recruited to sites of UV lesion repair through interactions with the NER apparatus and is required for the restoration of chromatin structure after repair.

Talbot DC, Ranson M, Davies J, Lahn M, Callies S, André V, Kadam S, Burgess M, Slapak C, Olsen AL et al. 2010. Tumor survivin is downregulated by the antisense oligonucleotide LY2181308: a proof-of-concept, first-in-human dose study. Clin Cancer Res, 16 (24), pp. 6150-6158. | Show Abstract | Read more

PURPOSE: Enhanced tumor cell survival through expression of inhibitors of apoptosis (IAP) is a hallmark of cancer. Survivin, an IAP absent from most normal tissues, is overexpressed in many malignancies and associated with a poorer prognosis. We report the first-in-human dose study of LY2181308, a second-generation antisense oligonucleotide (ASO) directed against survivin mRNA. PATIENTS AND METHODS: A dose-escalation study evaluating the safety, pharmacokinetics, and pharmacodynamics of LY2181308 administered intravenously for 3 hours as a loading dose on 3 consecutive days and followed by weekly maintenance doses. Patients were eligible after signing informed consent, had exhausted approved anticancer therapies and agreed to undergo pre- and posttreatment tumor biopsies to evaluate reduction of survivin protein and gene expression. RESULTS: A total of 40 patients were treated with LY2181308 at doses of 100 to 1,000 mg. Twenty-six patients were evaluated at the recommended phase 2 dose of 750 mg, at which level serial tumor sampling and [(11)C]LY2183108 PET (positron emission tomography) imaging demonstrated that ASO accumulated within tumor tissue, reduced survivin gene and protein expression by 20% and restored apoptotic signaling in tumor cells in vivo. Pharmacokinetics were consistent with preclinical modeling, exhibiting rapid tissue distribution, and terminal half-life of 31 days. CONCLUSIONS: The tumor-specific, molecularly targeted effects demonstrated by this ASO in man underpin confirmatory studies evaluating its therapeutic efficacy in cancer.

Cattell E, Sengerová B, McHugh PJ. 2010. The SNM1/Pso2 family of ICL repair nucleases: from yeast to man. Environ Mol Mutagen, 51 (6), pp. 635-645. | Show Abstract | Read more

Efficient interstrand crosslink (ICL) repair in yeast depends on the Pso2/Snm1 protein. Pso2 is a member of the highly conserved metallo-beta-lactamase structural family of nucleases. Mammalian cells possess three SNM1/Pso2 related proteins, SNM1A, SNM1B/Apollo, and SNM1C/Artemis. Evidence that SNM1A and SNM1B contribute to ICL repair is mounting, whereas Artemis appears to primarily contribute to non-ICL repair pathways, particularly some double-strand break repair events. Yeast Pso2 and all three mammalian SNM1-family proteins have been shown to possess nuclease activity. Here, we review the biochemical, genetic, and cellular evidence for the SNM1 family as DNA repair factors, focusing on ICL repair.

Medley L, West S, Davies J, Clelland C, Olsen A, McHugh P, Talbot D. 2009. Diagnostic bronchoscopy: a window of opportunity for timely biomarker driven therapy in non-small cell lung cancer (NSCLC) JOURNAL OF THORACIC ONCOLOGY, 4 (9), pp. S317-S317.

Talbot DC, Davies J, Olsen A, Andre V, Lahn M, Powell E, Kadam S, de Bono J, McHugh P, Ranson M. 2009. Pharmacodynamic (PD) evaluation of LY2181308 in patients with metastatic malignancies. J Clin Oncol, 27 (15_suppl), pp. 3507. | Show Abstract

3507 Background: Survivin is an inhibitor of apoptosis and its overexpression in cancer has been associated with poor survival. LY2181308 (LY), a novel modified antisense oligonucleotide is a specific inhibitor of survivin. The safety and PK profile of LY from this first-in-human study was presented at ASCO 2008. We now present the PD data from this trial. METHODS: Patients with advanced or metastatic malignancies who had failed previous anti-tumor treatments were enrolled. Three consecutive IV loading doses given over 3 hours were followed by weekly maintenance doses. Pre- and post-dosing biopsies were mandated to test for evidence of modification of the target at doses where a PD effect was expected. Biopsies were taken 48 hours after the last loading dose by CT-guided fine needle biopsy. Tumor tissue was paraffin-embedded for immunohistochemistry (IHC) and survivin gene expression. Given the finite amount of biopsy material, the quantification of survivin protein was prioritized. In addition, at one site, pre- and post-dosing endobronchial sampling was conducted in NSCLC patients, with the aim of quantifying levels of native survivin protein, and assessing changes in cell cycle profile in freshly isolated tumor cells using FACS analysis. RESULTS: Out of the 34 patients enrolled, 22 patients had a pre- and posttreatment biopsy. Results from IHC indicated that survivin expression was reduced in the nucleus and cytoplasm in 11/17 and 5/14 evaluable pairs, respectively. LY was detected in neoplastic and non-neoplastic tumor cells in 5/16 and 15/16 evaluable pairs respectively. Gene expression analysis indicated a reduction in survivin expression by 20% to 50% in 11/15 evaluable pairs. Analysis of the fresh tumor material from endobronchial sampling revealed that 2 out of 3 patients with NSCLC had a near-complete elimination of survivin-positive cells accompanied by an increase in the fraction of cells with a sub-G1 DNA content, consistent with cell death. CONCLUSIONS: In this study, we were able to demonstrate the presence of the ASO in tumor tissue and confirm a reduction in survivin protein and gene expression. These findings demonstrate the proof of principle of antitumor activity of LY and provide the rationale for phase II studies. [Table: see text].

Medley L, West S, Davies J, Clelland C, Olsen A, McHugh P, Talbot D. 2009. The potential for tailored therapy based on molecular characteristics of endobronchial tumour sampling at diagnosis LUNG CANCER, 63 pp. S13-S14.

Bhagwat N, Olsen AL, Wang AT, Hanada K, Stuckert P, Kanaar R, D'Andrea A, Niedernhofer LJ, McHugh PJ. 2009. XPF-ERCC1 participates in the Fanconi anemia pathway of cross-link repair. Mol Cell Biol, 29 (24), pp. 6427-6437. | Show Abstract | Read more

Interstrand cross-links (ICLs) prevent DNA strand separation and, therefore, transcription and replication, making them extremely cytotoxic. The precise mechanism by which ICLs are removed from mammalian genomes largely remains elusive. Genetic evidence implicates ATR, the Fanconi anemia proteins, proteins required for homologous recombination, translesion synthesis, and at least two endonucleases, MUS81-EME1 and XPF-ERCC1. ICLs cause replication-dependent DNA double-strand breaks (DSBs), and MUS81-EME1 facilitates DSB formation. The subsequent repair of these DSBs occurs via homologous recombination after the ICL is unhooked by XPF-ERCC1. Here, we examined the effect of the loss of either nuclease on FANCD2 monoubiquitination to determine if the nucleolytic processing of ICLs is required for the activation of the Fanconi anemia pathway. FANCD2 was monoubiquitinated in Mus81(-/-), Ercc1(-/-), and XPF-deficient human, mouse, and hamster cells exposed to cross-linking agents. However, the monoubiquitinated form of FANCD2 persisted longer in XPF-ERCC1-deficient cells than in wild-type cells. Moreover, the levels of chromatin-bound FANCD2 were dramatically reduced and the number of ICL-induced FANCD2 foci significantly lower in XPF-ERCC1-deficient cells. These data demonstrate that the unhooking of an ICL by XPF-ERCC1 is necessary for the stable localization of FANCD2 to the chromatin and subsequent homologous recombination-mediated DSB repair.

Wang AT, McHugh PJ. 2009. Apollo: a healer of the genome? Cell Cycle, 8 (13), pp. 1980-1981.

Plummer R, Jones C, Middleton M, Wilson R, Evans J, Olsen A, Curtin N, Boddy A, McHugh P, Newell D et al. 2008. Phase I study of the poly(ADP-ribose) polymerase inhibitor, AG014699, in combination with temozolomide in patients with advanced solid tumors. Clin Cancer Res, 14 (23), pp. 7917-7923. | Show Abstract | Read more

PURPOSE: One mechanism of tumor resistance to cytotoxic therapy is repair of damaged DNA. Poly(ADP-ribose) polymerase (PARP)-1 is a nuclear enzyme involved in base excision repair, one of the five major repair pathways. PARP inhibitors are emerging as a new class of agents that can potentiate chemotherapy and radiotherapy. The article reports safety, efficacy, pharmacokinetic, and pharmacodynamic results of the first-in-class trial of a PARP inhibitor, AG014699, combined with temozolomide in adults with advanced malignancy. EXPERIMENTAL DESIGN: Initially, patients with solid tumors received escalating doses of AG014699 with 100 mg/m2/d temozolomide x 5 every 28 days to establish the PARP inhibitory dose (PID). Subsequently, AG014699 dose was fixed at PID and temozolomide escalated to maximum tolerated dose or 200 mg/m2 in metastatic melanoma patients whose tumors were biopsied. AG014699 and temozolomide pharmacokinetics, PARP activity, DNA strand single-strand breaks, response, and toxicity were evaluated. RESULTS: Thirty-three patients were enrolled. PARP inhibition was seen at all doses; PID was 12 mg/m2 based on 74% to 97% inhibition of peripheral blood lymphocyte PARP activity. Recommended doses were 12 mg/m2 AG014699 and 200 mg/m2 temozolomide. Mean tumor PARP inhibition at 5 h was 92% (range, 46-97%). No toxicity attributable to AG014699 alone was observed. AG014699 showed linear pharmacokinetics with no interaction with temozolomide. All patients treated at PID showed increases in DNA single-strand breaks and encouraging evidence of activity was seen. CONCLUSIONS: The combination of AG014699 and temozolomide is well tolerated, pharmacodynamic assessments showing proof of principle of the mode of action of this new class of agents.

Hazrati A, Ramis-Castelltort M, Sarkar S, Barber LJ, Schofield CJ, Hartley JA, McHugh PJ. 2008. Human SNM1A suppresses the DNA repair defects of yeast pso2 mutants. DNA Repair (Amst), 7 (2), pp. 230-238. | Show Abstract | Read more

Pso2/Snm1 plays a key role in the repair of DNA interstrand cross-links in yeast. Human cells possess three orthologues of Pso2; SNM1A, SNM1B/Apollo and SNM1C/Artemis. Studies using mammalian cells disrupted or depleted for these genes have yielded equivocal evidence that any of these is a true functional homologues of the yeast gene. Here we show that ectopic expression of only one of the three human orthologues, hSNM1A, effectively suppresses the sensitivity of yeast pso2 (snm1) disruptants to cross-linking agents. Two other phenotypes of the pso2 mutants are also partially rescued by ectopic expression of hSNM1A, namely the double-strand repair break defect observed during cross-link processing in pso2 cells, as well as the spontaneous intrachromatid recombination defect of pso2 msh2 double mutants. Finally, we show that recombinant hSNM1A is a 5'-exonuclease, as also recently reported for the yeast Pso2 protein. Together our data suggest that hSnm1A is a functional homologue of yeast Pso2/Snm1.

Kiely R, Sarkar S, McHugh P. 2007. The INO80 and SWR complexes are both required for efficient nucleotide excision repair in chromatin MUTAGENESIS, 22 (6), pp. 445-446.

Kiakos K, Sato A, Asao T, McHugh PJ, Lee M, Hartley JA. 2007. DNA sequence selective adenine alkylation, mechanism of adduct repair, and in vivo antitumor activity of the novel achiral seco-amino-cyclopropylbenz[e]indolone analogue of duocarmycin AS-I-145. Mol Cancer Ther, 6 (10), pp. 2708-2718. | Show Abstract | Read more

AS-I-145 is a novel achiral seco-amino-cyclopropylbenz[e]indolone (seco-amino-CBI) analogue of duocarmycin that has evolved from an alternative strategy of designing CC-1065/duocarmycin agents lacking the characteristic chiral center of the natural agents. The sequence specificity of this compound was assessed by a Taq polymerase stop assay, identifying the sites of covalent modification on plasmid DNA. The adenine-N3 adducts were confirmed at AT-rich sequences using a thermally induced strand cleavage assay. These studies reveal that this compound retains the inherent sequence selectivity of the related natural compounds. The AS-I-145 sensitivity of yeast mutants deficient in excision and post-replication repair (PRR) pathways was assessed. The sensitivity profile suggests that the sequence-specific adenine-N3 adducts are substrates for nucleotide excision repair (NER) but not base excision repair (BER). Single-strand ligation PCR was employed to follow the induction and repair of the lesions at nucleotide resolution in yeast cells. Sequence specificity was preserved in intact cells, and adduct elimination occurred in a transcription-coupled manner and was dependent on a functional NER pathway and Rad18. The involvement of NER as the predominant excision pathway was confirmed in mammalian DNA repair mutant cells. AS-I-145 showed good in vivo antitumor activity in the National Cancer Institute standard hollow fiber assay and was active against the human breast MDA-MD-435 xenograft when administered i.v. or p.o. Its novel structure and in vivo activity renders AS-I-145 a new paradigm in the design of novel achiral analogues of CC-1065 and the duocarmycins.

Lehoczký P, McHugh PJ, Chovanec M. 2007. DNA interstrand cross-link repair in Saccharomyces cerevisiae. FEMS Microbiol Rev, 31 (2), pp. 109-133. | Show Abstract | Read more

DNA interstrand cross-links (ICL) present a formidable challenge to the cellular DNA repair apparatus. For Escherichia coli, a pathway which combines nucleotide excision repair (NER) and homologous recombination repair (HRR) to eliminate ICL has been characterized in detail, both genetically and biochemically. Mechanisms of ICL repair in eukaryotes have proved more difficult to define, primarily as a result of the fact that several pathways appear compete for ICL repair intermediates, and also because these competing activities are regulated in the cell cycle. The budding yeast Saccharomyces cerevisiae has proven a powerful tool for dissecting ICL repair. Important roles for NER, HRR and postreplication/translesion synthesis pathways have all been identified. Here we review, with reference to similarities and differences in higher eukaryotes, what has been discovered to date concerning ICL repair in this simple eukaryote.

McHugh PJ, Sarkar S. 2006. DNA interstrand cross-link repair in the cell cycle: a critical role for polymerase zeta in G1 phase. Cell Cycle, 5 (10), pp. 1044-1047. | Show Abstract | Read more

DNA interstrand cross-links (ICLs) present a formidable challenge to the cellular repair apparatus, but to date ICL repair pathways have proved difficult to dissect genetically. It now appears that this is partly the result of a high degree of cell cycle phase selectivity in the choice of ICL pathway employed. Here we review recent results showing that Polymerase zeta, specialized translesion plays an important role during ICL repair in G1 phase yeast cells, and that PCNA modification by ubiquitin is a key regulator of its activity. Given that this reaction can occur outside the context of S-phase, these results imply a more general role for PCNA modification in the control of DNA repair pathways through the cell cycle, which is dependent on the type of damage or repair intermediate encountered.

Sarkar S, Davies AA, Ulrich HD, McHugh PJ. 2006. DNA interstrand crosslink repair during G1 involves nucleotide excision repair and DNA polymerase zeta. EMBO J, 25 (6), pp. 1285-1294. | Show Abstract | Read more

The repair mechanisms acting on DNA interstrand crosslinks (ICLs) in eukaryotes are poorly understood. Here, we provide evidence for a pathway of ICL processing that uses components from both nucleotide excision repair (NER) and translesion synthesis (TLS) and predominates during the G1 phase of the yeast cell cycle. Our results suggest that repair is initiated by the NER apparatus and is followed by a thwarted attempt at gap-filling by the replicative Polymerase delta, which likely stalls at the site of the remaining crosslinked oligonucleotide. This in turn leads to ubiquitination of PCNA and recruitment of the damage-tolerant Polymerase zeta that can perform TLS. The ICL repair factor Pso2 acts downstream of the incision step and is not required for Polymerase zeta activation. We show that this combination of NER and TLS is the only pathway of ICL repair available to the cell in G1 phase and is essential for viability in the presence of DNA crosslinks.

Matsushita N, Kitao H, Ishiai M, Nagashima N, Hirano S, Okawa K, Ohta T, Yu DS, McHugh PJ, Hickson ID et al. 2005. A FancD2-monoubiquitin fusion reveals hidden functions of Fanconi anemia core complex in DNA repair. Mol Cell, 19 (6), pp. 841-847. | Show Abstract | Read more

In DNA damage responses, the Fanconi anemia (FA) protein, FancD2, is targeted to chromatin and forms nuclear foci following its monoubiquitination, a process likely catalyzed by the FA core complex. Here, we show that a chicken FancD2-ubiquitin fusion protein, carrying a Lys-Arg substitution removing the natural monoubiquitination site (D2KR-Ub), could reverse cisplatin hypersensitivity and localize to chromatin in FANCD2-deficient DT40 cells. Importantly, the chromatin targeting was dependent on three core complex components as well as the hydrophobic surface of ubiquitin that may direct protein-protein interactions. Furthermore, a constitutively chromatin bound fusion of D2KR-histone H2B could complement cisplatin sensitivity in FANCD2- but not FANCC-, FANCG-, or FANCL-deficient cells. Thus these core complex components have an additional function in the DNA repair, which is independent of the monoubiquitination and chromatin targeting of FancD2. These results define functional consequences of FancD2 monoubiquitination and reveal previously hidden functions for the FA protein core complex.

Clingen PH, De Silva IU, McHugh PJ, Ghadessy FJ, Tilby MJ, Thurston DE, Hartley JA. 2005. The XPF-ERCC1 endonuclease and homologous recombination contribute to the repair of minor groove DNA interstrand crosslinks in mammalian cells produced by the pyrrolo[2,1-c][1,4]benzodiazepine dimer SJG-136. Nucleic Acids Res, 33 (10), pp. 3283-3291. | Show Abstract | Read more

SJG-136, a pyrrolo[2,1-c][1,4]benzodiazepine (PBD) dimer, is a highly efficient interstrand crosslinking agent that reacts with guanine bases in a 5'-GATC-3' sequence in the DNA minor groove. SJG-136 crosslinks form rapidly and persist compared to those produced by conventional crosslinking agents such as nitrogen mustard, melphalan or cisplatin which bind in the DNA major groove. A panel of Chinese hamster ovary (CHO) cells with defined defects in specific DNA repair pathways were exposed to the bi-functional agents SJG-136 and melphalan, and to their mono-functional analogues mmy-SJG and mono-functional melphalan. SJG-136 was >100 times more cytotoxic than melphalan, and the bi-functional agents were much more cytotoxic than their respective mono-functional analogues. Cellular sensitivity of both SJG-136 and melphalan was dependent on the XPF-ERCC1 heterodimer, and homologous recombination repair factors XRCC2 and XRCC3. The relative level of sensitivity of these repair mutant cell lines to SJG-136 was, however, significantly less than with major groove crosslinking agents. In contrast to melphalan, there was no clear correlation between sensitivity to SJG-136 and crosslink unhooking capacity measured using a modified comet assay. Furthermore, repair of SJG-136 crosslinks did not involve the formation of DNA double-strand breaks. SJG-136 cytotoxicity is likely to result from the poor recognition of DNA damage by repair proteins resulting in the slow repair of both mono-adducts and more importantly crosslinks in the minor groove.

Barber LJ, Ward TA, Hartley JA, McHugh PJ. 2005. DNA interstrand cross-link repair in the Saccharomyces cerevisiae cell cycle: overlapping roles for PSO2 (SNM1) with MutS factors and EXO1 during S phase. Mol Cell Biol, 25 (6), pp. 2297-2309. | Show Abstract | Read more

Pso2/Snm1 is a member of the beta-CASP metallo-beta-lactamase family of proteins that include the V(D)J recombination factor Artemis. Saccharomyces cerevisiae pso2 mutants are specifically sensitive to agents that induce DNA interstrand cross-links (ICLs). Here we establish a novel overlapping function for PSO2 with MutS mismatch repair factors and the 5'-3' exonuclease Exo1 in the repair of DNA ICLs, which is confined to S phase. Our data demonstrate a requirement for NER and Pso2, or Exo1 and MutS factors, in the processing of ICLs, and this is required prior to the repair of ICL-induced DNA double-strand breaks (DSBs) that form during replication. Using a chromosomally integrated inverted-repeat substrate, we also show that loss of both pso2 and exo1/msh2 reduces spontaneous homologous recombination rates. Therefore, PSO2, EXO1, and MSH2 also appear to have overlapping roles in the processing of some forms of endogenous DNA damage that occur at an irreversibly collapsed replication fork. Significantly, our analysis of ICL repair in cells synchronized for each cell cycle phase has revealed that homologous recombination does not play a major role in the direct repair of ICLs, even in G2, when a suitable template is readily available. Rather, we propose that recombination is primarily involved in the repair of DSBs that arise from the collapse of replication forks at ICLs. These findings have led to considerable clarification of the complex genetic relationship between various ICL repair pathways.

Plummer ER, Middleton MR, Jones C, Olsen A, Hickson I, McHugh P, Margison GP, McGown G, Thorncroft M, Watson AJ et al. 2005. Temozolomide pharmacodynamics in patients with metastatic melanoma: dna damage and activity of repair enzymes O6-alkylguanine alkyltransferase and poly(ADP-ribose) polymerase-1. Clin Cancer Res, 11 (9), pp. 3402-3409. | Show Abstract | Read more

PURPOSE: Temozolomide, a DNA methylating agent used to treat melanoma, induces DNA damage, which is repaired by O6-alkylguanine alkyltransferase (ATase) and poly(ADP-ribose) polymerase-1 (PARP-1)-dependent base excision repair. The current study was done to define the effect of temozolomide on DNA integrity and relevant repair enzymes as a prelude to a phase I trial of the combination of temozolomide with a PARP inhibitor. EXPERIMENTAL DESIGN: Temozolomide (200 mg/m2 oral administration) was given to 12 patients with metastatic malignant melanoma. Peripheral blood lymphocytes (PBL) were analyzed for PARP activity, DNA single-strand breakage, ATase levels, and DNA methylation. PARP activity was also measured in tumor biopsies from 9 of 12 patients and in PBLs from healthy volunteers. RESULTS: Temozolomide pharmacokinetics were consistent with previous reports. Temozolomide therapy caused a substantial and sustained elevation of N7-methylguanine levels, a modest and sustained reduction in ATase activity, and a modest and transient increase in DNA strand breaks and PARP activity in PBLs. PARP-1 activity in tumor homogenates was variable (828 +/- 599 pmol PAR monomer/mg protein) and was not consistently affected by temozolomide treatment. CONCLUSIONS: The effect of temozolomide reported here are consistent with those documented in previous studies with temozolomide and similar drug, dacarbazine, demonstrating that a representative patient population was investigated. Furthermore, PARP activity was not inhibited by temozolomide treatment and this newly validated pharmacodynamic assay is therefore suitable for use in a proof-of-principle phase I trial a PARP-1 inhibitor in combination with temozolomide.

Lambert S, Mason SJ, Barber LJ, Hartley JA, Pearce JA, Carr AM, McHugh PJ. 2003. Schizosaccharomyces pombe checkpoint response to DNA interstrand cross-links. Mol Cell Biol, 23 (13), pp. 4728-4737. | Show Abstract | Read more

Drugs that produce covalent interstrand cross-links (ICLs) in DNA remain central to the treatment of cancer, but the cell cycle checkpoints activated by ICLs have received little attention. We have used the fission yeast, Schizosaccharomyces pombe, to elucidate the checkpoint responses to the ICL-inducing anticancer drugs nitrogen mustard and mitomycin C. First we confirmed that the repair pathways acting on ICLs in this yeast are similar to those in the main organisms studied to date (Escherichia coli, budding yeast, and mammalian cells), principally nucleotide excision repair and homologous recombination. We also identified and disrupted the S. pombe homologue of the Saccharomyces cerevisiae SNM1/PSO2 ICL repair gene and found that this activity is required for normal resistance to cross-linking agents, but not other forms of DNA damage. Survival and biochemical analysis indicated a key role for the "checkpoint Rad" family acting through the chk1-dependent DNA damage checkpoint in the ICL response. Rhp9-dependent phosphorylation of Chk1 correlates with G(2) arrest following ICL induction. In cells able to bypass the G(2) block, a second-cycle (S-phase) arrest was observed. Only a transient activation of the Cds1 DNA replication checkpoint factor occurs following ICL formation in wild-type cells, but this is increased and persists in G(2) arrest-deficient mutants. This likely reflects the fraction of cells escaping the G(2) damage checkpoint and arresting in the subsequent S phase due to ICL replication blocks. Disruption of cds1 confers increased resistance to ICLs, suggesting that this second-cycle S-phase arrest might be a lethal event.

Kiakos K, Howard TT, Lee M, Hartley JA, McHugh PJ. 2002. Saccharomyces cerevisiae RAD5 influences the excision repair of DNA minor groove adducts. J Biol Chem, 277 (46), pp. 44576-44581. | Show Abstract | Read more

Nucleotide excision repair (NER) is the primary pathway for the removal of DNA adducts that distort the double helix. In the yeast Saccharomyces cerevisiae the RAD6 epistasis group defines a more poorly characterized set of DNA damage response pathways, believed to be distinct from NER. Here we show that the elimination of the DNA minor groove adducts formed by an important class of anticancer antibiotic (CC-1065 family) requires NER factors in S. cerevisiae. We also demonstrate that the elimination of this class of minor groove adduct from the active MFA2 gene depends upon functional Rad18 and Rad6. This is most clear for the repair of adducts on the transcribed strand, where an absolute requirement for Rad6 and Rad18 was seen. Further experiments revealed that a specific RAD6-RAD18-controlled subpathway, the RAD5 branch, mediates these events. Cells disrupted for rad5 are highly sensitive to this minor groove binding agent, and rad5 cells exhibit an in vivo adduct elimination defect indistinguishable from that seen in rad6 and rad18 cells as well as in NER-defective cells. Our results indicate that the RAD5 subpathway may interact with NER factors during the repair of certain DNA adducts.

Erdal E, Haider S, Rehwinkel J, Harris AL, McHugh PJ. 2017. A prosurvival DNA damage-induced cytoplasmic interferon response is mediated by end resection factors and is limited by Trex1. Genes Dev, 31 (4), pp. 353-369. | Show Abstract | Read more

Radiotherapy and chemotherapy are effective treatment methods for many types of cancer, but resistance is common. Recent findings indicate that antiviral type I interferon (IFN) signaling is induced by these treatments. However, the underlying mechanisms still need to be elucidated. Expression of a set of IFN-stimulated genes comprises an IFN-related DNA damage resistance signature (IRDS), which correlates strongly with resistance to radiotherapy and chemotherapy across different tumors. Classically, during viral infection, the presence of foreign DNA in the cytoplasm of host cells can initiate type I IFN signaling. Here, we demonstrate that DNA-damaging modalities used during cancer therapy lead to the release of ssDNA fragments from the cell nucleus into the cytosol, engaging this innate immune response. We found that the factors that control DNA end resection during double-strand break repair, including the Bloom syndrome (BLM) helicase and exonuclease 1 (EXO1), play a major role in generating these DNA fragments and that the cytoplasmic 3'-5' exonuclease Trex1 is required for their degradation. Analysis of mRNA expression profiles in breast tumors demonstrates that those with lower Trex1 and higher BLM and EXO1 expression levels are associated with poor prognosis. Targeting BLM and EXO1 could therefore represent a novel approach for circumventing the IRDS produced in response to cancer therapeutics.

Lee SY, Brem J, Pettinati I, Claridge TDW, Gileadi O, Schofield CJ, McHugh PJ. 2016. Cephalosporins inhibit human metallo β-lactamase fold DNA repair nucleases SNM1A and SNM1B/apollo. Chem Commun (Camb), 52 (40), pp. 6727-6730. | Show Abstract | Read more

Bacterial metallo-β-lactamases (MBLs) are involved in resistance to β-lactam antibiotics including cephalosporins. Human SNM1A and SNM1B are MBL superfamily exonucleases that play a key role in the repair of DNA interstrand cross-links, which are induced by antitumour chemotherapeutics, and are therefore targets for cancer chemosensitization. We report that cephalosporins are competitive inhibitors of SNM1A and SNM1B exonuclease activity; both the intact β-lactam and their hydrolysed products are active. This discovery provides a lead for the development of potent and selective SNM1A and SNM1B inhibitors.

Broderick R, Nieminuszczy J, Baddock HT, Deshpande R, Gileadi O, Paull TT, McHugh PJ, Niedzwiedz W. 2016. EXD2 promotes homologous recombination by facilitating DNA end resection. Nat Cell Biol, 18 (3), pp. 271-280. | Show Abstract | Read more

Repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is critical for survival and genome stability of individual cells and organisms, but also contributes to the genetic diversity of species. A vital step in HR is MRN-CtIP-dependent end resection, which generates the 3' single-stranded DNA overhangs required for the subsequent strand exchange reaction. Here, we identify EXD2 (also known as EXDL2) as an exonuclease essential for DSB resection and efficient HR. EXD2 is recruited to chromatin in a damage-dependent manner and confers resistance to DSB-inducing agents. EXD2 functionally interacts with the MRN complex to accelerate resection through its 3'-5' exonuclease activity, which efficiently processes double-stranded DNA substrates containing nicks. Finally, we establish that EXD2 stimulates both short- and long-range DSB resection, and thus, together with MRE11, is required for efficient HR. This establishes a key role for EXD2 in controlling the initial steps of chromosomal break repair.

Pettinati I, Brem J, Lee SY, McHugh PJ, Schofield CJ. 2016. The Chemical Biology of Human Metallo-β-Lactamase Fold Proteins. Trends Biochem Sci, 41 (4), pp. 338-355. | Show Abstract | Read more

The αββα metallo β-lactamase (MBL) fold (MBLf) was first observed in bacterial enzymes that catalyze the hydrolysis of almost all β-lactam antibiotics, but is now known to be widely distributed. The MBL core protein fold is present in human enzymes with diverse biological roles, including cell detoxification pathways and enabling resistance to clinically important anticancer medicines. Human (h)MBLf enzymes can bind metals, including zinc and iron ions, and catalyze a range of chemically interesting reactions, including both redox (e.g., ETHE1) and hydrolytic processes (e.g., Glyoxalase II, SNM1 nucleases, and CPSF73). With a view to promoting basic research on MBLf enzymes and their medicinal targeting, here we summarize current knowledge of the mechanisms and roles of these important molecules.

Allerston CK, Lee SY, Newman JA, Schofield CJ, McHugh PJ, Gileadi O. 2015. The structures of the SNM1A and SNM1B/Apollo nuclease domains reveal a potential basis for their distinct DNA processing activities. Nucleic Acids Res, 43 (22), pp. 11047-11060. | Show Abstract | Read more

The human SNM1A and SNM1B/Apollo proteins are members of an extended family of eukaryotic nuclease containing a motif related to the prokaryotic metallo-β-lactamase (MBL) fold. SNM1A is a key exonuclease during replication-dependent and transcription-coupled interstrand crosslink repair, while SNM1B/Apollo is required for maintaining telomeric overhangs. Here, we report the crystal structures of SNM1A and SNM1B at 2.16 Å. While both proteins contain a typical MBL-β-CASP domain, a region of positive charge surrounds the active site of SNM1A, which is absent in SNM1B and explains the greater apparent processivity of SNM1A. The structures of both proteins also reveal a putative, wide DNA-binding groove. Extensive mutagenesis of this groove, coupled with detailed biochemical analysis, identified residues that did not impact on SNM1A catalytic activity, but drastically reduced its processivity. Moreover, we identified a key role for this groove for efficient digestion past DNA interstrand crosslinks, facilitating the key DNA repair reaction catalysed by SNM1A. Together, the architecture and dimensions of this groove, coupled to the surrounding region of high positive charge, explain the remarkable ability of SNM1A to accommodate and efficiently digest highly distorted DNA substrates, such as those containing DNA lesions.

Brem J, van Berkel SS, Zollman D, Lee SY, Gileadi O, McHugh PJ, Walsh TR, McDonough MA, Schofield CJ. 2015. Structural Basis of Metallo-β-Lactamase Inhibition by Captopril Stereoisomers. Antimicrob Agents Chemother, 60 (1), pp. 142-150. | Show Abstract | Read more

β-Lactams are the most successful antibacterials, but their effectiveness is threatened by resistance, most importantly by production of serine- and metallo-β-lactamases (MBLs). MBLs are of increasing concern because they catalyze the hydrolysis of almost all β-lactam antibiotics, including recent-generation carbapenems. Clinically useful serine-β-lactamase inhibitors have been developed, but such inhibitors are not available for MBLs. l-Captopril, which is used to treat hypertension via angiotensin-converting enzyme inhibition, has been reported to inhibit MBLs by chelating the active site zinc ions via its thiol(ate). We report systematic studies on B1 MBL inhibition by all four captopril stereoisomers. High-resolution crystal structures of three MBLs (IMP-1, BcII, and VIM-2) in complex with either the l- or d-captopril stereoisomer reveal correlations between the binding mode and inhibition potency. The results will be useful in the design of MBL inhibitors with the breadth of selectivity required for clinical application against carbapenem-resistant Enterobacteriaceae and other organisms causing MBL-mediated resistant infections.

Iyama T, Lee SY, Berquist BR, Gileadi O, Bohr VA, Seidman MM, McHugh PJ, Wilson DM. 2015. CSB interacts with SNM1A and promotes DNA interstrand crosslink processing. Nucleic Acids Res, 43 (1), pp. 247-258. | Show Abstract | Read more

Cockayne syndrome (CS) is a premature aging disorder characterized by photosensitivity, impaired development and multisystem progressive degeneration, and consists of two strict complementation groups, A and B. Using a yeast two-hybrid approach, we identified the 5'-3' exonuclease SNM1A as one of four strong interacting partners of CSB. This direct interaction was confirmed using purified recombinant proteins-with CSB able to modulate the exonuclease activity of SNM1A on oligonucleotide substrates in vitro-and the two proteins were shown to exist in a common complex in human cell extracts. CSB and SNM1A were also found, using fluorescently tagged proteins in combination with confocal microscopy and laser microirradiation, to be recruited to localized trioxsalen-induced ICL damage in human cells, with accumulation being suppressed by transcription inhibition. Moreover, SNM1A recruitment was significantly reduced in CSB-deficient cells, suggesting coordination between the two proteins in vivo. CSB-deficient neural cells exhibited increased sensitivity to DNA crosslinking agents, particularly, in a non-cycling, differentiated state, as well as delayed ICL processing as revealed by a modified Comet assay and γ-H2AX foci persistence. The results indicate that CSB coordinates the resolution of ICLs, possibly in a transcription-associated repair mechanism involving SNM1A, and that defects in the process could contribute to the post-mitotic degenerative pathologies associated with CS.

Hatch SB, Swift LP, Caporali S, Carter R, Hill EJ, Macgregor TP, D'Atri S, Middleton MR, McHugh PJ, Sharma RA. 2014. XPF protein levels determine sensitivity of malignant melanoma cells to oxaliplatin chemotherapy: Suitability as a biomarker for patient selection International Journal of Cancer, 134 (6), pp. 1495-1503. | Show Abstract | Read more

As the options for systemic treatment of malignant melanoma (MM) increase, the need to develop biomarkers to identify patients who might benefit from cytotoxic chemotherapy becomes more apparent. In preclinical models, oxaliplatin has activity in cisplatin-resistant cells. In this study, we have shown that oxaliplatin forms interstrand crosslinks (ICLs) in cellular DNA and that loss of the heterodimeric structure-specific endonuclease XPF-ERCC1 causes hypersensitivity to oxaliplatin in mammalian cells. XPF deficiency resulted in late S-phase arrest and persistence of double-strand breaks following oxaliplatin treatment. In a panel of 12 MM cell lines, oxaliplatin sensitivity correlated with XPF and ERCC1 protein levels. The knockdown of ERCC1 and XPF protein levels by RNA interference increased sensitivity of cancer cells to oxaliplatin; overexpression of exogenous ERCC1 significantly decreased drug sensitivity. Following immunohistochemical optimization, XPF protein levels were quantified in MM tissue samples from 183 patients, showing variation in expression and no correlation with prognosis. In 57 patients with MM treated with cisplatin or carboplatin, XPF protein levels did not predict the likelihood of clinical r esponse. We propose that oxaliplatin should not be discarded as a potential treatment for MM on the basis of the limited activity of cisplatin in unselected patients. Moreover, we show that XPF-ERCC1 protein levels are a key determinant of the sensitivity of melanoma cells to oxaliplatin in vitro. Immunohistochemical detection of XPF appears suitable for development as a tissue biomarker for potentially selecting patients for oxaliplatin treatment in a prospective clinical trial. What's new? With options for systemic treatment of malignant melanoma (MM) on the rise, there is an increasing need to develop biomarkers for patient selection. To that end, this study explored the possibility of a biomarker to improve objective response rates to the drug oxaliplatin. The study reveals a mechanism by which mammalian cells are rendered hypersensitive to oxaliplatin that centers around the loss of endonuclease XPF-ERCC1. Sensitivity to oxaliplatin was directly related to XPF and ERCC1 protein levels. The findings indicate that XPF may be a suitable biomarker for MM patient selection for oxaliplatin therapy. © 2013 UICC.

Srivas R, Costelloe T, Carvunis A-R, Sarkar S, Malta E, Sun SM, Pool M, Licon K, van Welsem T, van Leeuwen F et al. 2013. A UV-induced genetic network links the RSC complex to nucleotide excision repair and shows dose-dependent rewiring. Cell Rep, 5 (6), pp. 1714-1724. | Show Abstract | Read more

Efficient repair of UV-induced DNA damage requires the precise coordination of nucleotide excision repair (NER) with numerous other biological processes. To map this crosstalk, we generated a differential genetic interaction map centered on quantitative growth measurements of >45,000 double mutants before and after different doses of UV radiation. Integration of genetic data with physical interaction networks identified a global map of 89 UV-induced functional interactions among 62 protein complexes, including a number of links between the RSC complex and several NER factors. We show that RSC is recruited to both silenced and transcribed loci following UV damage where it facilitates efficient repair by promoting nucleosome remodeling. Finally, a comparison of the response to high versus low levels of UV shows that the degree of genetic rewiring correlates with dose of UV and reveals a network of dose-specific interactions. This study makes available a large resource of UV-induced interactions, and it illustrates a methodology for identifying dose-dependent interactions based on quantitative shifts in genetic networks.

Ward TA, Dudášová Z, Sarkar S, Bhide MR, Vlasáková D, Chovanec M, McHugh PJ. 2012. Components of a Fanconi-like pathway control Pso2-independent DNA interstrand crosslink repair in yeast. PLoS Genet, 8 (8), pp. e1002884. | Show Abstract | Read more

Fanconi anemia (FA) is a devastating genetic disease, associated with genomic instability and defects in DNA interstrand cross-link (ICL) repair. The FA repair pathway is not thought to be conserved in budding yeast, and although the yeast Mph1 helicase is a putative homolog of human FANCM, yeast cells disrupted for MPH1 are not sensitive to ICLs. Here, we reveal a key role for Mph1 in ICL repair when the Pso2 exonuclease is inactivated. We find that the yeast FANCM ortholog Mph1 physically and functionally interacts with Mgm101, a protein previously implicated in mitochondrial DNA repair, and the MutSα mismatch repair factor (Msh2-Msh6). Co-disruption of MPH1, MGM101, MSH6, or MSH2 with PSO2 produces a lesion-specific increase in ICL sensitivity, the elevation of ICL-induced chromosomal rearrangements, and persistence of ICL-associated DNA double-strand breaks. We find that Mph1-Mgm101-MutSα directs the ICL-induced recruitment of Exo1 to chromatin, and we propose that Exo1 is an alternative 5'-3' exonuclease utilised for ICL repair in the absence of Pso2. Moreover, ICL-induced Rad51 chromatin loading is delayed when both Pso2 and components of the Mph1-Mgm101-MutSα and Exo1 pathway are inactivated, demonstrating that the homologous recombination stages of ICL repair are inhibited. Finally, the FANCJ- and FANCP-related factors Chl1 and Slx4, respectively, are also components of the genetic pathway controlled by Mph1-Mgm101-MutSα. Together this suggests that a prototypical FA-related ICL repair pathway operates in budding yeast, which acts redundantly with the pathway controlled by Pso2, and is required for the targeting of Exo1 to chromatin to execute ICL repair.

Sengerová B, Allerston CK, Abu M, Lee SY, Hartley J, Kiakos K, Schofield CJ, Hartley JA, Gileadi O, McHugh PJ. 2012. Characterization of the human SNM1A and SNM1B/Apollo DNA repair exonucleases. J Biol Chem, 287 (31), pp. 26254-26267. | Show Abstract | Read more

Human SNM1A and SNM1B/Apollo have both been implicated in the repair of DNA interstrand cross-links (ICLs) by cellular studies, and SNM1B is also required for telomere protection. Here, we describe studies on the biochemical characterization of the SNM1A and SNM1B proteins. The results reveal some fundamental differences in the mechanisms of the two proteins. Both SNM1A and SNM1B digest double-stranded and single-stranded DNA with a 5'-to-3' directionality in a reaction that is stimulated by divalent cations, and both nucleases are inhibited by the zinc chelator o-phenanthroline. We find that SNM1A has greater affinity for single-stranded DNA over double-stranded DNA that is not observed with SNM1B. Although both proteins demonstrate a low level of processivity on low molecular weight DNA oligonucleotide substrates, when presented with high molecular weight DNA, SNM1A alone is rendered much more active, being capable of digesting kilobase-long stretches of DNA. Both proteins can digest past ICLs induced by the non-distorting minor groove cross-linking agent SJG-136, albeit with SNM1A showing a greater capacity to achieve this. This is consistent with the proposal that SNM1A and SNM1B might exhibit some redundancy in ICL repair. Together, our work establishes differences in the substrate selectivities of SNM1A and SNM1B that are likely to be relevant to their in vivo roles and which might be exploited in the development of selective inhibitors.

Sengerová B, Wang AT, McHugh PJ. 2011. Orchestrating the nucleases involved in DNA interstrand cross-link (ICL) repair. Cell Cycle, 10 (23), pp. 3999-4008. | Show Abstract | Read more

DNA interstrand cross-links (ICLs) pose a significant threat to genomic and cellular integrity by blocking essential cellular processes, including replication and transcription. In mammalian cells, much ICL repair occurs in association with DNA replication during S phase, following the stalling of a replication fork at the block caused by an ICL lesion. Here, we review recent work showing that the XPF-ERCC1 endonuclease and the hSNM1A exonuclease act in the same pathway, together with SLX4, to initiate ICL repair, with the MUS81-EME1 fork incision activity becoming important in the absence of the XPF-SNM1A-SLX4-dependent pathway. Another nuclease, the Fanconi anemia-associated nuclease (FAN1), has recently been implicated in the repair of ICLs, and we discuss the possible ways in which the activities of different nucleases at the ICL-stalled replication fork may be coordinated. In relation to this, we briefly speculate on the possible role of SLX4, which contains XPF and MUS81- interacting domains, in the coordination of ICL repair nucleases.

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Wang AT, Sengerová B, Cattell E, Inagawa T, Hartley JM, Kiakos K, Burgess-Brown NA, Swift LP, Enzlin JH, Schofield CJ et al. 2011. Human SNM1a and XPF-ERCC1 collaborate to initiate DNA interstrand cross-link repair Genes and Development, 25 (17), pp. 1859-1870. | Show Abstract | Read more

One of the major DNA interstrand cross-link (ICL) repair pathways in mammalian cells is coupled to replication, but the mechanistic roles of the critical factors involved remain largely elusive. Here, we show that purified human SNM1A (hSNM1A), which exhibits a 59-39 exonuclease activity, can load from a single DNA nick and digest past an ICL on its substrate strand. hSNM1A-depleted cells are ICL-sensitive and accumulate replicationassociated DNA double-strand breaks (DSBs), akin to ERCC1-depleted cells. These DSBs are Mus81-induced, indicating that replication fork cleavage by Mus81 results from the failure of the hSNM1A- and XPF-ERCC1-dependent ICL repair pathway. Our results reveal how collaboration between hSNM1A and XPF-ERCC1 is necessary to initiate ICL repair in replicating human cells. © 2011 by Cold Spring Harbor Laboratory Press.

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