DNA breaks are a significant source of errors and mutations that can lead to cancer. Cells possess DNA repair systems that try to correct these errors, although sometimes the repair process itself can create cancerous mutations. Understanding how DNA repair functions, or mis-functions in cancer, is therefore an important area of research. The Blackford group at the MRC WIMM is one of several research groups in Oxford tackling this issue.
Early markers of DNA repair
DNA repair mechanisms are known to be active in interphase, the part of the cell cycle when the cell is not actively dividing. It is currently believed that no DNA repair takes place in mitosis. However, cells can at least detect DNA breaks during mitosis, as early players in the DNA repair process are still recruited to DNA damage. But is this early recruitment of DNA repair players important for cells to survive DNA breaks in mitosis? Does it act as a flag to mark breaks that must be repaired later, or is it just the signs of an incomplete repair process?
To address this question, the Blackford lab and their collaborators in the Stucki lab at the University of Zurich examined cells lacking the last known DNA repair protein to be recruited to the DNA breaks in mitosis, the scaffolding protein MDC1. Specifically, they asked whether cells lacking MDC1 were able to cope when exposed to ionising radiation, which causes DNA breaks. The researchers showed that these cells were less able to survive DNA breaks in mitosis than their MDC1-containing counterparts, suggesting that MDC1 is indeed important for cell survival during cell division.
As MDC1 is known to be a scaffolding protein, the next question was to determine what other proteins it recruits to the DNA breaks. In conjunction with collaborators at the University of Oxford’s Target Discovery Institute, the team used mass spectrometry to identify an evolutionarily conserved player called TopBP1 as a binding partner for MDC1. With the help of the MRC WIMM’s Wolfson Imaging Facility, the team was then able to image TopBP1 at high resolution. What they observed was surprising:
“Most known DNA repair players are visible on DNA as very localised dots (foci), recruited to the precise areas where the DNA is broken” explains Dr Andrew Blackford, senior author on this study. “Our imaging shows that TopBP1 localises to DNA by forming filaments that bridge MDC1 foci, which, we hypothesise, may be holding together two sections of broken DNA. This process must be regulated by the cell in some way during mitosis, as we never observe these fibres in interphase”. This work is now published in Molecular Cell.
The team is now interested in understanding whether any other DNA repair proteins localise to these fibres, how the process is regulated and how it plays a role in DNA repair in mitosis. They also hope to explore whether this might be a new therapeutic target for cancer treatment.
Dr Andrew Blackford is a Group Leader in the Department of Oncology and the CRUK/MRC Oxford Institute for Radiation Oncology.