Our long-term objective is to delineate the mechanisms eukaryotic cells employ to overcome replication blockage caused by DNA lesions.
The specific aims of this project are to elucidate the mechanisms of translesion synthesis (TLS) by DNA polymerases eta and n in the yeast Saccharomyces cerevisiae.
In Aim 1, we will carry out biochemical and genetic studies to examine the hypothesis that the Rev1 DNA synthetic activity co-operates with Poleta_ to replicate past highly distorting DNA adducts originating from endogenous metabolism at the reactive N position of guanine.
Aim 2 will test the idea that Rev1, via its interaction with PCNA, plays a crucial role in the targeting of Poleta to the lesion site.
In Aim 3, proteins that are components of Poln, Revl, and Rev7 associated multi-protein assemblies will be purified, as will interacting proteins. Biochemical and genetic studies will define the roles of the newly identified protein factors in TLS.
In Aim 4, in vivo and in vitro studies will examine the ubiquitination of Poln, Rev1, Rev3, Rev7, and other proteins, and ascertain the roles of the Rad6-Radl8 and Mms2-Ubcl3 ubiquitin conjugating enzyme complexes in these ubiquitination events.
In Aim 5, the mechanism by which Poleta and Poln enter into the replication complex stalled at a lesion site will be determined. Our studies should yield important insights into the mechanisms of translesion synthesis by Poleta and Poln. The results will be highly relevant for cancer biology, as the manner in which DNA lesions are bypassed during replication has a major impact on genome stability, and, in fact, inactivation of Poln in humans leads to the cancer prone syndrome, the variant form of xeroderma pigmentosum. ? ?

National Institute of Health (NIH)
National Cancer Institute (NCI)
Research Project (R01)
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Special Emphasis Panel (ZRG1-CDF-2 (90))
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Okano, Paul
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University of Texas Medical Br Galveston
Schools of Medicine
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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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