The ultimate goal of this project is to understand the mechanism of eukaryotic DNA replication, in particular, lagging strand replication, using the yeast, Saccharomyces cerevisiae as a model system. Although there are several DNA polymerases in eukaryotic cells whose specific roles are not clearly defined, it is clear that DNA polymerase alpha is an important replicating polymerase that is involved in priming and Okazaki fragment synthesis. A high molecular weight multiprotein form of pol alpha has been purified from yeast, and its associated polymerase, primase, 5' -> 3' exonuclease, ssDNA dependent ATPase, and DNA helicase activities analyzed in this laboratory. Peptide sequencing of the purified 5' -> 3' exonuclease has identified it to be the RTH1 nuclease. A new DNA helicase which is uniquely homologous to DnaB helicase and SV40 T antigen helicase has been identified.
The specific aims of this project are as follows: (1) DNA helicase A activity co-purifies with three proteins of 90, 60, and 50 kDa, and the helicase activity is in the 90 kDa subunit. To understand the roles of the other two subunits in helicase function and DNA replication, the genes for these subunits will be cloned using the sequence information from the purified proteins, the helicase activity will be reconstituted, and the roles of these subunits in helicase function studied. The four subunits of DNA polymerase alpha will be purified and used for reconstituting the DNA polymerase activity and for analyzing their roles in DNA replication. (2) Two hybrid techniques will be employed to identify yeast genes whose encoded proteins interact with subunits of DNA polymerase alpha , with yeast DNA helicase A, and with RTH1 nuclease. For new genes, genetic studies will explore their function in DNA synthesis and in other related processes. (3) Genetic studies will be done to determine the role of yeast DNA helicase A. Since helicase A is essential for viability, temperature sensitive conditional lethal mutations will be generated, and their effects on DNA replication assessed by flow cytometry and other techniques. Biochemical studies of purified helicase A will characterize the nucleotidase and helicase activities of this protein.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM036002-12
Application #
6179688
Study Section
Physiological Chemistry Study Section (PC)
Program Officer
Rhoades, Marcus M
Project Start
1987-08-01
Project End
2003-03-31
Budget Start
2000-04-01
Budget End
2001-03-31
Support Year
12
Fiscal Year
2000
Total Cost
$274,091
Indirect Cost
Name
University of Medicine & Dentistry of NJ
Department
Biochemistry
Type
Schools of Osteopathy
DUNS #
City
Stratford
State
NJ
Country
United States
Zip Code
08084
Biswas, Subhasis B; Biswas-Fiss, Esther E (2006) Quantitative analysis of binding of single-stranded DNA by Escherichia coli DnaB helicase and the DnaB x DnaC complex. Biochemistry 45:11505-13
Biswas, Subhasis B; Khopde, Sujata M; Biswas-Fiss, Esther E (2005) Control of ATP-dependent binding of Saccharomyces cerevisiae origin recognition complex to autonomously replicating DNA sequences. Cell Cycle 4:494-500
Mitkova, Atanaska V; Biswas-Fiss, Esther E; Biswas, Subhasis B (2005) Modulation of DNA synthesis in Saccharomyces cerevisiae nuclear extract by DNA polymerases and the origin recognition complex. J Biol Chem 280:6285-92
Flowers, Stephen; Biswas, Esther E; Biswas, Subhasis B (2003) Conformational dynamics of DnaB helicase upon DNA and nucleotide binding: analysis by intrinsic tryptophan fluorescence quenching. Biochemistry 42:1910-21
Biswas, Subhasis B; Khopde, Sujata M; Zhu Fx, Fan xiu et al. (2003) Subunit interactions in the assembly of Saccharomyces cerevisiae DNA polymerase alpha. Nucleic Acids Res 31:2056-65
Biswas, Esther E; Chen, Pei-Hua; Biswas, Subhasis B (2002) Modulation of enzymatic activities of Escherichia coli DnaB helicase by single-stranded DNA-binding proteins. Nucleic Acids Res 30:2809-16
Suarez, Tatiana; Biswas, Subhasis B; Biswas, Esther E (2002) Biochemical defects in retina-specific human ATP binding cassette transporter nucleotide binding domain 1 mutants associated with macular degeneration. J Biol Chem 277:21759-67
Khopde, Sujata; Biswas, Esther E; Biswas, Subhasis B (2002) Affinity and sequence specificity of DNA binding and site selection for primer synthesis by Escherichia coli primase. Biochemistry 41:14820-30
Mitkova, Atanaska V; Biswas, Esther E; Biswas, Subhasis B (2002) Cell cycle specific plasmid DNA replication in the nuclear extract of Saccharomyces cerevisiae: modulation by replication protein A and proliferating cell nuclear antigen. Biochemistry 41:5255-65
Biswas, E E; Nagele, R G; Biswas, S (2001) A novel human hexameric DNA helicase: expression, purification and characterization. Nucleic Acids Res 29:1733-40

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