DNA lesions in the template strand block the continued progression of the replication fork. Human cells possess a number of DNA polymerases (Pols) with the ability to synthesize DNA opposite such blocking lesions, and structural, biochemical, and cellular studies have indicated that these Pols function in translesion synthesis (TLS) in a highly specialized manner. However, there exists little, if any, information on many of the important aspects of TLS. It remains unclear whether TLS occurs in conjunction with the stalled replication fork or occurs post-replicatively in gaps that are left behind opposite DNA lesions, and whether other important cellular processes also play an essential role in promoting lesion bypass by TLS. In this proposal we will carry out studies to examine the many aspects of the novel hypothesis that the cohesin complex assembles de novo at the replication fork stalled at a DNA lesion where it mediates the stabilization of the replication fork, and that TLS occurs in conjunction with the stalled replication fork.
In Aim 1, w will carry out studies in human cells to: (a) analyze the requirements of various proteins of the cohesin complex for TLS opposite UV induced DNA lesions. For these studies, we will use the SV40 origin-based based plasmid which harbors a site-specific lesion on one of the template strands. Mutagenesis studies will be done using the supF gene carried on a plasmid in human cells, and with the cII gene integrated into the chromosome in mouse cells;(b) determine the requirement of the cohesin complex for the localization of TLS Pols into replication foci in UV damaged cells;(c) determine the requirement of cohesin for complex formation with replication proteins and with TLS Pols in UV damaged cells;and (d) determine the requirement of Smc3 acetylation in TLS.
In Aim 2, we will carry out studies to: (a) examine the requirement of the cohesin complex for promoting replication through DNA lesions in UV damaged human cells;(b) using in situ ligation assays, examine whether direct interactions of cohesin proteins with TLS Pols and with the CMG complex occur in UV damaged human cells;(c) determine by ChIP analyses whether TLS occurs at a site-specific cis-syn TT dimer in conjunction with the CMG helicase complex at the replication fork and with the cohesin complex;and (d) analyze the role of cohesin in TLS in the Xenopus egg extract system. By coordinating TLS with the replication fork stalled at DNA lesions induced by environmental and cellular DNA damaging agents, the cohesin complex would play an important role in maintaining the integrity and fidelity of the genome. In keeping with this idea, defects in the cohesin proteins confer a high degree of genomic instability and are associated with cancers;thus, the cohesin proteins play an important cancer suppressor role in humans.
DNA lesions are generated in human cells from cellular oxidative damage and from exposure to environmental pollutants and carcinogens. Cellular processes that function in ameliorating the harmful consequences of DNA lesions play an essential role in maintaining genomic stability and preventing carcinogenesis. A comprehensive understanding of the means by which genomic stability is maintained is crucial for deciphering the underlying bases of carcinogenesis.