Molecular analysis of genetic recombination and DNA break repair

项目总结/文摘

Project Summary/Abstract The long-term goal of the research proposed here is to determine the molecular mechanism of homologous genetic recombination and DNA break repair. This objective is approached by a combination of genetic analysis of mutants and biochemical analysis of proteins and DNA from cells. This research uses the fission yeast Schizosaccharomyces pombe as well as the bacterium Escherichia coli and its phage lambda. All are widely studied, highly tractable model organisms with features common to all organisms, including humans. The studies are focused on meiotic recombination in S. pombe, whose high rates of recombination facilitate both genetic and biochemical analyses, and on the major pathway of recombination and DNA break repair in bacteria, promoted by RecBCD enzyme, a complex DNA repair machine, whose 3D structure allows us to determine at atomic level how recombination initiation is regulated. Building on past achievements, the research is currently focused on the following areas. 1) Studying how meiotic DNA double-strand break (DSB) hotspots form clusters, and how these clusters impart DSB interference and, consequently, crossover interference important for proper chromosome segregation. This research promises to solve the 100-year-old problem of crossover interference, a major genetic puzzle for which we have proposed a molecular mechanism and supported with many data. 2) Studying how RecBCD enzyme controls its potentially rampant nuclease activity and appropriately activates it by interaction with Chi hotspots of recombination (5’ GCTGGTGG 3’). This research promises to solve at near-atomic level the molecular mechanism of RecBCD enzyme, the principal controller of the major pathway of E. coli recombination, first observed 75 years ago, and a paradigm for chromosomal site control of other complex DNA enzymes. 3) Seeking more potent RecBCD inhibitors, which are promising antibiotics against a novel (unused) target. New antibiotics are needed to counter ever-more-frequent drug-resistant bacteria. These goals will be attacked by a combination of genetic analysis of mutants, fluorescence microscopy of intracellular proteins and chromosomal sites, physical analysis of DNA intermediates from meiotic cells, and enzymatic and biophysical analyses of isolated proteins. The results of these studies will elucidate the molecular mechanism of recombination and DNA break repair as well as the controls on recombination that ensure that it occurs at the proper time and place along chromosomes. Recombination is important for faithful meiotic chromosome segregation, error-free repair of frequently arising DNA double-strand breaks, and generation of cellular and organismal diversity. Aberrancies of recombination can generate chromosomal rearrangements, such as translocations, duplications, and deletions, which are often associated with or the cause of infertility, birth defects, and cancers.