Mechanisms of Palindrome-Mediated Chromosome Fragility

Georgia Tech Research Corporation, Atlanta GA

Investigators

Abstract

Chromosomal rearrangements play a major role in the evolution of eukaryotic genomes. The presence of repetitive sequences that can adopt non-canonical DNA structures is one of the factors which can predispose chromosomal regions to instability. Palindromic sequences (inverted repeats with or without a unique sequence between them) that can adopt hairpin or cruciform structures are frequently found in regions that are prone to gross chromosomal rearrangements (GCRs) in somatic and germ cells in different organisms. Direct physical evidence was obtained that double-strand breaks (DSBs) occur at the location of long inverted repeats, a triggering event for the genomic instability. However, the mechanisms by which palindromic sequences lead to chromosomal fragility are largely unknown. The overall goal of this research is to elucidate how palindromic sequences induce DSB and GCRs in the yeast Saccharomyces cerevisiae. With previous NSF support, the investigator demonstrated that the cruciform structure adopted by Alu inverted repeats is a target for a nuclease attack, leading to the formation of hairpin-capped breaks that lead to translocations, deletions and gene amplifications. Mutations in several genes that affect palindrome-mediated fragility were identified in preliminary studies. The following specific objectives will make possible in-depth analyses of the key players in cruciform resolution. Objective #1: To determine the role of DNA replication and the post-replicative repair pathway in fragility. Objective #2: To assess the role of Rvb2 and Rvb1 helicases in cruciform metabolism. Objective #3: To use genome-wide screening to identify mutants that are defective in, and prone to, DSB formation at the location of quasi-palindromes. Sensitive biological assays for monitoring DSB formation as well as direct physical analyses of structural intermediates will be used to achieve these objectives. The information resulting from this research will yield important insights into the causes of genome instability at fragile sites in eukaryotic chromosomes. In addition, this work will shed light on the structural organization and evolution of genomes that contain repetitive DNA. This research relies heavily on the active participation of undergraduate and graduate students. In addition, some of these studies will overlap with laboratory activities in an Honors Genetics course taught by the PI at the Georgia Institute of Technology. As in prior NSF-funded studies, the laboratory will continue to recruit female researchers, minority students, and high school teachers. This project will involve collaboration with other laboratories at Georgia Tech, Duke, and Columbia. The results of this work will be presented at various national and international scientific meetings.