Translesion synthesis (TLS) DNA polymerases (pols) promote replication through DNA lesions. Humans possess four TLS pols that belong to the Y-family, Pols eta, iota, kappa, and Rev1, and another Pol, Pol zeta, that belongs to the B-family. The overall objectives of this proposal are to elucidate the biological roles of these pols in TLS in human cells, to determine whether they function in an error-free or mutagenic manner, and to identify the means by which a TLS Pol gains access to PCNA and thereby to the replication fork stalled at a DNA lesion site. These and related questions will be studied in the following Specific Aims.
In Aim 1, the roles of TLS Pols in promoting replication through a diverse set of DNA lesions in human cells will be analyzed using an SV-40 based plasmid system that we have constructed. DNA lesions to be studied include UV photoproducts, and DNA adducts such as 1,N6-ethenodeoxyadenosine, 1,N2-propano-2'-deoxyguanosine, and (+) trans-dG-N2- benzo[a]pyrene diol epoxide, which result from cellular oxidative damage and from exposure to DNA damaging environmental carcinogens.
In Aim 2, studies will be done to test the hypothesis that in human cells, Rev1 functions as a structural element for Pols eta, iota, and kappa.
In Aim 3, the roles of TLS Pols in promoting replication through UV lesions and whether they do so in an error-free or mutagenic manner will be studied in the chromosomal cII transgene carried in a mouse embryonic fibroblast cell line. The contributions that cyclobutane pyrimidine dimers vs. (6-4) photoproducts make to UV mutations resulting from the action of different TLS Pols will be analyzed.
In Aim 4, the model that, in addition to their binding to PCNA via their PIP domain, TLS Pols bind the ubiquitin moiety on PCNA via their ubiquitin binding domain, will be tested by genetic and biochemical studies of mutations in these domains of Pol eta.
In Aim 5, biochemical studies will be undertaken to test the hypothesis that upon stalling at a DNA lesion site, the binding of the replicative pol to PCNA is attenuated as a result of PCNA ubiquitination, and that, in turn, promotes the access of the TLS pol to the primer-template junction via its binding to ubiquitinated PCNA (Ub-PCNA). Specifically, these studies will examine how Ub-PCNA promotes synthesis by Pol eta through a cis-syn TT dimer when a processively moving Pol delta has become stalled at the lesion site. By helping ensure the continuity of the replication fork, TLS Pols play an important role in the maintenance of genomic integrity. Furthermore, their proficient abilities for promoting error-free replication through a large variety of DNA adducts that result from cellular oxidative reactions and from exposure to chemical and environmental carcinogens will have a major impact on genome stability by keeping the rate of mutations low, reducing thereby the incidence of carcinogenesis in humans. In fact, the inactivation of Pol eta in humans results in highly elevated levels of skin cancers. The proposed studies are highly relevant for cancer etiology as the results will reveal how human cells minimize the mutagenic and carcinogenic consequences of DNA lesions.
DNA lesions are generated in human cells from cellular oxidative reactions and from exposure to a variety of environmental pollutants and carcinogens. By promoting error-free replication through such DNA lesions, translesion synthesis DNA polymerases would play an important role in maintaining genome stability by keeping the rate of mutations and hence the incidence of cancers low. The proposed studies will elucidate the roles of human DNA polymerases in promoting replication through a variety of DNA lesions and they will examine the mechanisms of this process. ? ? ?
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