Dr Andrew Blackford

Research Area: Cell and Molecular Biology
Technology Exchange: Bioinformatics, Ex vivo models, Human genetics, Microscopy (Confocal) and Protein interaction
Scientific Themes: Cancer and Molecular, Cell & Systems Biology
Keywords: Bloom Syndrome, Cancer, DNA Damage Response, DNA Repair, Homologous Recombination, Non-Homologous End-Joining, Phosphorylation and Ubiquitin

Mutations caused by DNA damage enable a normal cell to become cancerous. This is highlighted by the fact that individuals with mutations in many genes involved in DNA damage recognition, signalling and repair are predisposed to cancer, and that somatically acquired defects in such genes can drive tumour formation. Furthermore, some of the most effective cancer treatments work in tumour cells by inducing DNA damage, particularly DNA double-strand breaks, which are especially toxic and difficult to repair accurately without introducing mutations. Exploiting knowledge of DNA double-strand break repair is therefore likely to lead to more effective and personalised cancer therapies and treatments for patients with DNA repair disorders in future.

The aim of our research is to gain a greater understanding of the signalling mechanisms cells use to coordinate DNA double-strand break recognition and repair with cell cycle checkpoint activation and apoptosis. To achieve this, we are using cutting-edge bioinformatics, proteomics, microscopy and CRISPR-Cas9 gene-editing techniques to answer specific questions related to DNA damage signalling. In doing so, we hope to provide novel insights into carcinogenesis and how it is held at bay by the cell’s DNA damage response system. We also aim to translate our research to develop novel potential cancer treatments. In particular, we are interested in the potential utility of signalling events for use as biomarkers and to identify novel targets in the DNA damage response for anti-cancer drugs.

A major focus of our work is trying to understand how the products of genes mutated in DNA repair and cancer predisposition disorders such as Bloom syndrome are regulated by post-translational modifications and protein-protein interactions. Patients with Bloom syndrome suffer from a variety of symptoms including growth retardation, immunodeficiency, hypersensitivity to sunlight in addition to cancer predisposition. It is caused by mutations in BLM, a DNA helicase that plays important roles in coordinating DNA double-strand break repair and recombination, particularly during DNA replication and mitosis. However, we still know little about how this protein is regulated during the repair process and throughout the cell cycle. We are addressing this question by characterising novel post-translational modifications and protein-protein interactions of BLM and its binding partners.

 

Name Department Institution Country
Prof Sir Tom L Blundell Department of Biochemistry University of Cambridge United Kingdom
Prof Mark Glover Department of Biochemistry University of Alberta Canada
Prof Andrew P Jackson MRC Human Genetics Unit University of Edinburgh United Kingdom
Prof Stephen P Jackson Wellcome Trust/CRUK Gurdon Institute University of Cambridge United Kingdom
Sun L, Huang Y, Edwards RA, Yang S, Blackford AN, Niedzwiedz W, Glover JNM. 2017. Structural Insight into BLM Recognition by TopBP1. Structure, 25 (10), pp. 1582-1588.e3. | Show Abstract | Read more

Topoisomerase IIβ binding protein 1 (TopBP1) is a critical protein-protein interaction hub in DNA replication checkpoint control. It was proposed that TopBP1 BRCT5 interacts with Bloom syndrome helicase (BLM) to regulate genome stability through either phospho-Ser304 or phospho-Ser338 of BLM. Here we show that TopBP1 BRCT5 specifically interacts with the BLM region surrounding pSer304, not pSer338. Our crystal structure of TopBP1 BRCT4/5 bound to BLM reveals recognition of pSer304 by a conserved pSer-binding pocket, and interactions between an FVPP motif N-terminal to pSer304 and a hydrophobic groove on BRCT5. This interaction utilizes the same surface of BRCT5 that recognizes the DNA damage mediator, MDC1; however the binding orientations of MDC1 and BLM are reversed. While the MDC1 interactions are largely electrostatic, the interaction with BLM has higher affinity and relies on a mix of electrostatics and hydrophobicity. We suggest that similar evolutionarily conserved interactions may govern interactions between TopBP1 and 53BP1.

Henssen AG, Koche R, Zhuang J, Jiang E, Reed C, Eisenberg A, Still E, MacArthur IC, Rodríguez-Fos E, Gonzalez S et al. 2017. PGBD5 promotes site-specific oncogenic mutations in human tumors. Nat Genet, 49 (7), pp. 1005-1014. | Show Abstract | Read more

Genomic rearrangements are a hallmark of human cancers. Here, we identify the piggyBac transposable element derived 5 (PGBD5) gene as encoding an active DNA transposase expressed in the majority of childhood solid tumors, including lethal rhabdoid tumors. Using assembly-based whole-genome DNA sequencing, we found previously undefined genomic rearrangements in human rhabdoid tumors. These rearrangements involved PGBD5-specific signal (PSS) sequences at their breakpoints and recurrently inactivated tumor-suppressor genes. PGBD5 was physically associated with genomic PSS sequences that were also sufficient to mediate PGBD5-induced DNA rearrangements in rhabdoid tumor cells. Ectopic expression of PGBD5 in primary immortalized human cells was sufficient to promote cell transformation in vivo. This activity required specific catalytic residues in the PGBD5 transposase domain as well as end-joining DNA repair and induced structural rearrangements with PSS breakpoints. These results define PGBD5 as an oncogenic mutator and provide a plausible mechanism for site-specific DNA rearrangements in childhood and adult solid tumors.

Blackford AN, Jackson SP. 2017. ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response. Mol Cell, 66 (6), pp. 801-817. | Show Abstract | Read more

In vertebrate cells, the DNA damage response is controlled by three related kinases: ATM, ATR, and DNA-PK. It has been 20 years since the cloning of ATR, the last of the three to be identified. During this time, our understanding of how these kinases regulate DNA repair and associated events has grown profoundly, although major questions remain unanswered. Here, we provide a historical perspective of their discovery and discuss their established functions in sensing and responding to genotoxic stress. We also highlight what is known regarding their structural similarities and common mechanisms of regulation, as well as emerging non-canonical roles and how our knowledge of ATM, ATR, and DNA-PK is being translated to benefit human health.

Balmus G, Barros AC, Wijnhoven PWG, Lescale C, Hasse HL, Boroviak K, le Sage C, Doe B, Speak AO, Galli A et al. 2016. Synthetic lethality between PAXX and XLF in mammalian development. Genes Dev, 30 (19), pp. 2152-2157. | Show Abstract | Read more

PAXX was identified recently as a novel nonhomologous end-joining DNA repair factor in human cells. To characterize its physiological roles, we generated Paxx-deficient mice. Like Xlf-/- mice, Paxx-/- mice are viable, grow normally, and are fertile but show mild radiosensitivity. Strikingly, while Paxx loss is epistatic with Ku80, Lig4, and Atm deficiency, Paxx/Xlf double-knockout mice display embryonic lethality associated with genomic instability, cell death in the central nervous system, and an almost complete block in lymphogenesis, phenotypes that closely resemble those of Xrcc4-/- and Lig4-/- mice. Thus, combined loss of Paxx and Xlf is synthetic-lethal in mammals.

Lescale C, Lenden Hasse H, Blackford AN, Balmus G, Bianchi JJ, Yu W, Bacoccina L, Jarade A, Clouin C, Sivapalan R et al. 2016. Specific Roles of XRCC4 Paralogs PAXX and XLF during V(D)J Recombination. Cell Rep, 16 (11), pp. 2967-2979. | Show Abstract | Read more

Paralog of XRCC4 and XLF (PAXX) is a member of the XRCC4 superfamily and plays a role in nonhomologous end-joining (NHEJ), a DNA repair pathway critical for lymphocyte antigen receptor gene assembly. Here, we find that the functions of PAXX and XLF in V(D)J recombination are masked by redundant joining activities. Thus, combined PAXX and XLF deficiency leads to an inability to join RAG-cleaved DNA ends. Additionally, we demonstrate that PAXX function in V(D)J recombination depends on its interaction with Ku. Importantly, we show that, unlike XLF, the role of PAXX during the repair of DNA breaks does not overlap with ATM and the RAG complex. Our findings illuminate the role of PAXX in V(D)J recombination and support a model in which PAXX and XLF function during NHEJ repair of DNA breaks, whereas XLF, the RAG complex, and the ATM-dependent DNA damage response promote end joining by stabilizing DNA ends.

Harley ME, Murina O, Leitch A, Higgs MR, Bicknell LS, Yigit G, Blackford AN, Zlatanou A, Mackenzie KJ, Reddy K et al. 2016. TRAIP promotes DNA damage response during genome replication and is mutated in primordial dwarfism. Nat Genet, 48 (1), pp. 36-43. | Show Abstract | Read more

DNA lesions encountered by replicative polymerases threaten genome stability and cell cycle progression. Here we report the identification of mutations in TRAIP, encoding an E3 RING ubiquitin ligase, in patients with microcephalic primordial dwarfism. We establish that TRAIP relocalizes to sites of DNA damage, where it is required for optimal phosphorylation of H2AX and RPA2 during S-phase in response to ultraviolet (UV) irradiation, as well as fork progression through UV-induced DNA lesions. TRAIP is necessary for efficient cell cycle progression and mutations in TRAIP therefore limit cellular proliferation, providing a potential mechanism for microcephaly and dwarfism phenotypes. Human genetics thus identifies TRAIP as a component of the DNA damage response to replication-blocking DNA lesions.

Wijnhoven P, Konietzny R, Blackford AN, Travers J, Kessler BM, Nishi R, Jackson SP. 2015. USP4 Auto-Deubiquitylation Promotes Homologous Recombination. Mol Cell, 60 (3), pp. 362-373. | Show Abstract | Read more

Repair of DNA double-strand breaks is crucial for maintaining genome integrity and is governed by post-translational modifications such as protein ubiquitylation. Here, we establish that the deubiquitylating enzyme USP4 promotes DNA-end resection and DNA repair by homologous recombination. We also report that USP4 interacts with CtIP and the MRE11-RAD50-NBS1 (MRN) complex and is required for CtIP recruitment to DNA damage sites. Furthermore, we show that USP4 is ubiquitylated on multiple sites including those on cysteine residues and that deubiquitylation of these sites requires USP4 catalytic activity and is required for USP4 to interact with CtIP/MRN and to promote CtIP recruitment and DNA repair. Lastly, we establish that regulation of interactor binding by ubiquitylation occurs more generally among USP-family enzymes. Our findings thus identify USP4 as a novel DNA repair regulator and invoke a model in which ubiquitin adducts regulate USP enzyme interactions and functions.

Broderick R, Nieminuszczy J, Blackford AN, Winczura A, Niedzwiedz W. 2015. TOPBP1 recruits TOP2A to ultra-fine anaphase bridges to aid in their resolution. Nat Commun, 6 pp. 6572. | Show Abstract | Read more

During mitosis, sister chromatids must be faithfully segregated to ensure that daughter cells receive one copy of each chromosome. However, following replication they often remain entangled. Topoisomerase IIα (TOP2A) has been proposed to resolve such entanglements, but the mechanisms governing TOP2A recruitment to these structures remain poorly understood. Here, we identify TOPBP1 as a novel interactor of TOP2A, and reveal that it is required for TOP2A recruitment to ultra-fine anaphase bridges (UFBs) in mitosis. The C-terminal region of TOPBP1 interacts with TOP2A, and TOPBP1 recruitment to UFBs requires its BRCT domain 5. Depletion of TOPBP1 leads to accumulation of UFBs, the majority of which arise from centromeric loci. Accordingly, expression of a TOPBP1 mutant that is defective in TOP2A binding phenocopies TOP2A depletion. These findings provide new mechanistic insights into how TOP2A promotes resolution of UFBs during mitosis, and highlights a pivotal role for TOPBP1 in this process.

Blackford AN, Nieminuszczy J, Schwab RA, Galanty Y, Jackson SP, Niedzwiedz W. 2015. TopBP1 interacts with BLM to maintain genome stability but is dispensable for preventing BLM degradation. Mol Cell, 57 (6), pp. 1133-1141. | Show Abstract | Read more

The Bloom syndrome helicase BLM and topoisomerase-IIβ-binding protein 1 (TopBP1) are key regulators of genome stability. It was recently proposed that BLM phosphorylation on Ser338 mediates its interaction with TopBP1, to protect BLM from ubiquitylation and degradation (Wang et al., 2013). Here, we show that the BLM-TopBP1 interaction does not involve Ser338 but instead requires BLM phosphorylation on Ser304. Furthermore, we establish that disrupting this interaction does not markedly affect BLM stability. However, BLM-TopBP1 binding is important for maintaining genome integrity, because in its absence cells display increased sister chromatid exchanges, replication origin firing and chromosomal aberrations. Therefore, the BLM-TopBP1 interaction maintains genome stability not by controlling BLM protein levels, but via another as-yet undetermined mechanism. Finally, we identify critical residues that mediate interactions between TopBP1 and MDC1, and between BLM and TOP3A/RMI1/RMI2. Taken together, our findings provide molecular insights into a key tumor suppressor and genome stability network.

Higgs MR, Reynolds JJ, Winczura A, Blackford AN, Borel V, Miller ES, Zlatanou A, Nieminuszczy J, Ryan EL, Davies NJ et al. 2015. BOD1L Is Required to Suppress Deleterious Resection of Stressed Replication Forks. Mol Cell, 59 (3), pp. 462-477. | Show Abstract | Read more

Recognition and repair of damaged replication forks are essential to maintain genome stability and are coordinated by the combined action of the Fanconi anemia and homologous recombination pathways. These pathways are vital to protect stalled replication forks from uncontrolled nucleolytic activity, which otherwise causes irreparable genomic damage. Here, we identify BOD1L as a component of this fork protection pathway, which safeguards genome stability after replication stress. Loss of BOD1L confers exquisite cellular sensitivity to replication stress and uncontrolled resection of damaged replication forks, due to a failure to stabilize RAD51 at these forks. Blocking DNA2-dependent resection, or downregulation of the helicases BLM and FBH1, suppresses both catastrophic fork processing and the accumulation of chromosomal damage in BOD1L-deficient cells. Thus, our work implicates BOD1L as a critical regulator of genome integrity that restrains nucleolytic degradation of damaged replication forks.

Ochi T, Blackford AN, Coates J, Jhujh S, Mehmood S, Tamura N, Travers J, Wu Q, Draviam VM, Robinson CV et al. 2015. DNA repair. PAXX, a paralog of XRCC4 and XLF, interacts with Ku to promote DNA double-strand break repair. Science, 347 (6218), pp. 185-188. | Show Abstract | Read more

XRCC4 and XLF are two structurally related proteins that function in DNA double-strand break (DSB) repair. Here, we identify human PAXX (PAralog of XRCC4 and XLF, also called C9orf142) as a new XRCC4 superfamily member and show that its crystal structure resembles that of XRCC4. PAXX interacts directly with the DSB-repair protein Ku and is recruited to DNA-damage sites in cells. Using RNA interference and CRISPR-Cas9 to generate PAXX(-/-) cells, we demonstrate that PAXX functions with XRCC4 and XLF to mediate DSB repair and cell survival in response to DSB-inducing agents. Finally, we reveal that PAXX promotes Ku-dependent DNA ligation in vitro and assembly of core nonhomologous end-joining (NHEJ) factors on damaged chromatin in cells. These findings identify PAXX as a new component of the NHEJ machinery.

Polo SE, Blackford AN, Chapman JR, Baskcomb L, Gravel S, Rusch A, Thomas A, Blundred R, Smith P, Kzhyshkowska J et al. 2012. Regulation of DNA-end resection by hnRNPU-like proteins promotes DNA double-strand break signaling and repair. Mol Cell, 45 (4), pp. 505-516. | Show Abstract | Read more

DNA double-strand break (DSB) signaling and repair are critical for cell viability, and rely on highly coordinated pathways whose molecular organization is still incompletely understood. Here, we show that heterogeneous nuclear ribonucleoprotein U-like (hnRNPUL) proteins 1 and 2 play key roles in cellular responses to DSBs. We identify human hnRNPUL1 and -2 as binding partners for the DSB sensor complex MRE11-RAD50-NBS1 (MRN) and demonstrate that hnRNPUL1 and -2 are recruited to DNA damage in an interdependent manner that requires MRN. Moreover, we show that hnRNPUL1 and -2 stimulate DNA-end resection and promote ATR-dependent signaling and DSB repair by homologous recombination, thereby contributing to cell survival upon exposure to DSB-inducing agents. Finally, we establish that hnRNPUL1 and -2 function downstream of MRN and CtBP-interacting protein (CtIP) to promote recruitment of the BLM helicase to DNA breaks. Collectively, these results provide insights into how mammalian cells respond to DSBs.

Blackford AN, Schwab RA, Nieminuszczy J, Deans AJ, West SC, Niedzwiedz W. 2012. The DNA translocase activity of FANCM protects stalled replication forks. Hum Mol Genet, 21 (9), pp. 2005-2016. | Show Abstract | Read more

FANCM is the most highly conserved protein within the Fanconi anaemia (FA) tumour suppressor pathway. However, although FANCM contains a helicase domain with translocase activity, this is not required for its role in activating the FA pathway. Instead, we show here that FANCM translocaseactivity is essential for promoting replication fork stability. We demonstrate that cells expressing translocase-defective FANCM show altered global replication dynamics due to increased accumulation of stalled forks that subsequently degenerate into DNA double-strand breaks, leading to ATM activation, CTBP-interacting protein (CTIP)-dependent end resection and homologous recombination repair. Accordingly, abrogation of ATM or CTIP function in FANCM-deficient cells results in decreased cell survival. We also found that FANCM translocase activity protects cells from accumulating 53BP1-OPT domains, which mark lesions resulting from problems arising during replication. Taken together, these data show that FANCM plays an essential role in maintaining chromosomal integrity by promoting the recovery of stalled replication forks and hence preventing tumourigenesis.

Forrester NA, Sedgwick GG, Thomas A, Blackford AN, Speiseder T, Dobner T, Byrd PJ, Stewart GS, Turnell AS, Grand RJA. 2011. Serotype-specific inactivation of the cellular DNA damage response during adenovirus infection. J Virol, 85 (5), pp. 2201-2211. | Show Abstract | Read more

Adenovirus type 5 (Ad5) inactivates the host cell DNA damage response by facilitating the degradation of Mre11, DNA ligase IV, and p53. In the case of p53, this is achieved through polyubiquitylation by Ad5E1B55K and Ad5E4orf6, which recruit a Cul5-based E3 ubiquitin ligase. Recent evidence indicates that this paradigm does not apply to other adenovirus serotypes, since Ad12, but not Ad5, causes the degradation of TOPBP1 through the action of E4orf6 alone and a Cul2-based E3 ubiquitin ligase. We now have extended these studies to adenovirus groups A to E. While infection by Ad4, Ad5, and Ad12 (groups E, C, and A, respectively) cause the degradation of Mre11, DNA ligase IV, and p53, infection with Ad3, Ad7, Ad9, and Ad11 (groups B1, B1, D, and B2, respectively) only affects DNA ligase IV levels. Indeed, Ad3, Ad7, and Ad11 cause the marked accumulation of p53. Despite this, MDM2 levels were very low following infection with all of the viruses examined here, regardless of whether they increase p53 expression. In addition, we found that only Ad12 causes the degradation of TOPBP1, and, like Ad5, Ad4 recruits a Cul5-based E3 ubiquitin ligase to degrade p53. Surprisingly, Mre11 and DNA ligase IV degradation do not appear to be significantly affected in Ad4-, Ad5-, or Ad12-infected cells depleted of Cul2 or Cul5, indicating that E1B55K and E4orf6 recruit multiple ubiquitin ligases to target cellular proteins. Finally, although Mre11 is not degraded by Ad3, Ad7, Ad9, and Ad11, no viral DNA concatemers could be detected. We suggest that group B and D adenoviruses have evolved mechanisms based on the loss of DNA ligase IV and perhaps other unknown molecules to disable the host cell DNA damage response to promote viral replication.

Blackford AN, Schwab RA, Niedzwiedz W. 2010. A novel ATRibute of FANCM. Cell Cycle, 9 (8), pp. 1453-1455. | Read more

Schwab RA, Blackford AN, Niedzwiedz W. 2010. ATR activation and replication fork restart are defective in FANCM-deficient cells. EMBO J, 29 (4), pp. 806-818. | Show Abstract | Read more

Fanconi anaemia is a chromosomal instability disorder associated with cancer predisposition and bone marrow failure. Among the 13 identified FA gene products only one, the DNA translocase FANCM, has homologues in lower organisms, suggesting a conserved function in DNA metabolism. However, a precise role for FANCM in DNA repair remains elusive. Here, we show a novel function for FANCM that is distinct from its role in the FA pathway: promoting replication fork restart and simultaneously limiting the accumulation of RPA-ssDNA. We show that in DT40 cells this process is controlled by ATR and PLK1, and that in the absence of FANCM, stalled replication forks are unable to resume DNA synthesis and genome duplication is ensured by excess origin firing. Unexpectedly, we also uncover an early role for FANCM in ATR-mediated checkpoint signalling by promoting chromatin retention of TopBP1. Failure to retain TopBP1 on chromatin impacts on the ability of ATR to phosphorylate downstream molecular targets, including Chk1 and SMC1. Our data therefore indicate a fundamental role for FANCM in the maintenance of genome integrity during S phase.

Blackford AN, Patel RN, Forrester NA, Theil K, Groitl P, Stewart GS, Taylor AMR, Morgan IM, Dobner T, Grand RJA, Turnell AS. 2010. Adenovirus 12 E4orf6 inhibits ATR activation by promoting TOPBP1 degradation. Proc Natl Acad Sci U S A, 107 (27), pp. 12251-12256. | Show Abstract | Read more

Activation of the cellular DNA damage response is detrimental to adenovirus (Ad) infection. Ad has therefore evolved a number of strategies to inhibit ATM- and ATR-dependent signaling pathways during infection. Recent work suggests that the Ad5 E4orf3 protein prevents ATR activation through its ability to mislocalize the MRN complex. Here we provide evidence to indicate that Ad12 has evolved a different strategy from Ad5 to inhibit ATR. We show that Ad12 utilizes a CUL2/RBX1/elongin C-containing ubiquitin ligase to promote the proteasomal degradation of the ATR activator protein topoisomerase-IIbeta-binding protein 1 (TOPBP1). Ad12 also uses this complex to degrade p53 during infection, in contrast to Ad5, which requires a CUL5-based ubiquitin ligase. Although Ad12-mediated degradation of p53 is dependent upon both E1B-55K and E4orf6, Ad12-mediated degradation of TOPBP1 is solely dependent on E4orf6. We propose that Ad12 E4orf6 has two principal activities: to recruit the CUL2-based ubiquitin ligase and to act as substrate receptor for TOPBP1. In support of the idea that Ad12 E4orf6 specifically prevents ATR activation during infection by targeting TOPBP1 for degradation, we demonstrate that Ad12 E4orf6 can inhibit the ATR-dependent phosphorylation of CHK1 in response to replication stress. Taken together, these data provide insights into how Ad modulates ATR signaling pathways during infection.

Shimwell NJ, Martin A, Bruton RK, Blackford AN, Sedgwick GG, Gallimore PH, Turnell AS, Grand RJA. 2009. Adenovirus 5 E1A is responsible for increased expression of insulin receptor substrate 4 in established adenovirus 5-transformed cell lines and interacts with IRS components activating the PI3 kinase/Akt signalling pathway. Oncogene, 28 (5), pp. 686-697. | Show Abstract | Read more

Using mass spectrometric analysis insulin receptor substrate 4 (IRS-4) has been identified as a novel adenovirus 5 early region 1A (Ad5E1A)-binding protein. IRS-4 interacts with both the transcriptional activation domain (conserved region 3) and the N-terminal region of Ad5E1A13S. Prolonged expression of Ad5E1A13S is required for the observed dramatic increase in the levels of IRS-4 mRNA and protein in Ad5E1-transformed human cell lines. Once expressed, as well as binding to E1A and the insulin receptor, IRS-4 remains tyrosine phosphorylated and constitutively associates with the regulatory p85 subunit of phosphoinositide 3 kinase, resulting in the phosphorylation of Akt (causing activation) and GSK-3beta (causing inhibition). Reducing IRS-4 expression using small interfering RNA (siRNA) in established Ad5E1A-expressing cell lines decreases the activation of Akt and cellular proliferation. During Ad5 infection, IRS-4 is not expressed. However, Ad5E1A associates with IRS-1, increasing Akt and GSK-3beta phosphorylation and tyrosine phosphorylation of IRS-1 itself. We conclude that the association and altered regulation of IRS proteins by Ad5E1A contribute to the adenovirus-transformed phenotype and modulates viral infection in an Akt-dependent manner.

Townsend K, Mason H, Blackford AN, Miller ES, Chapman JR, Sedgwick GG, Barone G, Turnell AS, Stewart GS. 2009. Mediator of DNA damage checkpoint 1 (MDC1) regulates mitotic progression. J Biol Chem, 284 (49), pp. 33939-33948. | Show Abstract | Read more

Human mediator of DNA damage checkpoint 1 (hMDC1) is an essential component of the cellular response to DNA double strand breaks. Recently, hMDC1 has been shown to associate with a subunit of the anaphase-promoting complex/cyclosome (APC/C) (Coster, G., Hayouka, Z., Argaman, L., Strauss, C., Friedler, A., Brandeis, M., and Goldberg, M. (2007) J. Biol. Chem. 282, 32053-32064), a key regulator of mitosis, suggesting a possible role for hMDC1 in controlling normal cell cycle progression. Here, we extend this work to show that hMDC1 regulates normal metaphase-to-anaphase transition through its ability to bind directly to the APC/C and modulate its E3 ubiquitin ligase activity. In support of a role for hMDC1 in controlling mitotic progression, depletion of hMDC1 by small interfering RNA results in a metaphase arrest that appears to be independent of both BubR1-dependent signaling pathways and ATM/ATR activation. Mitotic cells lacking hMDC1 exhibit markedly reduced levels of APC/C activity characterized by reduced levels of Cdc20, and a failure of Cdc20 to bind the APC/C and CREB-binding protein. We suggest therefore that hMDC1 functionally regulates the normal metaphase-to-anaphase transition by modulating the Cdc20-dependent activation of the APC/C.

Blackford AN, Grand RJA. 2009. Adenovirus E1B 55-kilodalton protein: multiple roles in viral infection and cell transformation. J Virol, 83 (9), pp. 4000-4012. | Read more

Blackford AN, Bruton RK, Dirlik O, Stewart GS, Taylor AMR, Dobner T, Grand RJA, Turnell AS. 2008. A role for E1B-AP5 in ATR signaling pathways during adenovirus infection. J Virol, 82 (15), pp. 7640-7652. | Show Abstract | Read more

E1B-55K-associated protein 5 (E1B-AP5) is a cellular, heterogeneous nuclear ribonucleoprotein that is targeted by adenovirus (Ad) E1B-55K during infection. The function of E1B-AP5 during infection, however, remains largely unknown. Given the role of E1B-55K targets in the DNA damage response, we examined whether E1B-AP5 function was integral to these pathways. Here, we show a novel role for E1B-AP5 as a key regulator of ATR signaling pathways activated during Ad infection. E1B-AP5 is recruited to viral replication centers during infection, where it colocalizes with ATR-interacting protein (ATRIP) and the ATR substrate replication protein A 32 (RPA32). Indeed, E1B-AP5 associates with ATRIP and RPA complex component RPA70 in both uninfected and Ad-infected cells. Additionally, glutathione S-transferase pull-downs show that E1B-AP5 associates with RPA components RPA70 and RPA32 directly in vitro. E1B-AP5 is required for the ATR-dependent phosphorylation of RPA32 during infection and contributes to the Ad-induced phosphorylation of Smc1 and H2AX. In this regard, it is interesting that Ad5 and Ad12 differentially promote the phosphorylation of RPA32, Rad9, and Smc1 during infection such that Ad12 promotes a significant phosphorylation of RPA32 and Rad9, whereas Ad5 only weakly promotes RPA32 phosphorylation and does not induce Rad9 phosphorylation. These data suggest that Ad5 and Ad12 have evolved different strategies to regulate DNA damage signaling pathways during infection in order to promote viral replication. Taken together, our results define a role for E1B-AP5 in ATR signaling pathways activated during infection. This might have broader implications for the regulation of ATR activity during cellular DNA replication or in response to DNA damage.

Sun L, Huang Y, Edwards RA, Yang S, Blackford AN, Niedzwiedz W, Glover JNM. 2017. Structural Insight into BLM Recognition by TopBP1. Structure, 25 (10), pp. 1582-1588.e3. | Show Abstract | Read more

Topoisomerase IIβ binding protein 1 (TopBP1) is a critical protein-protein interaction hub in DNA replication checkpoint control. It was proposed that TopBP1 BRCT5 interacts with Bloom syndrome helicase (BLM) to regulate genome stability through either phospho-Ser304 or phospho-Ser338 of BLM. Here we show that TopBP1 BRCT5 specifically interacts with the BLM region surrounding pSer304, not pSer338. Our crystal structure of TopBP1 BRCT4/5 bound to BLM reveals recognition of pSer304 by a conserved pSer-binding pocket, and interactions between an FVPP motif N-terminal to pSer304 and a hydrophobic groove on BRCT5. This interaction utilizes the same surface of BRCT5 that recognizes the DNA damage mediator, MDC1; however the binding orientations of MDC1 and BLM are reversed. While the MDC1 interactions are largely electrostatic, the interaction with BLM has higher affinity and relies on a mix of electrostatics and hydrophobicity. We suggest that similar evolutionarily conserved interactions may govern interactions between TopBP1 and 53BP1.

Henssen AG, Koche R, Zhuang J, Jiang E, Reed C, Eisenberg A, Still E, MacArthur IC, Rodríguez-Fos E, Gonzalez S et al. 2017. PGBD5 promotes site-specific oncogenic mutations in human tumors. Nat Genet, 49 (7), pp. 1005-1014. | Show Abstract | Read more

Genomic rearrangements are a hallmark of human cancers. Here, we identify the piggyBac transposable element derived 5 (PGBD5) gene as encoding an active DNA transposase expressed in the majority of childhood solid tumors, including lethal rhabdoid tumors. Using assembly-based whole-genome DNA sequencing, we found previously undefined genomic rearrangements in human rhabdoid tumors. These rearrangements involved PGBD5-specific signal (PSS) sequences at their breakpoints and recurrently inactivated tumor-suppressor genes. PGBD5 was physically associated with genomic PSS sequences that were also sufficient to mediate PGBD5-induced DNA rearrangements in rhabdoid tumor cells. Ectopic expression of PGBD5 in primary immortalized human cells was sufficient to promote cell transformation in vivo. This activity required specific catalytic residues in the PGBD5 transposase domain as well as end-joining DNA repair and induced structural rearrangements with PSS breakpoints. These results define PGBD5 as an oncogenic mutator and provide a plausible mechanism for site-specific DNA rearrangements in childhood and adult solid tumors.

Blackford AN, Jackson SP. 2017. ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response. Mol Cell, 66 (6), pp. 801-817. | Show Abstract | Read more

In vertebrate cells, the DNA damage response is controlled by three related kinases: ATM, ATR, and DNA-PK. It has been 20 years since the cloning of ATR, the last of the three to be identified. During this time, our understanding of how these kinases regulate DNA repair and associated events has grown profoundly, although major questions remain unanswered. Here, we provide a historical perspective of their discovery and discuss their established functions in sensing and responding to genotoxic stress. We also highlight what is known regarding their structural similarities and common mechanisms of regulation, as well as emerging non-canonical roles and how our knowledge of ATM, ATR, and DNA-PK is being translated to benefit human health.

Balmus G, Barros AC, Wijnhoven PWG, Lescale C, Hasse HL, Boroviak K, le Sage C, Doe B, Speak AO, Galli A et al. 2016. Synthetic lethality between PAXX and XLF in mammalian development. Genes Dev, 30 (19), pp. 2152-2157. | Show Abstract | Read more

PAXX was identified recently as a novel nonhomologous end-joining DNA repair factor in human cells. To characterize its physiological roles, we generated Paxx-deficient mice. Like Xlf-/- mice, Paxx-/- mice are viable, grow normally, and are fertile but show mild radiosensitivity. Strikingly, while Paxx loss is epistatic with Ku80, Lig4, and Atm deficiency, Paxx/Xlf double-knockout mice display embryonic lethality associated with genomic instability, cell death in the central nervous system, and an almost complete block in lymphogenesis, phenotypes that closely resemble those of Xrcc4-/- and Lig4-/- mice. Thus, combined loss of Paxx and Xlf is synthetic-lethal in mammals.

Lescale C, Lenden Hasse H, Blackford AN, Balmus G, Bianchi JJ, Yu W, Bacoccina L, Jarade A, Clouin C, Sivapalan R et al. 2016. Specific Roles of XRCC4 Paralogs PAXX and XLF during V(D)J Recombination. Cell Rep, 16 (11), pp. 2967-2979. | Show Abstract | Read more

Paralog of XRCC4 and XLF (PAXX) is a member of the XRCC4 superfamily and plays a role in nonhomologous end-joining (NHEJ), a DNA repair pathway critical for lymphocyte antigen receptor gene assembly. Here, we find that the functions of PAXX and XLF in V(D)J recombination are masked by redundant joining activities. Thus, combined PAXX and XLF deficiency leads to an inability to join RAG-cleaved DNA ends. Additionally, we demonstrate that PAXX function in V(D)J recombination depends on its interaction with Ku. Importantly, we show that, unlike XLF, the role of PAXX during the repair of DNA breaks does not overlap with ATM and the RAG complex. Our findings illuminate the role of PAXX in V(D)J recombination and support a model in which PAXX and XLF function during NHEJ repair of DNA breaks, whereas XLF, the RAG complex, and the ATM-dependent DNA damage response promote end joining by stabilizing DNA ends.

Blackford AN, Nieminuszczy J, Schwab RA, Galanty Y, Jackson SP, Niedzwiedz W. 2015. TopBP1 interacts with BLM to maintain genome stability but is dispensable for preventing BLM degradation. Mol Cell, 57 (6), pp. 1133-1141. | Show Abstract | Read more

The Bloom syndrome helicase BLM and topoisomerase-IIβ-binding protein 1 (TopBP1) are key regulators of genome stability. It was recently proposed that BLM phosphorylation on Ser338 mediates its interaction with TopBP1, to protect BLM from ubiquitylation and degradation (Wang et al., 2013). Here, we show that the BLM-TopBP1 interaction does not involve Ser338 but instead requires BLM phosphorylation on Ser304. Furthermore, we establish that disrupting this interaction does not markedly affect BLM stability. However, BLM-TopBP1 binding is important for maintaining genome integrity, because in its absence cells display increased sister chromatid exchanges, replication origin firing and chromosomal aberrations. Therefore, the BLM-TopBP1 interaction maintains genome stability not by controlling BLM protein levels, but via another as-yet undetermined mechanism. Finally, we identify critical residues that mediate interactions between TopBP1 and MDC1, and between BLM and TOP3A/RMI1/RMI2. Taken together, our findings provide molecular insights into a key tumor suppressor and genome stability network.

Ochi T, Blackford AN, Coates J, Jhujh S, Mehmood S, Tamura N, Travers J, Wu Q, Draviam VM, Robinson CV et al. 2015. DNA repair. PAXX, a paralog of XRCC4 and XLF, interacts with Ku to promote DNA double-strand break repair. Science, 347 (6218), pp. 185-188. | Show Abstract | Read more

XRCC4 and XLF are two structurally related proteins that function in DNA double-strand break (DSB) repair. Here, we identify human PAXX (PAralog of XRCC4 and XLF, also called C9orf142) as a new XRCC4 superfamily member and show that its crystal structure resembles that of XRCC4. PAXX interacts directly with the DSB-repair protein Ku and is recruited to DNA-damage sites in cells. Using RNA interference and CRISPR-Cas9 to generate PAXX(-/-) cells, we demonstrate that PAXX functions with XRCC4 and XLF to mediate DSB repair and cell survival in response to DSB-inducing agents. Finally, we reveal that PAXX promotes Ku-dependent DNA ligation in vitro and assembly of core nonhomologous end-joining (NHEJ) factors on damaged chromatin in cells. These findings identify PAXX as a new component of the NHEJ machinery.

2861