of Work: Repair of double-strand breaks (DSBs) in the chromosomal DNA of humans and other eukaryotic organisms involves a complex interplay between proteins associated with repair of broken DNA ends, maintenance of chromatin structure and DNA damage-sensitive checkpoints. Mutations within genes involved in the two major pathways of DSB repair, referred to as homologous recombination and nonhomologous end-joining (NHEJ), and in cell cycling responses to DNA strand breaks have been implicated in the etiology of several human cancers and in processes leading to cell senescence. Our research efforts have focused on DSB repair in the genetically tractable yeast Saccharomyces cerevisiae. Most genes comprising the two major pathways of DSB repair are structurally and functionally conserved in yeast and human cells. We have developed an in vivo system for enyzmatically generating defined DSBs and conducted a systematic analysis of genes involved in repair using multiple assay systems. We have a) provided the first demonstration that yeast genes involved in recombination-independent NHEJ play an essential role in the repair of EcoRI endonuclease-induced DSBs in vivo; b) shown that the functions of two repair complexes (involving Ku70:Ku80 and Rad50:Mre11:Xrs2) in NHEJ are genetically separable; c) developed the first functional assay for yeast DNA Ligase IV based on quantitation of endonuclease-induced checkpoint activation in dnl4 strains; d) confirmed that the mismatch repair exonuclease encoded by EXO1 and the excision repair endonuclease Rad1/Rad10 are involved in the DSB recombinational repair pathway; e) revealed that low-level expression of PvuII endonuclease (generating DSBs with blunt termini) is lethal in haploid cells, but not in diploid cells. This latter result establishes that the structures at the ends of DSBs are critical for determining their mechanism(s) of repair.

Agency
National Institute of Health (NIH)
Institute
National Institute of Environmental Health Sciences (NIEHS)
Type
Intramural Research (Z01)
Project #
1Z01ES021016-17
Application #
6106558
Study Section
Special Emphasis Panel (LMG)
Project Start
Project End
Budget Start
Budget End
Support Year
17
Fiscal Year
1998
Total Cost
Indirect Cost
City
State
Country
United States
Zip Code
Andres, Sara N; Appel, C Denise; Westmoreland, James W et al. (2015) Tetrameric Ctp1 coordinates DNA binding and DNA bridging in DNA double-strand-break repair. Nat Struct Mol Biol 22:158-66
Ma, Wenjian; Westmoreland, Jim W; Gordenin, Dmitry A et al. (2011) Alkylation base damage is converted into repairable double-strand breaks and complex intermediates in G2 cells lacking AP endonuclease. PLoS Genet 7:e1002059
Nakai, Wataru; Westmoreland, Jim; Yeh, Elaine et al. (2011) Chromosome integrity at a double-strand break requires exonuclease 1 and MRX. DNA Repair (Amst) 10:102-10
Argueso, Juan Lucas; Westmoreland, James; Mieczkowski, Piotr A et al. (2008) Double-strand breaks associated with repetitive DNA can reshape the genome. Proc Natl Acad Sci U S A 105:11845-50
Chen, Ling; Trujillo, Kelly M; Van Komen, Stephen et al. (2005) Effect of amino acid substitutions in the rad50 ATP binding domain on DNA double strand break repair in yeast. J Biol Chem 280:2620-7
Resnick, Michael A (2005) Reduced replication: a call to ARMS. Cell 120:569-70
Lewis, L Kevin; Karthikeyan, G; Cassiano, Jared et al. (2005) Reduction of nucleosome assembly during new DNA synthesis impairs both major pathways of double-strand break repair. Nucleic Acids Res 33:4928-39
Lewis, L Kevin; Lobachev, Kirill; Westmoreland, James W et al. (2005) Use of a restriction endonuclease cytotoxicity assay to identify inducible GAL1 promoter variants with reduced basal activity. Gene 363:183-92
Lewis, L Kevin; Storici, Francesca; Van Komen, Stephen et al. (2004) Role of the nuclease activity of Saccharomyces cerevisiae Mre11 in repair of DNA double-strand breaks in mitotic cells. Genetics 166:1701-13
Lobachev, Kirill; Vitriol, Eric; Stemple, Jennifer et al. (2004) Chromosome fragmentation after induction of a double-strand break is an active process prevented by the RMX repair complex. Curr Biol 14:2107-12

Showing the most recent 10 out of 11 publications