Abstract

Our long term objectives are to understand the roles of human DNA polymerases, Pol-eta, Pol-iota, and Pol-kappa, in the replication of damaged DNA.
In Specific Aim 1, it is proposed that for replication to occur through highly distorting DNA lesions, two different DNA polymerases are required, wherein Pol-iota carries out nucleotide incorporation opposite the lesion and Pol-kappa extends from the inserted nucleotide. By contrast, Pol-eta is suggested to replicate only through moderately distorting lesions. These ideas will be tested using propano adducts that are highly distorting and affect Watson-Crick base pairing, and which have been shown to be present at high levels in human DNA. Propano adducts are formed in DNA from the reaction of the reactive N2 of guanine with alpha,beta-unsaturated aldehydes or enals, which include acrolein and crotonaldehyde, that are ubiquitious pollutants in the environment and which are also formed in cells as the end product of peroxidation of lipids, and trans-4-hydroxy-2-nonenal, a unique oxidation product of omega-6 polyunsaturated fatty acids.
In Specific Aim 2, it is proposed that Pol-kappa can efficiently extend mispaired primer termini either by incorporating the next correct nucleotide or by using a specific primer-template misalignment mechanism that generates -1 deletions. The proficiency of Pol-kappa to extend mispaired primer termini by these two means will be examined by steady-state kinetic analyses.
In Specific Aim 3, it is proposed that because of their proficient ability to extend from mispaired primer termini, both Pol-kappa and Pol-zeta would contribute to mutagenesis in undamaged as well as UV damaged cells in humans. This idea will be tested by carrying out genetic studies in the fission yeast S. pombe to examine the relative contributions of Pol-kappa and Pol-zeta to spontaneous and UV induced mutagenesis.
In Specific Aim 4, the manner by which the switch from the replicative polymerase, Pol-delta, to a translesion synthesis polymerase occurs at the lesion site will be studied and the role of PCNA as the key intermediary in this process delineated. Our understanding of the manner in which human cells overcome blocks to DNA replication should be greatly enhanced by these studies.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Research Project (R01)
Project #
5R01ES012411-02
Application #
6796160
Study Section
Chemical Pathology Study Section (CPA)
Program Officer
Mcallister, Kimberly A
Project Start
2003-09-01
Project End
2008-05-31
Budget Start
2004-06-01
Budget End
2005-05-31
Support Year
2
Fiscal Year
2004
Total Cost
$358,625
Indirect Cost

  Institution

Name
University of Texas Medical Br Galveston
Department
Biochemistry
Type
Schools of Medicine
DUNS #
800771149
City
Galveston
State
TX
Country
United States
Zip Code
77555

  Publications

Yoon, Jung-Hoon; Roy Choudhury, Jayati; Park, Jeseong et al. (2014) A role for DNA polymerase ? in promoting replication through oxidative DNA lesion, thymine glycol, in human cells. J Biol Chem 289:13177-85
Yoon, Jung-Hoon; Acharya, Narottam; Park, Jeseong et al. (2014) Identification of two functional PCNA-binding domains in human DNA polymerase ?. Genes Cells 19:594-601
Yoon, Jung-Hoon; Prakash, Satya; Prakash, Louise (2012) Genetic control of translesion synthesis on leading and lagging DNA strands in plasmids derived from Epstein-Barr virus in human cells. MBio 3:e00271-12
Yoon, Jung-Hoon; Prakash, Satya; Prakash, Louise (2012) Requirement of Rad18 protein for replication through DNA lesions in mouse and human cells. Proc Natl Acad Sci U S A 109:7799-804
Ummat, Ajay; Rechkoblit, Olga; Jain, Rinku et al. (2012) Structural basis for cisplatin DNA damage tolerance by human polymerase ýý during cancer chemotherapy. Nat Struct Mol Biol 19:628-32
Yoon, Jung-Hoon; Bhatia, Gita; Prakash, Satya et al. (2010) Error-free replicative bypass of thymine glycol by the combined action of DNA polymerases kappa and zeta in human cells. Proc Natl Acad Sci U S A 107:14116-21
Yoon, Jung-Hoon; Prakash, Louise; Prakash, Satya (2010) Error-free replicative bypass of (6-4) photoproducts by DNA polymerase zeta in mouse and human cells. Genes Dev 24:123-8
Acharya, Narottam; Yoon, Jung-Hoon; Hurwitz, Jerard et al. (2010) DNA polymerase eta lacking the ubiquitin-binding domain promotes replicative lesion bypass in humans cells. Proc Natl Acad Sci U S A 107:10401-5
Yoon, Jung-Hoon; Prakash, Louise; Prakash, Satya (2009) Highly error-free role of DNA polymerase eta in the replicative bypass of UV-induced pyrimidine dimers in mouse and human cells. Proc Natl Acad Sci U S A 106:18219-24
Acharya, Narottam; Yoon, Jung-Hoon; Gali, Himabindu et al. (2008) Roles of PCNA-binding and ubiquitin-binding domains in human DNA polymerase eta in translesion DNA synthesis. Proc Natl Acad Sci U S A 105:17724-9

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