Roles For The Rad1-Rad10-Slx4, Msh2-Msh6, And Sgs1 Dna Repair Factors In Ensuring The Efficiency And Fidelity Of Homologous Recombination In S. Cerevisiae
Nearly half of the human genome consists of repetitive DNA, and these sequences are a threat to genome stability because of their potential to recombine. The work presented here centers on two pathways that contribute to genome stability by ensuring the efficiency and fidelity of homologous recombination processes: 3' nonhomologous tail removal by the Rad1-Rad10-Slx4 endonuclease complex, and heteroduplex rejection by the Msh2-Msh6 mismatch recognition complex and the Sgs1 helicase. To study the coordination of checkpoint signaling and enzymatic repair functions during repair of a single chromosomal break, mating type switching was monitored in S. cerevisiae strains defective for the Rad1-Rad10-Slx4 3' endonuclease complex. Mutant strains displayed RAD9- and MAD2-dependent cell cycle delays and decreased viability during mating type switching. In particular, these mutants defective for Rad1-Rad10-Slx4 exhibited a unique pattern of dead and switched daughter cells arising from the same DSB-containing cell. Thus, Rad1-Rad10-Slx4 promotes efficient repair during gene conversion events involving a single 3' nonhomologous tail, and it is proposed that the rad1-delta and slx4-delta mutant phenotypes result from inefficient repair of a lesion at the MAT locus that is bypassed by replication-mediated repair. DNA mismatch repair proteins actively inhibit recombination between divergent sequences while allowing recombination between identical substrates. This process, known as heteroduplex rejection, is thought to occur when the Msh2-Msh6 DNA mismatch recognition complex recognizes and binds to mismatches in heteroduplex DNA, and either recruits or stimulates the Sgs1 helicase to unwind inappropriate recombination intermediates. Purification of the Msh2-Msh6 complex and a soluble fragment of Sgs1, Sgs1400-1268, in this study allowed characterization of their physical and functional interactions in vitro. Msh2-Msh6 and Sgs1400-1268 physically interact as demonstrated in coimmunoprecipitation experiments, and Msh2Msh6 appears to inhibit Sgs1-dependent unwinding of short 3' overhang substrates. Studies of the effect of Msh2-Msh6 on Sgs1 helicase activity are ongoing. Together, this work contributes to our molecular understanding of how recombination events are both regulated and carried out, as well as how defects in these DNA repair genes contribute to a variety of disease states in humans.
Dissertation or Thesis