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.
Intellectual Merit Eukaryotic chromosomes include regions that are susceptible for breakage and rearrangements. Repeats that can adopt non-B form DNA secondary structure are often found to be responsible for the induction of instability that underlies evolutionary changes, polymorphisms and diseases. We have studied the mechanisms of chromosomal breakage and the resulting instabilities caused by palindromic sequences that can adopt hairpin and cruciform structures using yeast as a model organism. We have identified 28 mutants that predispose the genome to breakage at these repeats and at GAA/TTC repeats that can adopt triplex structure. Deficiencies in the DNA replication machinery were found to destabilize both repeats. We also found mutations in the structure specific helicases, SGS1 and PIF1 and the proteins involved in Fe-S cluster biogenesis specifically amplified the palindrome-mediated fragility. The fragility associated with DNA replication defects relies on the activity of homologous-recombination. In addition to breakage, these repeats are also a source of mutagenesis that can spread to the genes located at long distances from the repeats. Remarkably, repair of the break involving error-prone synthesis restores inverted repeats making them a long-term resource of mutations. These results demonstrate that chromosomal regions with breakage motifs have a high potential for structural rearrangements as well as a tendency to accumulate nucleotide polymorphisms. Accelerated rate of the genetic changes in such regions predisposes them for evolution and can contribute to the development of diseases. Broader Impacts These studies involved the participation of six undergraduate students, among them two female students and one minority female. We also trained a female high-school student during the course of these studies. The experiments pertaining to this research were incorporated into the Independent Research Project Lab taught by the PI. This project is part of the dissertation for three female graduate students and one minority MS student. We disseminated the results of this work at multiple international and domestic meetings. Additional Information The results of these studies were published in Molecular Cell, Biochimie and in Environmental and Molecular Mutagenesis. In addition, we anticipate 3 new publications resulting from this work in the near future.
