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Abstract
DNA interstrand cross-links (ICLs) are a unique type of damage in which complementary DNA strands are covalently linked, and therefore require a distinct pathway for repair. There is evidence for multiple ICL repair pathways, which has inhibited the understanding of this type of repair. Both nucleotide excision repair (NER) and homologous recombination (HR) play important roles in ICL repair. Translesion synthesis (TLS) is also involved in certain pathways, specifically when filling in a gap across from an unhooked ICL. The goal of this dissertation was to identify the polymerase(s) involved in this process.
The A-family polymerases have been suggested to be involved in ICL repair. In E. coli, the main ICL repair pathway involves incisions by NER proteins to unhook the ICL, gap repair by HR, incisions by NER to release the ICL, and gap repair by the A-family polymerase Pol I. If HR is unable to repair the gap across from an unhooked ICL, a TLS polymerase could bypass the ICL. We proposed this bypass could be done by Pol I since it is already present at the ICL site. To examine this hypothesis, we developed an in vitro DNA polymerase assay to monitor the bypass of an unhooked ICL. We found that Pol I had the ability to bypass the ICL and preferred to insert the correct nucleotide across from the ICL.
In humans, the A-family polymerases, PolQ and PolN, have been implicated in ICL repair due to their homology to Drosophila mus308, whose mutant is sensitive to DNA cross-linking agents. Additionally, both PolQ and PolN have TLS activity in vitro. To determine the role of PolQ and PolN in human ICL repair, we suppressed PolQ or PolN expression in HeLa cells using siRNA. Both PolQ- and PolN-depleted cells were specifically sensitive to the DNA cross-linking agent mitomycin C (MMC). Furthermore, we found purified PolN bypassed an unhooked ICL in vitro, and showed preference to incorporate the correct nucleotide across from the ICL. Overall, the results presented here demonstrate that the A-family polymerases accurately bypass an unhooked ICL.
