Researchers at Tokyo Metropolitan University have identified key factors in the mechanism behind DNA repair in our body. For the first time, they showed that the “proofreading” part of the DNA replication enzyme epsilon ensures safe termination of replication at damaged parts of the DNA strand, thereby saving the DNA serious damage. This new knowledge gives scientists the means to make cancer drugs more effective and could provide new diagnostic methods.
Our DNA is under attack. Every day, approximately 55,000 single-strand breaks (SSBs) appear in the strands constituting the DNA helices of individual cells. When polymerases, molecules that replicate DNA strands, attempt to create new helices from broken strands, they can break the helix, creating what is called a single-end double-strand break (seDSB).
Fortunately, cells have their own ways of dealing with strand damage. One is homology-directed repair (HDR), where double-strand breaks are corrected. Another solution is “fork reversal”, where the replication process is reversed, thereby preventing single-strand breaks from transforming into DSBs.
The exact mechanism behind fork inversion remains unknown. Understanding how to prevent DNA damage is essential not only for preventing cancers, but also for ensuring the effectiveness of anticancer drugs that rely on DNA damage. Take camptothecin (CPT), an anticancer drug that introduces many single-strand breaks; Since cancer cells tend to replicate more quickly, they create a lot of seDSBs and die, leaving normal cells less damaged.
Now, an international team led by Professor Kouji Hirata of Tokyo Metropolitan University has shed new light on how fork inversion works. They focused on epsilon polymerase, an enzyme responsible for making new DNA from an unzipped part of the DNA. They found that exonuclease, the “proofreading” part of the polymerase that ensures copy accuracy, played a key role, a new and rare insight into the largely unknown molecular mechanism behind fork reversal.
The article is published in the journal Nucleic acid research.
First, the team found that exonuclease-deficient cells showed high susceptibility to CPT exposure. Deleting a factor known as PARP, the only other player known to affect fork reversal, also led to increased cell death. However, when both were removed, there was no further increase in cell death beyond what was observed with PARP. This suggests that PARP and the exonuclease polymerase epsilon work together to trigger fork reversal.
In addition, the team studied cells whose gene encoding BRCA1 (the breast cancer susceptibility protein) was disrupted; additional exonuclease deficiency resulted in significantly increased sensitivity to CPT, much more than would be expected from either defect. Since BRCA1 deficiency is linked to a high risk of breast cancer, the exonuclease could be targeted to make drug treatments more effective.
Researchers have shown that drugs targeting the exonuclease polymerase epsilon can amplify the effect of anticancer drugs. Equally important, exonuclease defects have previously been observed in a wide range of cancers, including bowel cancer; it is therefore likely that these cells have impaired fork reversal capacity, a promising target for future diagnostics as well as treatments.
Tasnim Ahmad et al, Leading strand epsilon DNA polymerase proofreading exonuclease prevents replication fork collapse at broken template strands, Nucleic acid research (2023). DOI: 10.1093/nar/gkad999
Provided by Tokyo Metropolitan University
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