Abstract

I propose here setting up an in vitro system for the study of plasmid RSF1030 replication. I plan to rapidly establish the correspondence of in vivo and in vitro events and to go on to purify and establish the role of new proteins in Escherichia coli and plasmid replication. It is the identification and characterization of host and plasmid proteins involved in replication that is the main thrust of the proposed studies. RSF1030 is a small (5.6 x 10 to the 6th power MW) (drug resistance) plasmid that replicates under relaxed control and requires DNA polymerase I for replication. After developing the in vitro system and characterizing the DNA products synthesized, I plan to work out protocols for dissecting the replication reaction into initiation, elongation, and termination reactions. This will aid in the study of the involvement of the enzymes, termination reactions. This will aid in the study of the involvement of the enzymes, DNA polymerase I, DNA polymerase III, RNA polymerases, ligases, DNA gyrases and other enzymes involved. In addition plasmid replication proteins will be identified and studied. A second aspect of the work will be an investigation of the relationship of RNA synthesis to DNA replication. Ribonucleotides in RSF1030 will be mapped; the mechanism of removal of ribonucleotides will be investigated; enzymes involved will be elucidated.

  Funding Agency

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM025508-08
Application #
3273089
Study Section
(MG)
Project Start
1978-07-01
Project End
1986-08-31
Budget Start
1985-09-01
Budget End
1986-08-31
Support Year
8
Fiscal Year
1985
Total Cost
Indirect Cost

  Institution

Name
California Institute of Technology
Department
Type
DUNS #
078731668
City
Pasadena
State
CA
Country
United States
Zip Code
91125

  Publications

Jaszczur, Malgorzata; Rudzka, Justyna; Kraszewska, Joanna et al. (2009) Defective interaction between Pol2p and Dpb2p, subunits of DNA polymerase epsilon, contributes to a mutator phenotype in Saccharomyces cerevisiae. Mutat Res 669:27-35
Lou, Huiqiang; Komata, Makiko; Katou, Yuki et al. (2008) Mrc1 and DNA polymerase epsilon function together in linking DNA replication and the S phase checkpoint. Mol Cell 32:106-17
Jaszczur, Malgorzata; Flis, Krzysztof; Rudzka, Justyna et al. (2008) Dpb2p, a noncatalytic subunit of DNA polymerase epsilon, contributes to the fidelity of DNA replication in Saccharomyces cerevisiae. Genetics 178:633-47
Boronat, Susanna; Campbell, Judith L (2007) Mitotic Cdc6 stabilizes anaphase-promoting complex substrates by a partially Cdc28-independent mechanism, and this stabilization is suppressed by deletion of Cdc55. Mol Cell Biol 27:1158-71
Reis, Clara C; Campbell, Judith L (2007) Contribution of Trf4/5 and the nuclear exosome to genome stability through regulation of histone mRNA levels in Saccharomyces cerevisiae. Genetics 175:993-1010
Masuda-Sasa, Taro; Polaczek, Piotr; Campbell, Judith L (2006) Single strand annealing and ATP-independent strand exchange activities of yeast and human DNA2: possible role in Okazaki fragment maturation. J Biol Chem 281:38555-64
Masuda-Sasa, Taro; Imamura, Osamu; Campbell, Judith L (2006) Biochemical analysis of human Dna2. Nucleic Acids Res 34:1865-75
Stewart, Jason A; Campbell, Judith L; Bambara, Robert A (2006) Flap endonuclease disengages Dna2 helicase/nuclease from Okazaki fragment flaps. J Biol Chem 281:38565-72
Budd, Martin E; Reis, Clara C; Smith, Stephanie et al. (2006) Evidence suggesting that Pif1 helicase functions in DNA replication with the Dna2 helicase/nuclease and DNA polymerase delta. Mol Cell Biol 26:2490-500
Budd, Martin E; Tong, Amy Hin Yan; Polaczek, Piotr et al. (2005) A network of multi-tasking proteins at the DNA replication fork preserves genome stability. PLoS Genet 1:e61

Showing the most recent 10 out of 51 publications