Copyright Ó 2008 by the Genetics Society of America DOI: 10.1534/genetics.108.090233 Sequence Divergence Impedes Crossover More Than Noncrossover Events During Mitotic Gap Repair in Yeast Caroline Welz-Voegele and Sue Jinks-Robertson1 Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, North Carolina 27710 Manuscript received April 11, 2008 Accepted for publication May 8, 2008 ABSTRACT Homologous recombination between dispersed repeated sequences is important in shaping eukaryotic genome structure, and such ectopic interactions are affected by repeat size and sequence identity. A transformation-based, gap-repair assay was used to examine the effect of 2% sequence divergence on the efficiency of mitotic double-strand break repair templated by chromosomal sequences in yeast. Because the repaired plasmid could either remain autonomous or integrate into the genome, the effect of sequence divergence on the crossover–noncrossover (CO–NCO) outcome was also examined. Finally, proteins important for regulating the CO–NCO outcome and for enforcing identity requirements during recombination were examined by transforming appropriate mutant strains. Results demonstrate that the basic CO–NCO outcome is regulated by the Rad1-Rad10 endonuclease and the Sgs1 and Srs2 helicases, that sequence divergence impedes CO to a much greater extent than NCO events, that an intact mismatch repair system is required for the discriminating identical and nonidentical repair templates, and that the Sgs1 and Srs2 helicases play additional, antirecombination roles when the interacting sequences are not identical. ECOMBINATION is a high-fidelity process that persed throughout the genome. Such ectopic inter- R copies information from one DNA duplex to re- actions are important for shaping genome structure, pair single- or double-strand discontinuities in another with NCO events likely driving the concerted evolution DNA duplex (reviewed by Krogh and Symington of multigene families and COs leading to various types 2004). Repair events involve either the unidirectional of genome rearrangements. In mitotic studies using transfer of information between duplexes or the recip- model recombination substrates in the yeast Saccharo- rocal exchange of information, which will be referred myces cerevisiae, the rate of ectopic interactions is directly to here as noncrossover (NCO) and crossover (CO) proportional to repeat size (Ahn et al. 1988; Hayden events, respectively. In classic models of homologous and Byers 1992; Jinks-Robertson et al. 1993; Inbar recombination, NCO and CO events derive from et al. 2000) and inversely proportional to the level of alternative cleavage of a common intermediate known sequence divergence (Datta et al. 1997; Chen and as a Holliday junction (HJ), which corresponds to the Jinks-Robertson 1998). point where the single strands of the interacting The major barrier to recombination between di- duplexes switch pairing partners. In more recent re- verged sequences in yeast derives from antirecombina- combination models, however, some NCO events pro- tion activity of the mismatch repair (MMR) system ceed through an intermediate that cannot be processed (reviewed in Surtees et al. 2004), which is best known into a CO. The template for recombinational repair is for its roles in removing DNA replication errors and typically a homologous chromosome or sister chroma- repairing mismatches in meiotic recombination inter- tid, but because recombination is a homology-driven mediates (reviewed in Harfe and Jinks-Robertson process, it also can engage repetitive sequences dis- 2000; Kunkel and Erie 2005). There are three major yeast complexes involved in mismatch removal and in antirecombination: MutSa, MutSb, and MutLa com- This article is dedicated to the memory of Caroline Welz-Voegele, who posed of Msh2-Msh6, Msh2-Msh3, and Mlh1-Pms1, died on September 12, 2007. During her 10 years with the Jinks-Robertson respectively. MutSa and MutSb bind directly to mis- group, Caroline made numerous experimental and intellectual contribu- a a b tions, she was generous with her time and knowledge, and she served as a matches, while MutL couples MutS / -mediated mis- mentor and role model for all who passed through the lab. Caroline is match recognition to the appropriate downstream greatly missed and fondly remembered by her friends and colleagues at processing steps, the precise mechanism(s) of which Emory University and Duke University. remain obscure. Additional proteins implicated in 1Corresponding author: Department of Molecular Genetics and Microbi- ology, Duke University Medical Center 3020, 228 Jones Bldg., Research the repair of mismatches include the Rad1-Rad10 Dr., Durham, NC 27710. E-mail: [email protected] endonuclease (Kirkpatrick and Petes 1997), the Genetics 179: 1251–1262 ( July 2008) 1252 C. Welz-Voegele and S. Jinks-Robertson Exo1 exonuclease (Tishkoff et al. 1997), and the derivative containing a 1.7-kb BamHI genomic HIS3 fragment. PCNA sliding clamp ( Johnson et al. 1996; Umar et al. Following treatment with T4 DNA polymerase and T4 kinase, the fragment was inserted at the SmaI site of the LEU2-CEN 1996). Rad1-Rad10 and Exo1 are also important in ikorski ieter icholson vector pRS315 (S and H 1989). The resulting antirecombination (N et al. 2000), but plasmids pSR515 and pSR516 contain the HIS3 gene in the PCNA plays little, if any, role in this process (Stone opposite and same orientation, respectively, as the vector lacZ et al. 2008). In contrast, the helicase Sgs1 is important gene. The HIS3 gene of these plasmids contains ,10 bp of in antirecombination (Myung et al. 2001; Spell and identity to the his3D200 allele at the endogenous HIS3 locus. Jinks-Robertson 2004), but has no known role in The HIS3-18 allele of pSR612 contains 18 silent, randomly distributed mutations (see supplemental Figure 1) and is 98% the repair of mismatches. Thus, although there are identical to the standard HIS3 gene. The mutations were similarities in the MMR-directed editing of replication introduced by subjecting pSR515 to sequential rounds of site- and recombination intermediates, there are genetic directed mutagenesis using the Chameleon double-stranded, and presumably mechanistic differences as well. site-directed mutagenesis kit (Stratagene). All mutations were In yeast, chromosomal sequences can serve as a tem- confirmed by sequencing, and pSR612 was able to fully complement the HisÀ phenotype of a his3D200 strain. plate for the faithful repair of a linear, gap-containing pSR800 contains a 39-truncated, but otherwise wild-type plasmid and such gap-repair reactions were instrumen- (WT), his3 allele inserted into the CAN1 coding sequence. tal in the development of the double-strand break Because this allele contains no polymorphisms, it is referred to (DSB) repair model of recombination (Szostak et al. as his3-0,D39 to distinguish it from the similarly truncated allele 1983). Of particular significance was the observation that contains the engineered silent changes (his3-18,D39). The can1This3D39 allele was constructed as follows. First, the that the repaired plasmid either remains autonomous smallest KpnI fragment of pSR515 was deleted, thereby or integrates into the host genome (Orr-Weaver and truncating HIS3 at an internal KpnI site and eliminating the Szostak 1983), outcomes presumed to reflect cleavage last 11 amino acids of the encoded protein. An 834-bp BamHI- of a Holliday junction intermediate to generate NCO or KpnI fragment from the resulting plasmid (pSR798) was then CO products, respectively. Subsequent studies have treated with T4 DNA polymerase and inserted at the MscI site of pSR797. pSR797 is a pUC9 derivative containing an 1122-bp confirmed that plasmid-based repair assays generally CAN1 fragment (121 to 11141 of the 1773 nt CAN1 ORF) recapitulate the genetic requirements and features of inserted at the SmaI site of the vector polylinker. The resulting DSB-initiated chromosomal recombination (Bartsch plasmid pSR800 contains the his3-0,D39 allele in an orientation et al. 2000) and hence are a useful model for studying opposite to that of the CAN1 sequences. pSR801 contains the T basic recombination processes. In the experiments can1 his3-18,D39 allele and was constructed in the same manner as pSR800, but starting from pSR612. reported here, a gap-repair assay was used to examine pSR840 contains the his3DBgl allele and was used as the the regulation of mitotic recombination fidelity, with template for producing the linear ‘‘gapped vector’’ PCR an emphasis on how sequence divergence affects the fragment for transformation assays. pSR840 was derived by CO–NCO decision in different genetic backgrounds. first inserting a SacI-SalI fragment containing the full-length HIS3 gene (from pSR516) into SacI/SalI-digested pRS306, an These studies support roles for the Srs2 and Sgs1 ikorski ieter helicases, the Rad1-Rad10 endonuclease, and MutSb integrating URA3 vector (S and H 1989). The internal 60-bp BglII fragment was then deleted to generate the in regulating the CO–NCO outcome, confirm that the his3DBgl allele. Finally, a Klenow-treated HinfI-EcoO109 frag- regulation of recombination fidelity depends on activity ment containing the ARS4 replication origin of pRS315 of the MMR machinery, and demonstrate that sequence (Sikorski and Hieter 1989) was ligated to AatII-digested divergence impedes CO events to a much greater extent plasmid that had been treated with T4 DNA polymerase. than NCO events. Yeast strains: Transformation