The long term objectives are to delineate the action mechanisms of human DNA polymerase i (Poll) on undamaged and damaged DNAs.
The specific aims of this project are to examine the role of Hoogsteen base pairing in the replication of undamaged and damaged DNAs by Poll, and the proposed studies will use a combined biochemical and structural approach, hi Aim 1, biochemical studies will be done with base analogs of adenine and guanine to analyze the contributions of different hydrogen bond acceptors/donors in template purines to their Hoogsteen pairing with the incoming pyrimidine nucleotide (nt).
Aim 2 will test the hypothesis that Hoogsteen base pairing enables Poll to incorporate nts opposite DNA lesions that severely impinge upon the minor groove of the template base or which impair its Watson-Crick base pairing ability. The proficiency of Poll for incorporating nts opposite such lesions and to extend there from will be determined by steady-state kinetic methods.
Aim 3 will determine the structural bases of Hoogsteen base pairing in Poll's active site by analyzing the effects that mutational changes in the residues of the fingers and palm domains, that are seen to interact with the templating nt or with the incoming nt in Poll ternary structure, have on the efficiency and fidelity of nt incorporation, hi Aim 4, the X-ray structures of ternary complexes of Poll with undamaged templates G, T, and C, in the presence of different incoming nts will be determined, and in Aim 5, the ternary structures of Poll with DNA containing lesions at the templating site that severely impinge upon the minor groove, or which affect Watson-Crick base pairing, and others, in the presence of different incoming nts will be determined. 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, the inactivation of Polr in humans causes the cancer prone syndrome, the variant form of xeroderma pigmentosum.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA115856-04
Application #
7449723
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Okano, Paul
Project Start
2005-09-01
Project End
2010-06-30
Budget Start
2008-07-01
Budget End
2009-06-30
Support Year
4
Fiscal Year
2008
Total Cost
$329,839
Indirect Cost
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
Nair, Deepak T; Johnson, Robert E; Prakash, Louise et al. (2009) DNA synthesis across an abasic lesion by human DNA polymerase iota. Structure 17:530-7
Jain, Rinku; Nair, Deepak T; Johnson, Robert E et al. (2009) Replication across template T/U by human DNA polymerase-iota. Structure 17:974-80
Nair, Deepak T; Johnson, Robert E; Prakash, Louise et al. (2008) Protein-template-directed synthesis across an acrolein-derived DNA adduct by yeast Rev1 DNA polymerase. Structure 16:239-45
Howell, Craig A; Prakash, Satya; Washington, M Todd (2007) Pre-steady-state kinetic studies of protein-template-directed nucleotide incorporation by the yeast Rev1 protein. Biochemistry 46:13451-9
Nair, Deepak T; Johnson, Robert E; Prakash, Louise et al. (2006) An incoming nucleotide imposes an anti to syn conformational change on the templating purine in the human DNA polymerase-iota active site. Structure 14:749-55
Nair, Deepak T; Johnson, Robert E; Prakash, Louise et al. (2006) Hoogsteen base pair formation promotes synthesis opposite the 1,N6-ethenodeoxyadenosine lesion by human DNA polymerase iota. Nat Struct Mol Biol 13:619-25
Johnson, Robert E; Haracska, Lajos; Prakash, Louise et al. (2006) Role of hoogsteen edge hydrogen bonding at template purines in nucleotide incorporation by human DNA polymerase iota. Mol Cell Biol 26:6435-41
Nair, Deepak T; Johnson, Robert E; Prakash, Louise et al. (2005) Human DNA polymerase iota incorporates dCTP opposite template G via a G.C + Hoogsteen base pair. Structure 13:1569-77