Date of Degree
PhD (Doctor of Philosophy)
M. Todd Washington
Sites of unrepaired DNA damage block DNA synthesis by replicative polymerases and interrupt replication fork progression. This is a problem because stalled replication forks can collapse leading to DNA double-stranded breaks, chromosome instability, and eventually cell death. The translesion DNA synthesis (TLS) pathway rescues stalled replication forks by using polymerases that are specialized for replicating damaged DNA, called non-classical polymerases. The use of TLS is risky however, because it can result in replication errors leading to mutations and eventually carcinogenesis. Genetic studies have shown that the non-classical polymerase Rev1 plays a key role in error-prone TLS, but not much is known about its function. Rev1 is a modular protein that has three functional regions: an N-terminal BRCT (BRCA1 C-terminal) domain, a polymerase domain, and a C-terminal domain (CTD). Rev1 promotes replication fork rescue by other non-classical polymerases through interactions mediated by its CTD; however, the roles of the other two domains in promoting TLS are not well understood.
Non-classical polymerases have evolved to be specific for the bypass of a small number of lesions or families of closely related lesions called cognate lesions. Genetic studies using yeast have suggested that the abasic site, one of the most common forms of DNA damage, is a cognate lesion of Rev1. However, steady state kinetic studies suggest Rev1 is very inefficient at incorporating nucleotides opposite an abasic site. To resolve this controversy, I examined the pre-steady state kinetics of nucleotide incorporation by yeast Rev1. I found that Rev1 is capable of rapid nucleotide incorporation, but that only a small fraction of the protein molecules possessed this robust activity. I characterized the nucleotide incorporation activity of the catalytically robust fraction of Rev1 and found that it efficiently incorporated dCTP opposite a template abasic site. My studies strongly suggest that the abasic site is a cognate lesion for Rev1.
The rev1-1 allele encodes a mutant form of yeast Rev1 with a G193R substitution in the BRCT domain that reduces error-prone TLS and results in low levels of DNA damage-induced mutagenesis. Unfortunately, little is known about the function of the Rev1 BRCT domain and how this mutation disrupts this pathway. To gain insight into the function of the Rev1 BRCT domain, I solved the X-ray crystal structure of the domain and showed that substitutions in residues constituting its phosphate-binding pocket do not affect error-prone TLS. This suggests that the role of the Rev1 BRCT domain does not involve binding a phosphorylated interaction partner. I also found that the G193 residue is located in a conserved turn of the BRCT domain, and my in vivo and in vitro studies suggest that the G193R substitution disrupts Rev1 function by destabilizing the fold of the BRCT domain.
Copyright 2012 John Michael Pryor
Pryor, John Michael. "Studies of the Rev1 protein and its role in the replication of damaged DNA." PhD diss., University of Iowa, 2012.