Abstract

This proposal has as its major goal the elucidation of the mechanisms by which the fidelity of DNA synthesis is maintained in mammalian cells. We will approach this problem by: 1) purifying to homogeneity the high molecular weight DNA polymerases of calf thymus: DNA polymerases Delta1 and Delta2, which contain a proofreading 3' to 5' exonuclease activity, and DNA polymerase Alpha, which is devoid of an exonuclease activity; 2) characterizing the physical and enzymatic properties of these polymerases, and investigating their possible interrelationships; 3) investigating the physiological roles of the polymerases by determining the relative levels of these enzymes in resting and dividing cells, and by determining the relative sensitivities of the polymerases to inhibitors of DNA replication; 4) determining the error frequency during DNA synthesis with these DNA polymerases using both synthetic polynucleotides and primed PhiX174 DNA as template/primers; 5) determining the effects of selective inhibition of the 3' to 5' exonuclease activity of DNA polymerases Delta1 and Delta2 on the fidelity of DNA synthesis; 6) investigating the interaction of the DNA polymerases with other replication proteins, and the effects of such interaction on the fidelity of DNA synthesis.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK026206-13
Application #
3227775
Study Section
Biochemistry Study Section (BIO)
Project Start
1979-04-01
Project End
1993-03-31
Budget Start
1992-04-01
Budget End
1993-03-31
Support Year
13
Fiscal Year
1992
Total Cost
Indirect Cost

  Institution

Name
University of Miami School of Medicine
Department
Type
Schools of Medicine
DUNS #
City
Miami
State
FL
Country
United States
Zip Code
33146

  Publications

Lu, Xiaoqing; Tan, Cheng-Keat; Zhou, Jin-Qiu et al. (2002) Direct interaction of proliferating cell nuclear antigen with the small subunit of DNA polymerase delta. J Biol Chem 277:24340-5
Meyer, Peter R; Matsuura, Suzanne E; Tolun, Adviye A et al. (2002) Effects of specific zidovudine resistance mutations and substrate structure on nucleotide-dependent primer unblocking by human immunodeficiency virus type 1 reverse transcriptase. Antimicrob Agents Chemother 46:1540-5
Carastro, L Michael; Tan, Cheng-Keat; Selg, Manuel et al. (2002) Identification of delta helicase as the bovine homolog of HUPF1: demonstration of an interaction with the third subunit of DNA polymerase delta. Nucleic Acids Res 30:2232-43
He, H; Tan, C K; Downey, K M et al. (2001) A tumor necrosis factor alpha- and interleukin 6-inducible protein that interacts with the small subunit of DNA polymerase delta and proliferating cell nuclear antigen. Proc Natl Acad Sci U S A 98:11979-84
Meyer, P R; Matsuura, S E; Schinazi, R F et al. (2000) Differential removal of thymidine nucleotide analogues from blocked DNA chains by human immunodeficiency virus reverse transcriptase in the presence of physiological concentrations of 2'-deoxynucleoside triphosphates. Antimicrob Agents Chemother 44:3465-72
Meyer, P R; Matsuura, S E; Mian, A M et al. (1999) A mechanism of AZT resistance: an increase in nucleotide-dependent primer unblocking by mutant HIV-1 reverse transcriptase. Mol Cell 4:35-43
Mozzherin, D J; Tan, C K; Downey, K M et al. (1999) Architecture of the active DNA polymerase delta.proliferating cell nuclear antigen.template-primer complex. J Biol Chem 274:19862-7
Zaika, A; Mozzherin, D J; Tan, C K et al. (1999) A two-dimensional support for selective binding of polyhistidine-tagged proteins: identification of a proliferating cell nuclear antigen point mutant with altered function in vitro. Anal Biochem 268:193-200
Kramata, P; Downey, K M (1999) 9-(2-phosphonylmethoxyethyl) derivatives of purine nucleotide analogs: A comparison of their metabolism and interaction with cellular DNA synthesis. Mol Pharmacol 56:1262-70
Terashima, I; Suzuki, N; Dasaradhi, L et al. (1998) Translesional synthesis on DNA templates containing an estrogen quinone-derived adduct: N2-(2-hydroxyestron-6-yl)-2'-deoxyguanosine and N6-(2-hydroxyestron-6-yl)-2'-deoxyadenosine. Biochemistry 37:13807-15

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