Genomic approaches and the discovery that E. coli UmuC and DinB, yeast RAD30 and Xeroderma pigmentosum variant (XPV) encode DNA polymerases have led to an explosion in the number of known DNA polymerases. We have discovered, originally via a two hybrid screen, that the essential but non-catalytic C terminal domain of yeast DNA polymerase (pol) epsilon, a polymerase required for DNA replication, repair, and the DNA replication checkpoint, interacts both physically and functionally with one of the most novel of the new polymerases, pol sigma. Yeast pol sigma mutants are defective in completion of S phase, in DNA repair, in chromosome condensation, and in sister chromatid cohesion. The discovery of this interaction lends a new dimension to our continuing search for the precise role of pot epsilon in chromosome dynamics during S phase. We find that pol epsilon mutants are defective in cohesion. The overall goat of our studies is to determine how pol epsilon and pol sigma interact in these various cellular processes. First, to define the in vivo role of the interaction, structure/function analysis will be carried out by using site directed mutations and correlating effects on interaction between the two proteins with phenotypes of resulting mutants, especially with respect to sister chromatid cohesion. Second, we will characterize pol sigma biochemically- definition of substrates, fidelity, processivity, and stimulation by PCNA. A defining aspect of our studies is focus on the interaction between pol sigma and pot epsilon. We will also address pol epsilon's interaction with another cohesion protein,Ctf18, a component of an alternative polymerase clamp loader. The key questions in the nascent field of sister chromatid cohesion are what is the nature of the links between the chromosomes and what is the mechanism by which DNA replication contributes to forming these links. With this in mind, we will determine if pol epsilon is required for establishing cohesion and/or maintenance. We will study the fate of replication forks at sites of cohesion in vivo and the association of polymerases with these sites. We will use chromatin immunoprecipitation, 2D gel electrophoresis of DNA replication intermediates, and microscopic analysis of chromosome dynamics using fluorescence microscopy and the GFP-repressor/operator tagging strategy, techniques set up by us during the previous granting period. Several collaborations are in place to insure progress.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM025508-26
Application #
6734658
Study Section
Biochemistry Study Section (BIO)
Program Officer
Wolfe, Paul B
Project Start
1986-09-01
Project End
2007-03-31
Budget Start
2004-04-01
Budget End
2005-03-31
Support Year
26
Fiscal Year
2004
Total Cost
$397,923
Indirect Cost
Name
California Institute of Technology
Department
Type
Schools of Arts and Sciences
DUNS #
009584210
City
Pasadena
State
CA
Country
United States
Zip Code
91125
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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

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