DOUBLE STRAND BREAK REPAIR AND RECOMBINATION

Environmental Health Sciences

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Abstract

Summary of Work: The repair of DNA double-strand breaks (DSBs) and stabilization of telomeric sequences at chromosome ends involves homologous recombination and nonhomologous end-joining (NHEJ) genes in yeast. RAD50, MRE11 and XRS2 encode subunits of a nuclease complex functioning in both pathways. A search for highly expressed cDNAs that suppress the rad50 DNA repair deficiency yielded EXO1 and TLC1. Plasmids expressing EXO1 or TLC1 increased the resistance of rad50, mre11 and xrs2 strains to ionizing radiation and MMS, but did not suppress the sensitivities of recombination-defective rad51, rad52, rad54 or rad59 mutants. Elevated levels of Exo1 rescued spontaneous recombination defects and suppressed HO and EcoRI endonuclease-induced growth inhibition of rad50, but not rad52 cells. Increased EXO1 or TLC1 expression did not alter the kinetics of DSB-induced G2 arrest or restore plasmid NHEJ. The DNA metabolic yku70 and rad27 (Fen1) mutants were differentially suppressed by EXO1 and TLC1. Results with Exo1 suggest that the 5?->3? exonuclease can substitute for the Rad50/Mre11/Xrs2 nuclease complex in rescission of DSB ends prior to repair by homologous recombination, but not NHEJ. Suppression by TLC1, the template RNA subunit of yeast telomerase, required an intact recombination apparatus, but did not require other NHEJ genes. A new approach has been initiated for the in vivo characterization of a specific chromosome double-strand break in real-time. This is the first attempt to follow directly the fate a broken chromosomes in live cells. A set of novel plasmids and strains were created that permit monitoring the fate of a broken yeast. The strains contain fusions of green fluorescent protein (GFP) to the E. coli LacI repressor that can bind to an integrated binding sequence array near the site of the break. Recent experiments assessed chromosome movement in repair-proficient haploid cells. Direct visualization of a borken chromosome will be compared between recombination- and/or NHEJ-deficient vs wild type cells.