The general goal of the proposed research is to describe the molecular architecture of the eukaryotic DNA replication fork, its function during normal replication, and its response to stress or DNA damage, using S. cerevisiae as a model system. Recent studies have delineated the activities of DNA polymerase d and DNA polymerase e at the standard replication fork, and the role of DNA polymerase d in lagging strand DNA replication. Translesion synthesis (TLS) in response to DNA damage allows the cell to overcome damage that forms a terminal block for the regular replication machinery;TLS is associated with an increased frequency of mutations, hence mutagenesis. A ubiquitinated form of the replication clamp PCNA initiates its essential roles in TLS through recruitment of the Rev1 protein, a factor that organizes the TLS machinery. The DNA damage checkpoint is a signal transduction pathway that temporarily holds the cell cycle in response to DNA damage. The checkpoint clamp 9-1-1, when brought together onto DNA with the sensor kinase Mec1 (human ATR), activates Mec1 to phosphorylate downstream targets including Rad53 (human Chk1/2). Preliminary studies indicate that the replication initiation protein kinase Cdc7-Dbf4 uniquely phosphorylates critical factors in the mutagenesis and checkpoint pathways. These data suggest that the three pathways are more interconnected than they at first appear to be. The proposal will test specific hypotheses central to genome replication and maintenance.
In aim 1, the mechanistic and structural nature of strand displacement synthesis by DNA polymerase d, and its regulation by the flap endonuclease FEN1 will be investigated.
In aim 2, the efficiency and fidelity of TLS by DNA polymerase ? and associated factors, and the role of the Cdc7-Dbf4 protein kinase in TLS and mutagenesis will be studied.
In aim 3, the role of the checkpoint clamp 9-1-1 in checkpoint function and in mutagenesis will be addressed by biochemical studies and mutational analysis. The regulation of the 9-1-1 pathway by Cdc7-Dbf4 phosphorylation will also be studied. Proper replication of cellular DNA, and responses to stress and DNA damage is of the highest importance in maintaining the integrity of our genetic information. Patients with known defects in these pathways are at a highly increased risk for developing cancer. These pathways are conserved from human to yeast. The proposed studies will be carried out in yeast because this model organism is more approachable to genetic and biochemical analysis.

Public Health Relevance

Proper replication of cellular DNA is of the highest importance in maintaining the integrity of our genetic information. In this application, we are proposing to study the process of DNA replication, and the responses of the cell to DNA damage that can lead to the generation of mutations and to cell death. Patients with known defects in these pathways are at a highly increased risk for developing cancer. These pathways are conserved from human to yeast, and we are proposing to study these pathways in yeast, because this model organism is more approachable to genetic and biochemical analysis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM032431-28
Application #
8323564
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Reddy, Michael K
Project Start
1984-04-01
Project End
2014-08-31
Budget Start
2012-09-01
Budget End
2014-08-31
Support Year
28
Fiscal Year
2012
Total Cost
$527,286
Indirect Cost
$180,387
Name
Washington University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
068552207
City
Saint Louis
State
MO
Country
United States
Zip Code
63130
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