Abstract

The goal of the proposed research is to identify and characterize the proteins involved in DNA replication in mammalian cells. We are approaching this problem by analyzing the cellular protein requirements for replication of the SV40 genome in vitro. During the past grant period we developed methods for the fractionation of the crude SV40 DNA replication system and demonstrated that multiple cellular factors are required to reconstitute efficient DNA synthesis in vitro. A number of the required proteins were purified to near homogeneity and partially characterized. During the next grant period we plan to complete the identification of the components of the mammalian replication machinery and to reconstitute the complete replication reaction with purified proteins. We also plan to analyze the functional roles of the individual cellular replication proteins with particular emphasis on the proteins involved in the initiation reaction.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA040414-09
Application #
3180308
Study Section
Experimental Virology Study Section (EVR)
Project Start
1985-07-01
Project End
1995-04-30
Budget Start
1993-05-01
Budget End
1994-04-30
Support Year
9
Fiscal Year
1993
Total Cost
Indirect Cost

  Institution

Name
Johns Hopkins University
Department
Type
Schools of Medicine
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218

  Publications

Wu, Min; Lu, Wenyan; Santos, Ruth E et al. (2014) Geminin inhibits a late step in the formation of human pre-replicative complexes. J Biol Chem 289:30810-21
Dai, Jianli; Chuang, Ray-Yuan; Kelly, Thomas J (2005) DNA replication origins in the Schizosaccharomyces pombe genome. Proc Natl Acad Sci U S A 102:337-42
Vashee, S; Simancek, P; Challberg, M D et al. (2001) Assembly of the human origin recognition complex. J Biol Chem 276:26666-73
Collins, K L; Russo, A A; Tseng, B Y et al. (1993) The role of the 70 kDa subunit of human DNA polymerase alpha in DNA replication. EMBO J 12:4555-66
Moarefi, I F; Small, D; Gilbert, I et al. (1993) Mutation of the cyclin-dependent kinase phosphorylation site in simian virus 40 (SV40) large T antigen specifically blocks SV40 origin DNA unwinding. J Virol 67:4992-5002
Virshup, D M; Russo, A A; Kelly, T J (1992) Mechanism of activation of simian virus 40 DNA replication by protein phosphatase 2A. Mol Cell Biol 12:4883-95
Randall, S K; Kelly, T J (1992) The fate of parental nucleosomes during SV40 DNA replication. J Biol Chem 267:14259-65
Erdile, L F; Collins, K L; Russo, A et al. (1991) Initiation of SV40 DNA replication: mechanism and control. Cold Spring Harb Symp Quant Biol 56:303-13
Scheidtmann, K H; Virshup, D M; Kelly, T J (1991) Protein phosphatase 2A dephosphorylates simian virus 40 large T antigen specifically at residues involved in regulation of DNA-binding activity. J Virol 65:2098-101
Weinberg, D H; Collins, K L; Simancek, P et al. (1990) Reconstitution of simian virus 40 DNA replication with purified proteins. Proc Natl Acad Sci U S A 87:8692-6

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