| dc.description.abstract | Genome integrity is established through the high fidelity and efficient replication of cellular DNA as well as the ability to repair DNA when DNA damage or errors occur. A wide array of genotoxins can interfere with DNA synthesis by blocking DNA helicase or polymerase activity at replication forks, either directly or indirectly. Failure to employ repair mechanisms in a proper manner can result in genetic mutations, genomic instability, and cancer. A primary response to genotoxins is replication fork reversal, which involves reannealing of parental DNA strands and extrusion of nascent DNA strands to form a reversed fork. Fork reversal is thought to support error-free bypass of DNA lesions and regulate the rate of DNA synthesis. The nascent DNA strands of replication forks are degraded by exonucleases in a process known as ‘Nascent Strand Degradation' (NSD), which is thought to convert the reversed fork back to a replication fork so that DNA synthesis can resume. Although NSD is important for genome stability, too much NSD causes genome instability. Thus, NSD must be triggered efficiently but not overly triggered to ensure genome stability. Our lab has previously used Xenopus egg extracts to show that uncoupling causes NSD and fork reversal. However, a major question from this work was how do these events occur naturally in response to damaged DNA? To study this, we utilized 6-oxo-M1dG, a model DNA lesion that blocks polymerase function while also being resistant to more canonical DNA repair pathways, such as Translesion Synthesis. Our data shows that the non-bulky polymerase-blocking DNA lesion, 6-oxo-M1dG, is able to block DNA synthesis and stall DNA replication in the presence of a singular replication fork. Strikingly, the convergence of two replication forks allows for the efficient bypass of 6-oxo-M1dG in a CMG unloading-independent mechanism. Additionally, 6-oxo-M1dG is not readily repaired by any active mechanism in Xenopus egg extracts, making it a model DNA lesion to study replication-coupled DNA repair pathways. | |
