Translesion synthesis (TLS) DNA polymerases (Pols) help maintain the continuity of the replication fork by mediating the bypass of DNA lesions. To elucidate the mechanisms that govern replicative bypass of DNA adducts in eukaryotes including in humans, the proposed studies will utilize the yeast Saccharomyces cerevisiae. Since DNA repair and lesion bypass processes have been highly conserved between yeast and humans, the knowledge gained from these studies will be directly applicable to the understanding of TLS mechanisms in human cells.
In Aim 1, the role of DNA damage-induced checkpoint kinase-mediated Rev1 phosphorylation in Pol6-dependent TLS will be studied using a combined genetic and biochemical approach. In particular, the hypothesis that Rev1 phosphorylation enhances the efficiency of TLS by Pol6 will be tested.
In Aim 2, studies will be done with the novel 4-subunit Pol6, comprised of Rev3, Rev7, Pol31, and Pol32 subunits, that we have purified. Specifically, we will examine its lesion bypass properties and will determine whether it physically and functionally interacts with PCNA.
In Aim 3, the role of Slx4, which we have recently identified as a component of Pol6-dependent TLS and which associates with Rev1 to form the Rev1-Slx4 complex, will be defined. Genetic and biochemical studies will be conducted to determine the manner in which the Rev1-Slx4 complex modulates Pol6 function.
In Aim 4, studies will carried out to test the hypothesis that the Rad6-Rad18-Rad5 complex functions as a structural element in TLS to promote the assembly of TLS Pols and their associated protein factors at DNA lesions.
In Aim 5, TLS through site-specific DNA lesions carried on a plasmid will be examined so as to determine the contributions of different protein complexes to the TLS reaction. In particular, we will test the hypothesis that in addition to its structural role in the assembly of TLS Pols, the Rad6-Rad18-Rad5 complex functions as a mediator in TLS wherein it coordinates the actions of the TLS Pols with that of the replicative Pol. The roles of PCNA ubiquitination and of the PCNA binding and ubiquitin binding domains present in TLS Pols will also be analyzed in these studies. By helping ensure the continuity of the replication fork and by promoting error-free replication through a large variety of DNA adducts that result from the attack of free radicals and reactive oxygen species generated during normal cellular oxidative reactions and from exposure to chemical and environmental carcinogens, the TLS processes provide an important means for minimizing the mutagenic effects of DNA damage and for the suppression of carcinogenesis in humans. For instance, the inactivation of Pol7 in humans causes highly elevated levels of skin cancers. The proposed studies are highly relevant for cancer etiology, as they will be important for elucidating the mechanisms of TLS in human cells and for revealing the contributions that TLS processes make to genomic integrity and to the prevention of the mutagenic and carcinogenic consequences of DNA lesions.

Public Health Relevance

DNA lesions are generated in human cells from attack of free radicals and reactive oxygen species resulting from cellular oxidative reactions and from exposure to chemical and environmental carcinogens. By promoting replication through a diverse array of DNA adducts, translesion synthesis DNA polymerases play important roles in maintaining genomic integrity and fidelity. The proposed studies will be important for the elucidation of mechanisms that govern the replicative bypass of DNA lesions in human cells and for delineating the contributions of these processes to the avoidance of mutagenesis and carcinogenesis.

National Institute of Health (NIH)
National Cancer Institute (NCI)
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Cancer Etiology Study Section (CE)
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Okano, Paul
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University of Texas Medical Br Galveston
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Johnson, Robert E; Klassen, Roland; Prakash, Louise et al. (2015) A Major Role of DNA Polymerase ? in Replication of Both the Leading and Lagging DNA Strands. Mol Cell 59:163-175
Jain, Rinku; Rajashankar, Kanagalaghatta R; Buku, Angeliki et al. (2014) Crystal structure of yeast DNA polymerase ? catalytic domain. PLoS One 9:e94835
Jain, Rinku; Vanamee, Eva S; Dzikovski, Boris G et al. (2014) An iron-sulfur cluster in the polymerase domain of yeast DNA polymerase ?. J Mol Biol 426:301-8
Gómez-Llorente, Yacob; Malik, Radhika; Jain, Rinku et al. (2013) The architecture of yeast DNA polymerase ?. Cell Rep 5:79-86
Johnson, Robert E; Prakash, Louise; Prakash, Satya (2012) Pol31 and Pol32 subunits of yeast DNA polymerase ? are also essential subunits of DNA polymerase ?. Proc Natl Acad Sci U S A 109:12455-60
Ai, Yongxing; Wang, Jialiang; Johnson, Robert E et al. (2011) A novel ubiquitin binding mode in the S. cerevisiae translesion synthesis DNA polymerase ?. Mol Biosyst 7:1874-82
Gangavarapu, Venkateswarlu; Santa Maria, Sergio R; Prakash, Satya et al. (2011) Requirement of replication checkpoint protein kinases Mec1/Rad53 for postreplication repair in yeast. MBio 2:e00079-11
Acharya, Narottam; Klassen, Roland; Johnson, Robert E et al. (2011) PCNA binding domains in all three subunits of yeast DNA polymerase ? modulate its function in DNA replication. Proc Natl Acad Sci U S A 108:17927-32
Silverstein, Timothy D; Jain, Rinku; Johnson, Robert E et al. (2010) Structural basis for error-free replication of oxidatively damaged DNA by yeast DNA polymerase ?. Structure 18:1463-70
Silverstein, Timothy D; Johnson, Robert E; Jain, Rinku et al. (2010) Structural basis for the suppression of skin cancers by DNA polymerase eta. Nature 465:1039-43

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