The goals of this proposal are to determine how a histone deposition protein promotes DNA replication fork stability, and how the DNA replication machinery communicates the status of the replication fork to the replication checkpoint surveillance system, using yeast as a model system.
In Aim 1, I will investigate how specific mutations in the DNA polymerase processivity clamp, PCNA, impair activation of the replication checkpoint in response to replication stress. To do this, I will determine which steps in the replication checkpoint signaling pathway are defective in these PCNA mutants. Additionally, I will perform protein and chromatin immunoprecipitation experiments to test the hypothesis that interactions between checkpoint proteins, fork-associated factors and the DNA replication fork will be perturbed in the PCNA mutants.
In Aims 2 and 3, the mechanism behind replication fork collapse in cells lacking histone deposition protein Asf1 will be examined. Asfl mutant alleles specifically defective for fork stability, and a regulated Asf1 N-degron will be developed to address this question. Using these tools, fate of DNA replication forks and DNA replication proteins at stalled and moving replication forks will be examined by ChIP and 2D gel electrophoresis.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Postdoctoral Individual National Research Service Award (F32)
Project #
1F32GM075601-01
Application #
6999489
Study Section
Special Emphasis Panel (ZRG1-F08 (20))
Program Officer
Portnoy, Matthew
Project Start
2005-09-01
Project End
2008-08-31
Budget Start
2005-09-01
Budget End
2006-08-31
Support Year
1
Fiscal Year
2005
Total Cost
$48,296
Indirect Cost
Name
University of Massachusetts Medical School Worcester
Department
Genetics
Type
Schools of Medicine
DUNS #
603847393
City
Worcester
State
MA
Country
United States
Zip Code
01655
Erkmann, Judith A; Kaufman, Paul D (2009) A negatively charged residue in place of histone H3K56 supports chromatin assembly factor association but not genotoxic stress resistance. DNA Repair (Amst) 8:1371-9
Kaplan, Tommy; Liu, Chih Long; Erkmann, Judith A et al. (2008) Cell cycle- and chaperone-mediated regulation of H3K56ac incorporation in yeast. PLoS Genet 4:e1000270