Mechanisms and regulation of mitotic recombination in Saccharomyces cerevisiae - PubMed (original) (raw)

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Mechanisms and regulation of mitotic recombination in Saccharomyces cerevisiae

Lorraine S Symington et al. Genetics. 2014 Nov.

Abstract

Homology-dependent exchange of genetic information between DNA molecules has a profound impact on the maintenance of genome integrity by facilitating error-free DNA repair, replication, and chromosome segregation during cell division as well as programmed cell developmental events. This chapter will focus on homologous mitotic recombination in budding yeast Saccharomyces cerevisiae. However, there is an important link between mitotic and meiotic recombination (covered in the forthcoming chapter by Hunter et al. 2015) and many of the functions are evolutionarily conserved. Here we will discuss several models that have been proposed to explain the mechanism of mitotic recombination, the genes and proteins involved in various pathways, the genetic and physical assays used to discover and study these genes, and the roles of many of these proteins inside the cell.

Keywords: budding yeast; recombination; repair.

Copyright © 2014 by the Genetics Society of America.

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Figures

Figure 1

Figure 1

Genetic outcomes of homologous recombination. The letters A/a and B/b indicate heteroalleles. Circles indicate centromeres. Colors red and black indicate homologous chromosomes in diploid cells. (A) Sister-chromatid crossover. A crossover between sister chromatids results in two genetically identical cells. (B) Unequal sister-chromatid exchange (USCE). Within repetitive sequence elements (boxes), a crossover between misaligned repeats results in repeat copy number expansion and contraction. (C) Interhomolog crossover. A crossover between homologs leads to loss of hetorozygosity (LOH), if the recombinant molecules segregate to different cells in the ensuing cell division. (D) Gene conversion. A nonreciprocal genetic exchange between homologs leads to LOH in one of the resulting cells. (E) Productive ectopic translocation. A crossover between homologous sequences (boxes with arrows) with the same orientation relative to the centromere (circles) on different chromosomes in gray and black results in a productive ectopic translocation. Cosegregation of the recombinant molecules results in genetically balanced cells, shown on the right. Segregation of the recombinant molecules to different cells, shown on the left, leads to lethality if the regions represented by B and D are essential. (F) Nonproductive ectopic translocation. If the recombining sequences have opposite orientation with respect to the centromere, the reciprocal translocation results in inviable dicentric and acentric chromosomes.

Figure 2

Figure 2

Models for homology-dependent DSB repair. Recombinational repair of a DSB is initiated by 5′ to 3′ resection of the DNA end(s). The resulting 3′ single-stranded end(s) invades an intact homologous duplex (in red) to prime leading strand DNA synthesis. For one-ended breaks, a migrating D-loop is established to facilitate break-induced replication (BIR) to the end of the chromosome and the complementary strand is synthesized by conservative replication. For two-ended breaks, the classical double-strand break repair (DSBR) model predicts that the displaced strand from the donor duplex pairs with the 3′ ssDNA tail at the other side of the break and primes a second round of leading strand synthesis. After ligation of the newly synthesized DNA to the resected 5′ strands, a double Holliday junction intermediate (dHJ) is generated. The dHJ can be either dissolved by branch migration into a hemicatenane (HC) leading to noncrossover (NCO) products or resolved by endonucleolytic cleavage to produce NCO (positions 1, 2, 3, and 4) or CO (positions 1, 2, 5, and 6) products. In mitotic cells, the invading strand is often displaced after limited synthesis and the nascent complementary strand anneals with the 3′ single-stranded tail of the other end of the DSB and after fill-in synthesis and ligation generate exclusively NCO products (synthesis-dependent strand annealing, SDSA).

Figure 3

Figure 3

Single-strand annealing. Repair of a DSB flanked by direct repeat sequences (boxes) can occur by single-strand annealing (SSA), if 5′ to 3′ resection is allowed to progress past the repeats. The complementary single-stranded repeats can anneal, leaving heterologous flaps to be removed by the Rad1–Rad10 endonuclease, before gap-filling and ligation completes the repair thereby deleting one of the repeats and the intervening sequence.

Figure 4

Figure 4

Resection of DSB ends. Resection of DSB ends progress 5′ to 3′ and in two steps. First, MRX and Sae2 catalyze short-range resection of ∼100 nt. The initial resection by MRX and Sae2 is particularly important for cleaning up “dirty” ends harboring chemical adducts, secondary structures, or covalently attached proteins. The Yku complex inhibits initial end resection by competing with MRX for binding to ends. Second, extensive resection for up to 50 kb is catalyzed by Exo1 and/or STR–Dna2. Phosphorylation of Sae2 at serine 267 is required for resection. Sae2 is degraded upon acetylation. Dna2 nuclear localization and recruitment to DSBs require its phosphorylation at threonine 4 and serines 17 and 237 (see text for details).

Figure 5

Figure 5

Rad51 filament dynamics. During Rad51-catalyzed strand invasion, Rad52 mediates the loading of Rad51 onto RPA-coated ssDNA to facilitate formation of a Rad51 nucleoprotein filament. The Rad51 filament is further stabilized by Rad55–Rad57. In contrast, the Srs2 helicase counteracts the Rad51 mediators by displacing Rad51 from ssDNA to disrupt toxic recombination intermediates. Similarly, Rad54 can displace Rad51 from dsDNA to allow loading of PCNA–Polδ at the 3′ end of the invading strand. Phosphorylation of Rad51 at serine 192 is required for ATP hydrolysis and DNA binding. Rad55 is phosphorylated at serines 2, 8, and 14. Sumoylation of Rad52 at lysines 43, 44, and 253 mediates its dissociation from ssDNA. Phosphorylation and sumoylation of Srs2 have pro- and antirecombination functions, respectively (see text for details).

Figure 6

Figure 6

Recombination at replication forks. Parental strands are shown in dark blue and nascent strands in light blue. Polymerase-blocking DNA lesion indicated by a filled triangle. (A) Error-free bypass of leading strand blockage. The replication fork stalled at a DNA lesion on the leading strand template may be regressed in a Rad5-dependent manner to expose the lesion for excision repair after which the regressed fork is reversed and replication resumed. Alternatively, leading strand synthesis may transiently switch templates within the regressed fork. Upon fork reversal and reanneling of the extended leading strand to its parental template, the DNA lesion is bypassed and can subsequently be repaired by excision repair. (B) Fork collapse and rescue by passive replication. Fork collapse may result if the replication fork encounters a nick on the leading strand template or if a regressed fork is endonucleolytically cleaved to form a one-ended DSB, which is most often rescued by passive replication from an adjacent replication fork that can anneal to the end and be resolved into two intact sister chromatids. (C) Error-free bypass of lagging strand blockage. Lagging strand synthesis can be completed by postreplicative recombination to reestablish strand continuity at the lesion using the nascent sister chromatid as a template. The remaining lesion on the parental lagging strand can subsequently be removed by excision repair.

Figure 7

Figure 7

Assay for heteroallelic recombination. Nonfunctional ade2-I and ade2-n heteroalleles and wild-type ADE2 give rise to red and white colonies, respectively. (A) I-SceI-induced heteroallelic recombination. A DSB induced by the I-SceI endonuclease in the ade2-I allele in G2 can be repaired from the intact homolog by short-tract or long-tract gene conversion to give rise to ADE2 and ade2-n, respectively. Red-white half-sectored colonies are indicative of a recombination event that occurred in the first generation after plating. Markers MET22 and URA3 on the other side of the centromeres (filled circles) facilitate the scoring of chromosome nondisjunction events. Markers HPH and NAT adjacent to the ade2 locus facilitate the scoring of CO events. (B) Scoring CO, NCO, and BIR events associated with gene conversion. Genotyping of red-white half-sectored colonies with respect to the HPH (H) and NAT (N) markers described in panel A allows the distinction of CO and BIR events as reciprocal and nonreciprocal LOH, respectively. The remaining events are NCOs.

Figure 8

Figure 8

Genetic assays. (A) Direct-repeat recombination. Spontaneous homologous recombination between ade2-n and ade2-a alleles can occur by gene conversion to produce Ade+ Ura+ cells or by SSA to produce Ade+ Ura− cells (Fung et al. 2009). (B) Inverted repeat recombination. Inverted repeat recombination assays exclusively Ade+ recombinants arising from gene conversion since SSA will not produce viable recombinants. CO and NCO events will lead to inversion and noninversion of the TRP1 marker, respectively (Mott and Symington 2011). (C) Plasmid gap repair assay. The efficiency of plasmid–chromosome recombination, crossover frequency, and conversion tract length is assayed by transformation of the gapped pSB110 plasmid into yeast containing the chromosomal met17-sna mutant allele in which a SnaBI site is eliminated 216 bp downstream of the gap in the plasmid (Symington et al. 2000). When the plasmid gap is repaired by noncrossover gene conversion, the result is unstable (u) Ura+ transformants, which will be Met+ (class I) or Met− (class II), depending on the absence or presence of co-conversion of the met17-sna mutation, respectively. If the gene conversion event is associated with a crossover, the result is a stable (s) Ura+ phenotype (classes III and IV). ARS, autonomously replicating sequence. (D) Break-induced replication. In this assay, BIR is initiated by induction of an HO-mediated DSB adjacent to a 3′ truncated lys2 gene (lys) on chromosome V. The lys fragment has 2.1 kb of homology to a 5′ truncation of lys2 (ys2) close to the telomere on chromosome XI (Donnianni and Symington 2013), which serves as a donor for BIR. BIR results in deletion of the KanMX gene and all nonessential genes telomere proximal to the HO cut site and loss of G418 resistance (G418R). The strain has the MATa-inc allele to prevent cleavage at the endogenous HO cut site.

Figure 9

Figure 9

Model for the role of endonucleases in the resolution of recombination intermediates. Invasion by a 3′ end of a gapped vector into a chromosomal donor sequence generates a D-loop, which is extended by DNA synthesis. Initially, second end capture results in a structure that is a potential substrate for Mus81–Mms4 cleavage to produce a nicked HJ and subsequently a CO upon further cleavage. If, on the other hand, the captured D-loop is gap filled and ligated, a dHJ is formed, which in most cases is converted to a hemicatene and dissolved by STR to yield a NCO, but could also be resolved by Mus81–Mms4 to produce a CO or NCO. Alternatively, if resection and DNA synthesis proceed beyond the heterology boundary, the D-loop can branch migrate to create a region of single-stranded DNA adjacent to the branch point, which could be cleaved by Rad1–Rad10 to generate a single HJ (sHJ). The sHJ can be resolved by either Mus81–Mms4 or Yen1 cleavage to produce a CO or NCO, or converted to a CO and a NCO product during the next S phase.

Figure 10

Figure 10

Choreography of HR focus assembly. (A) Focus formation of HR proteins. The high local concentration of Rad52 and Rad59 at DSBs induced by treatment with 200 µg/ml zeocin for 2 hr at 25°. Strain NEB110-25B is a MATa haploid containing RAD52-CFP and RAD59-YFP. Arrowheads mark foci. (B) Order of assembly of HR proteins at foci. Proteins are recruited from the left to right starting with MRX binding at DSB ends and later replaced by proteins recruited to ssDNA at resected DSBs.

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