Template Switching During Break-Induced Replication Is Promoted by the Mph1 Helicase in Saccharomyces cerevisiae

Abstract

Chromosomal double-strand breaks (DSBs) that have only one end with homology to a donor duplex undergo repair by strand invasion followed by replication to the chromosome terminus (break-induced replication, BIR). Using a transformation-based assay system, it was previously shown that BIR could occur by several rounds of strand invasion, DNA synthesis, and dissociation. Here we describe a modification of the transformation-based assay to facilitate detection of switching between donor templates during BIR by genetic selection in diploid yeast. In addition to the expected recovery of template switch products, we found a high frequency of recombination between chromosome homologs during BIR, suggesting transfer of the DSB from the transforming linear DNA to the donor chromosome, initiating secondary recombination events. The frequency of BIR increased in the mph1Δ mutant, but the percentage of template switch events was significantly decreased, revealing an important role for Mph1 in promoting BIR-associated template switching. In addition, we show that the Mus81, Rad1, and Yen1 structure-selective nucleases act redundantly to facilitate BIR.

MTE1 Functions with MPH1 in Double-Strand Break Repair

“…In homologous BIR, where the sequences that recombine share 108 bp of homology, deletion of mte1 results in increased BIR ( Figure 6E). Deletion of mph1 also results in increased BIR ( Figure 6E), as previously reported (Stafa et al 2014). The double mutant mph1D mte1D displays increased BIR, much like mph1D ( Figure 6E), indicating that MPH1 and MTE1 function in the same genetic pathway, and that mph1D is epistatic to mte1D.…”

“…MPH1 suppresses BIR during double-strand break repair (Luke-Glaser and Luke 2012;Stafa et al 2014). Given the physical and genetic interactions between Mte1 and Mph1 that our work has revealed, we tested whether MTE1 also plays a role in suppressing BIR.…”

“…Crossovers can also be prevented if the D-loop structure that results from the first strand invasion by one end of a resected DSB into the homologous chromosome is unwound before capture of the second end to form the dHJ. Unwinding of D-loops is catalyzed in vitro and in vivo by the 39-to-59 DNA helicase Mph1 (Sun et al 2008;Prakash et al 2009) to prevent loss of heterozygosity due to crossovers and break-induced replication (BIR) (Luke-Glaser and Luke 2012;Mazon and Symington 2013;Stafa et al 2014).The Mph1 DNA helicase was first identified as a deletion mutant with an increased mutation frequency (Entian et al 1999). Subsequent characterization revealed that mph1 mutants are sensitive to the alkylating agent MMS and to a lesser degree to ionizing radiation (Scheller et al 2000), and that mph1 mutants are proficient for mitotic recombination (Schurer et al 2004 Here we leverage intracellular protein location data to identify the complement of proteins that colocalize with the recombination repair protein Rad52 in nuclear foci during the response to DNA double-strand breaks.…”

“…Crossovers can also be prevented if the D-loop structure that results from the first strand invasion by one end of a resected DSB into the homologous chromosome is unwound before capture of the second end to form the dHJ. Unwinding of D-loops is catalyzed in vitro and in vivo by the 39-to-59 DNA helicase Mph1 (Sun et al 2008;Prakash et al 2009) to prevent loss of heterozygosity due to crossovers and break-induced replication (BIR) (Luke-Glaser and Luke 2012; Mazon and Symington 2013;Stafa et al 2014).…”

Perinuclear tethers license telomeric DSBs for a broad kinesin- and NPC-dependent DNA repair process

“…However, the relationship between perinuclear telomere tethers and nuclear pore subcomplexes in subtelomeric DNA repair remains unknown 12,23 . In addition, it is unclear whether survival is achieved through bona fide DNA repair processes such as non-homologous end joining (NHEJ) or rather via genome patching mechanisms that may help propagate a compromised genome [24][25][26] . Moreover, how DSB sites move within the nucleus is unclear.…”