Rev1 is unique among DNA polymerases in that the protein itself rather than the DNA template determines the specificity for both the templating and the incoming nucleotide. The Rev1 crystal structure that we have solved suggests an elegant mechanism by which this polymerase could promote proficient and error-free replication through a large variety of N2-adducted guanines that result from endogenous oxidative damage and from exposure to a number of widespread DNA damaging chemical and environmental carcinogens such as butadiene epoxides and anti-benzo[a]pyrene diol epoxides. Such a role for yeast Rev1 will be examined using a combined biochemical, genetic, and structural approach.
In Aim 1, key amino acid residues involved in the pairing with the incoming dCTP and in the eviction and stabilization of templating G will be mutated and their effects on nucleotide incorporation specificity and catalytic efficiency determined.
In Aim 2, the conformational changes that occur in Rev1 upon DNA binding and upon dNTP binding will be analyzed through crystal structures of the Rev1 apoenzyme and Rev1.DNA binary complex and their comparison to the structure of Rev1.DNA.dCTP ternary complex.
In Aim 3, biochemical studies will be undertaken to test the hypothesis that a major role of the Rev1 DNA synthetic activity is to promote efficient and error-free replication through various N2adducts of guanine that sterically impinge upon the minor groove, and which result from cellular oxidative damage or from exposure to DNA damaging environmental carcinogens. Also as part of this aim, we will examine the means by which complex formation between Rev1 and the extender polymerase coordinates the nucleotide insertion and the subsequent extension steps in the bypass of these adducts. As a complement to these biochemical studies, in Aim 4, crystal structures of Rev1 with DNAs containing a variety of N2 guanine adducts will be determined, as well as the structure of Rev1 with an abasic lesion.
In Aim 5, genetic studies will be done to establish the requirement of the Rev1 DNA synthetic activity in promoting error-free replication through the various N2-adducts of guanine in yeast cells. Rev1 as well as the other DNA repair proteins are highly conserved between yeast and humans. The proficient and accurate ability of Rev1 for promoting replication through the large variety of DNA adducts that form at the N2 of guanine will have a major impact on genome stability by keeping the rate of mutations low, reducing thereby the incidence of carcinogenesis in humans. The results of this study are highly relevant for cancer biology and etiology, as error-free replication through DNA lesions provides for an important means of cancer prevention.
DNA lesions generated from cellular oxidative damage and from exposure to environmental pollutants affect the stability and integrity of genomic DNA. Error-free replication through such lesions reduces their adverse impact by keeping the rate of mutations low and by reducing the incidence of cancer formation. The proposed studies will examine the role of Rev1 DNA polymerase in promoting error-free replication through DNA lesions.
|Nair, Deepak T; Johnson, Robert E; Prakash, Louise et al. (2011) DNA synthesis across an abasic lesion by yeast REV1 DNA polymerase. J Mol Biol 406:18-28|
|Vasquez-Del Carpio, Rodrigo; Silverstein, Timothy D; Lone, Samer et al. (2009) Structure of human DNA polymerase kappa inserting dATP opposite an 8-OxoG DNA lesion. PLoS ONE 4:e5766|
|Swan, Michael K; Johnson, Robert E; Prakash, Louise et al. (2009) Structure of the human Rev1-DNA-dNTP ternary complex. J Mol Biol 390:699-709|