DNA damage or errors during DNA replication may trigger large scale changes to chromosome structure;viewed as deletions, additions, and duplications of genomic DNA, called gross chromosomal rearrangements (GCRs). Our lab has identified a fragile chromosomal region in Saccharomyces cerevisiae, which initiates GCRs. Both yeast fragile sites as well as fragile sites in mammals exhibit GCRs following the disruption of DNA replication and these events are associated with genome instability in cancer cells. Investigation of these highly conserved mechanisms and phenomena in yeast may yield important insights into human diseases, including cancer. The goal of the proposed research is to develop a better understanding of the specific cis elements which perturb replication at specific fragile loci and the trans cellular machinery that maintains genome integrity or that intervenes once problems arise. The overarching goal of this research is to study replication fork biology at a natural fragile site to better understand why replication forks stall within these regions at high frequency, and determine what proteins promote or prevent stalled forks from collapsing.
Specific Aim 1 : DNA determinants of chromosome fragility - We hypothesize that unstable replication intermediates at the fragile site induce genome instability. Using genetic and molecular approaches I will determine the role of transcription, replication and termination. I will characterize abnormal replication fork intermediates through the fragile site by genomic 2D agarose electrophoresis gels.
Specific Aim 2 : Proteins required for replication fork progression and stability through a yeast fragile site - Our initial genetic analysis has defined roles for DNA replication, repair and checkpoint proteins in fragile site stability. Using 2D gels in combination with Chromatin Immunoprecipitation (ChIP) I will isolate particular replication fork states, map and identify protein distributions and functions at the fragile site. PUBLIC HEALTH RELEVENCE: Of this research to public health - Little is known about the identity and function of DNA repair, checkpoint and replication proteins that act at natural pause sites;including fragile sites. Identification of frequent fragile site instability in tumor cells and their link to important cell cycle checkpoint pathways strongly supports the need for greater understanding of fragile site biology. The fragile site in S. cerevisiae can provide a novel molecular model to help further delineate the genomic structure and mechanisms of instability, including the dynamics between replication checkpoint and DNA repair pathways.
| Paek, Andrew L; Jones, Hope; Kaochar, Salma et al. (2010) The role of replication bypass pathways in dicentric chromosome formation in budding yeast. Genetics 186:1161-73 |
| Paek, Andrew L; Kaochar, Salma; Jones, Hope et al. (2009) Fusion of nearby inverted repeats by a replication-based mechanism leads to formation of dicentric and acentric chromosomes that cause genome instability in budding yeast. Genes Dev 23:2861-75 |
