REVIEW ARTICLE

Felix Prado Æ Felipe Cortes-Ledesma

Pablo Huertas Æ Andres Aguilera

Mitotic recombination in Saccharomyces cerevisiae

Received: 29 May 2002 / Revised: 15 October 2002 /Accepted: 15 October 2002 / Published online: 29 November 2002� Springer-Verlag 2002

Abstract Mitotic homologous recombination (HR) is animportant mechanism for the repair of double-strandbreaks and errors occurring during DNA replication. Itis likely that the recombinational repair of DNA lesionsoccurs preferentially by sister chromatid exchanges thathave no genetic consequences. However, most geneti-cally detectable HR events occur between homologousDNA sequences located at allelic positions in homolo-gous chromosomes, or between DNA repeats located atectopic positions in either the same, homologous orheterologous chromosomes. Mitotic recombination mayoccur by multiple mechanisms, including double-strandbreak repair, synthesis-dependent strand annealing,break-induced replication and single-strand annealing.The occurrence of one recombination mechanism versusanother depends on different elements, including theposition of the homologous partner, the initiation event,the length of homology of the recombinant moleculesand the genotype. The genetics and molecular biology ofthe yeast Saccharomyces cerevisiae have proved essentialfor the understanding of mitotic recombination mecha-nisms in eukaryotes. Here, we review recent genetic yeastdata that contribute to our understanding of thedifferent mechanisms of mitotic recombination and thein vivo role of the recombination proteins.

Keywords Mitotic recombination Æ DSB repair ÆGene conversion Æ Crossover Æ RAD genes

Introduction

Homologous recombination (HR) refers to anyexchange of genetic information between homologous

DNA sequences. Illegitimate recombination and site-specific recombination do not involve homologous DNAsequences. HR is essential during meiosis for properchromosome segregation and preservation of geneticdiversity (Roeder 1997) and is necessary during mitosisfor the repair of different types of DNA lesions gener-ated either by exposure to chemicals and ionizing radi-ation, by the action of nucleases (Aguilera et al. 2000)or by replication errors (Kogoma 1997; Cox 2001;Kuzminov 2001).

Different DNA partners can be used as donors ofinformation during a recombinational repair event.HR can occur between sister chromatids, betweenhomologous chromosomes or between repeated DNAsequences, regardless of their chromosomal location.In all cases, HR can involve non-reciprocal transferof information from one DNA molecule to another(gene conversion) and/or reciprocal transfer of infor-mation between the two DNA molecules (crossover;Fig. 1A).

HR is a major double-strand break-repair process inyeast, e.g. Saccharomyces cerevisiae. In this organism,HR events can be detected and analyzed either genet-ically or at the molecular level (Fig. 1B). Here, wereview the mechanisms of mitotic HR between dif-ferent DNA substrates in S. cerevisiae. Recent reviewson the connection between HR and replica-tion (Kogoma 1997; Kowalczykowski 2000; Cox 2001;Kraus et al. 2001; Kuzminov 2001; Michel et al. 2001;Forsburg 2002) or transcription (Aguilera 2002)have been recently published and will not be discussedhere.

Mechanisms of homologous recombination

The most accepted models on double-strand break(DSB) repair are shown in Fig. 2 (see Paques and Haber1999; Aguilera et al. 2000; Kupiec 2000; Sung et al. 2000;Prado et al. 2001). A short explanation of each modelfollows.

Curr Genet (2003) 42: 185–198DOI 10.1007/s00294-002-0346-3

Communicated by S. Hohmann

F. Prado Æ F. Cortes-Ledesma Æ P. Huertas Æ A. Aguilera (&)Departamento de Genetica, Facultad de Biologıa,Universidad de Sevilla, 41012 Sevilla, SpainE-mail: aguilo@us.es

Double-strand break repair

Double-strand break repair (DSBR) was first proposedto explain plasmid gap repair and is the most acceptedmechanism to explain the genetic relationship betweengene conversion and crossover in meiotic recombination(Szostak et al. 1983). Recombination is initiated by aDSB (Sun et al. 1989; Cao et al. 1990) and the 5¢ ends ateach side of the DSB are resected, exposing long 3¢-ended single-stranded tails (Sun et al. 1991), which in-vade the homologous double-strand DNA (dsDNA) andprime DNA synthesis. After pairing of the invadingsingle-strand DNA (ssDNA) and the invaded dsDNA,the reaction is followed by strand exchange. This gen-erates a DNA heteroduplex (Goyon and Lichten 1993;Nag and Petes 1993), which is a key feature for geneconversion in all recombination reactions. The double-strand exchange leads to the formation of two four-stranded intermediates, termed Holliday junctions (HJs;Collins and Newlon 1994; Schwacha and Kleckner1994). The cleavage of these two HJs in either the sameor the opposite direction results in non-crossover orcrossover products, respectively.

Synthesis-dependent strand annealing

It has been shown that, from yeast to animal cells, manymitotic gene conversion events are not associated withcrossovers (Gloor et al. 1991; Nassif et al. 1994; Johnsonand Jasin 2000; Virgin et al. 2001). Such findings led toan alternative model, termed SDSA (Hastings 1988;McGill et al. 1989), in which, after 3¢ end invasion,the newly synthesized DNA strands are releasedfrom the invaded DNA template and returned to theacceptor molecule. Consequently, the only possible

Fig. 1A, B Products of homologous recombination (HR). A HRcan lead to the unidirectional transfer of information (geneconversion) whether or not associated with the reciprocal exchangeof the adjacent sequences (crossover). B HR can lead to a selectablerecombinant product, which can be scored genetically as colony-forming units on selective medium (left) or molecularly bySouthern analysis (right). Each line represents a single-strandDNA molecule. r Restriction site, P parental, R recombinant

Fig. 2 Mechanisms of HR. Initiation of recombination by adouble-strand break (DSB) is followed by DNA resection of the5¢ ends. This generates 3¢-ended single strands that can invade ahomologous DNA molecule and prime new DNA synthesis. In thedouble-strand break repair (DSBR) mechanism, the newly repli-cated ends are captured by the resected 5¢ ends and two cruciformor Holliday junctions (HJs) are formed. Resolution of the HJs leadsto gene conversion whether or not associated with crossovers. Inthe synthesis-dependent strand annealing (SDSA) mechanism, thenewly replicated ends are displaced from the template and re-annealed to each other. Consequently, all outcomes are noncross-overs. In the break-induced replication (BIR) mechanism, thenewly replicated DNA is not captured by a second end andreplication may progress along the rest of the invaded molecule.Each line represents a single-strand DNA molecule

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recombination product is a gene conversion not associ-ated with crossover. SDSA is an important mechanismof mitotic recombination and it can also participate inthe formation of meiotic gene conversions (Allers andLichten 2001).

Break-induced replication

Based on the recombination-dependent replicationmodels proposed for T4 phage (Mosig 1998) and Esc-herichia coli (Kogoma 1997), break-induced replication(BIR) has been proposed as an explanation for the co-conversion of markers involving up to several hundredkilobases in yeast (Voelkel-Meiman and Roeder 1990;Malkova et al. 1996). In this mechanism, the invading 3¢end primes DNA synthesis that can cover long DNAfragments.

Genes involved in homologous recombination

Many mutants involved in yeast mitotic recombinationwere isolated as defective in radiation-induced DNAdamage (Petes et al. 1991). Further genetic and bio-chemical characterization allowed us to get a betterunderstanding of their molecular role during therecombination process (Table 1).

Initiation

The nature of the events leading to spontaneousrecombination remains to be determined. However,most recombination events seem to be associated withDSB formation. It is well established that a DSB caninitiate a HR event (Paques and Haber 1999). Single-strand breaks or mutagenic lesions may also inducerecombination, but they require the passage of the rep-lication fork, suggesting that such lesions are furtherprocessed into DSBs (Galli and Schiestl 1998, 1999).

DNA resection

Upon DSB formation, 5¢-ended tails are resected,leaving single-stranded 3¢ tails (White and Haber 1990;Sun et al. 1991). In meiosis, this step is carried out by amultifunctional complex formed by Rad50, Mre11 andXrs2, with roles in mitotic DSB repair (either by non-homologous end joining –NHEJ– or by HR), telomeremaintenance and cell-cycle checkpoint (Haber 1998).Another gene that might participate in DNA resectionis SAE2, whose null mutation confers mitotic andmeiotic defects similar to rad50 (Keeney and Kleckner1995; McKee and Kleckner 1997). In mitosis, in addi-tion to the Mre11/Rad50/Xrs2 complex, there may beredundant nuclease activities, since mutation of theexonuclease activity of Mre11 hardly affects the repair

of DSBs induced by the HO endonuclease (Moreauet al. 1999). One of these activities could be the exon-uclease Exo1 protein (Moreau et al. 2001). The Rad50/Mre11/Xrs2 complex may be required for additionalfunctions during DSB repair, as null mre11 but notnuclease-deficient mre11 mutants are defective in HR(Bressan et al. 1999). Interestingly, it has recently beenshown that Mre11/Rad50/Xrs2 is involved in prevent-ing genome instability through its capacity to repairDSBs with terminal hairpin structures that result fromthe processing of inverted repeats (Lobachev et al.2002).

DNA invasion

Two partially homologous proteins, Rad52 and Rad59,display DNA strand-annealing activity in vitro (Mor-tensen et al. 1996; Petukhova et al. 1999). Rad52interacts with the trimeric DNA-binding complex RPA(Hays et al. 1998). RPA stimulates the DNA-annealingactivity of Rad52, but not of Rad59 (Shinohara et al.1998; Petukhova et al. 1999), likely by removing sec-ondary structures in the ssDNA (Sung et al. 2000).Rad52 and Rad59 are good candidates to participate inDNA invasion. They interact in vitro (Davis andSymington 2001). Rad52 is the key protein in yeast HR,essential for most if not all recombination processes(Paques and Haber 1999). In contrast, Rad59 becomesessential for recombination only in the absence of Rad51(Bai and Symington 1996).

Strand exchange

Rad51 is the functional and structural homologue ofRecA (Aboussekhra et al. 1992; Shinohara et al. 1992).It has ATP-dependent DNA strand exchange activityin vitro (Sung 1994; Sung and Robberson 1995; Sungand Stratton 1996; Namsaraev and Berg 1997). In a firststep, Rad51 forms a nucleofilament on ssDNA, in areaction that is stimulated by RPA (Sung and Robber-son 1995; Sugiyama et al. 1997). Later, this nucleofila-ment invades a homologous dsDNA (Sung et al. 2000).Strand invasion is promoted by Rad52, likely targetingRad51 to a ssDNA/RPA complex (New et al. 1998;Shinohara and Ogawa 1998). Despite the importance ofthe strand-exchange reaction in E. coli recombination(Eggleston and West 1996) and in yeast meiotic recom-bination (Roeder 1997), the effects of the null rad51mutation on mitotic recombination vary, depending onthe type of recombination event studied. Thus, whileRad52 is essential for initiation of most types ofrecombination events (deletions, inversions, allelicrecombination, ectopic recombination), this is not thecase for Rad51 (see below). Only the double-mutantrad51 rad59 displays recombination defects as severe asrad52 (Bai and Symington 1996). A possible interpre-tation for this result is that Rad51 and Rad59 are

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necessary and enough to stabilize a putative DNA in-vasion event catalyzed by Rad52, but neither Rad51 norRad59 is able to bypass the requirement for Rad52.Consistent with this view, Rad59 and Rad51 have beenshown to physically interact with Rad52 (Shinohara andOgawa 1998; Davis and Symington 2001).

Rad55, Rad57, Rad54 and Rdh54 also participate inthe strand-exchange reaction. They interact physicallywith Rad51 in vitro (Krejci et al. 2001). Rad55 andRad57 share sequence similarities with Rad51 (Kansand Mortimer 1991; Lovett 1994) and form a hetero-meric complex that stimulates Rad51-mediated strand

exchange in vitro (Sung 1997; Benson et al. 1998; Pet-ukhova et al. 1998). They are essential for recombina-tion at 22 �C, in agreement with a structural rather thanan enzymatic role (Lovett and Mortimer 1987). Rad54and Rdh54 belong to the SWI2/MOT1 family of ATP-dependent chromatin-remodeling proteins (Emery et al.1991; Klein 1997; Shinohara et al. 1997) and stimulateRad51-dependent pairing in vitro (Petukhova et al.1998, 2000). In addition, Rad54 stimulates the ability ofRad51 to form D-loop structures in the invadeddsDNA, a step required for proper strand exchange(Petukhova et al. 2000; Van Komen et al. 2000).

Table 1 Biochemical and genetic features of the yeast mitotic recombination genes. ds double-strand, HJ Holliday junction, HUhydroxyurea, MMS methyl methanesulfonate, SCE sister chromatid exchange, ss single-strand, UV ultraviolet light

Gene Protein function Proteininteractions

Mutant phenotypes

MMS4 Heteromeric complex Mus81/Mms4 Mus81 MMS sensitiveMRE11 ssDNA endonuclease Rad50, Xrs2 Defective 5¢ to 3¢ DNA resection

dsDNA 3¢ to 5¢ exonuclease Retarded deletion formationHeteromeric complex Rad50/Mre11/Xrs2 Decreased SCE

MMS sensitiveMSH2 Mismatched dsDNA binding Msh3 Decreased deletion frequency

Heteromeric complex Msh2/Msh3 Decreased nonhomologous end recombinationMSH3 Mismatched dsDNA binding Msh2 Decreased deletion frequency

Heteromeric complex Msh2/Msh3 Decreased nonhomologous end recombinationHU sensitive

MUS81 Heteromeric complex Mus81/Mms4 Mms4, Rad54 HU, UV and MMS sensitiveCleavage of HJs and Y structures

RAD1 ss- and dsDNA endonuclease Rad10 Decreased wild-type deletion and Rad51-independentinversion frequencies

Cleavage of Y structures decreased nonhomologous end recombinationHeteromeric complex Rad1/Rad10 UV sensitive

RAD10 Heteromeric complex Rad1/Rad10 Rad1 similar to rad1RAD50 ATP-dependent DNA binding Mre11, Xrs2 Defective 5¢ to 3¢ DNA resection

Heteromeric complex Rad50/Mre11/Xrs2 Retarded deletion formationDecreased SCEX-ray and MMS sensitive

RAD51 ATP-dependent DNA pairing and strand exchange Rad52, Rad54,Rad55, Rdh57,Rdh54

Decreased allelic and ectopic recombinationDecreased DSB-induced SCEIncreased deletion frequencyX-ray and MMS sensitive

RAD52 ssDNA annealing activity Rad51, Rad59,Rpa1

General defect in spontaneous and DSB-inducedmitotic recombination

Stimulation of Rad51-dependent strand exchange X-ray and MMS sensitiveRAD54 dsDNA-dependent ATPase Rad51 Similar to rad51

ATP-dependent DNA remodelingstimulation of Rad51-dependent strand exchange

RAD55 Stimulation of Rad51-dependent strand exchange Rad51, Rad57 Similar to rad51RAD57 Stimulation of Rad51-dependent strand exchange Rad51, Rad55 Similar to rad51RAD59 ssDNA annealing activity Rad52 Increased allelic recombination

Decreased deletion and inversion frequenciesMMS sensitive

RDH54/TID1

dsDNA-dependent ATPase Rad51 Decreased allelic recombinationATP-dependent DNA remodeling MMS sensitiveStimulation of Rad51-dependent DNA pairing

RFA1 ssDNA binding (Rpa1) Rad52 InviableStimulation of Rad52-dependent ssDNA annealing Increased deletion formation and SCE in rfa1 allelesStimulation of ssDNA-Rad51 assembly Decreased plasmid gap repair in rfa1 alleles

SAE2 Not determined Unknown Defective 5¢ to 3¢ DNA resectionRetarded deletion formationDecreased SCE

XRS2 Heteromeric complex Rad50/Mre11/Xrs2 Rad50, Mre11 Defective 5¢ to 3¢ DNA resectionRetarded deletion formationDecreased SCEX-ray sensitive

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Resolution of HJs

There are no yeast orthologous proteins of the bacterialRuvA/RuvB and RuvC/RusA responsible for migrationand resolution of HJs in E. coli. Resolution of HJs maybe required for meiotic recombination, in which HJs areobserved (Schwacha and Kleckner 1994, 1995). In mi-tosis, HJs may arise from the rescue of stalled replicationforks (see Fig. 3 for details; Higgins et al. 1976; Postowet al. 2001). HJs have been observed during S-phase ofmitotic cells (Zou and Rothstein 1997).

It has been suggested that the Mus81/Mms4 complexis a HJ resolvase (Boddy et al. 2001; Chen et al. 2001;Kaliraman et al. 2001). Mus81 was identified in a screenfor proteins interacting with Rad54 (Interthal and Heyer2000), whereas Mms4 was identified in a screen formethyl methanesulfonate-sensitive mutants (Prakashand Prakash 1977). Mus81 is required for DNA repairduring replication, as supported by the sensitivity ofmus81 cells to DNA-damaging agents that impair rep-lication (Interthal and Heyer 2000), and the increase inMus81 abundance upon treatment with replication in-hibitors (Chen et al. 2001). Importantly, mus81 mutants

are defective in meiosis; and this defect is suppressed byoverexpression of the bacterial resolvase RusA (Boddyet al. 2001). Mus81/Mms4 cleaves four-branched and Ystructures in vitro. However, Mus81/Mms4 has not beenshown to be able either to religate HJ cleavage productsor to catalyze migration of HJs in vitro (Boddy et al.2001; Chen et al. 2001; Kaliraman et al. 2001). Alto-gether, these results suggest that Mus81/Mms4 partici-pates in the resolution of HJs that arise when thereplication fork is blocked (Fig. 3; Chen et al. 2001;Kaliraman et al. 2001). In addition, Mus81/Mms4 couldbe required for the cleavage of the ssDNA tails formedduring the re-annealing step of SDSA. This can occur inthe presence of heterologies (Kaliraman et al. 2001) orwhen a DNA fragment longer than the gap is synthe-sized (de los Santos et al. 2001).

It is worth noting that Mus81 is partially homologousto Rad1 which, together with Rad10, forms a heterodi-mer with endonuclease activity that participates innucleotide-excision repair (Tomkinson et al. 1993) anddifferent types of mitotic recombination events (Klein1988; Schiestl and Prakash 1988; Aguilera and Klein1989a; Ivanov and Haber 1995; Prado and Aguilera1995). Interestingly, Rad1/Rad10 was shown to cleaveY, but not HJ structures in vitro (Bardwell et al. 1994).This is consistent with a role in clipping off overhangingssDNA 3¢ tails (Fishman-Lobell and Haber 1992).

Mitotic recombination between differenthomologous partners

Allelic recombination

Allelic recombination refers to those events occurring atallelic positions between homologous chromosomes. Itcan easily be monitored by measuring the frequency ofgene conversion of a mutant allele and reciprocalexchange of their adjacent markers. In general, bothspontaneous and DSB-induced allelic gene conversionexhibit crossover association at frequencies of lowerthan 20% (Esposito 1978; Haber and Hearn 1985;Kupiec and Petes 1988). This value contrasts with thatof meiotic recombination, where crossovers associatewith gene conversion at frequencies reaching up to 66%,depending on the locus studied (Fogel et al. 1981).

Allelic recombination induced by the HO endonuc-lease is reduced 100-fold in rad52 cells, 3–10-fold inrad51, rad54, rdh54, rad55 and rad57 and is not affectedin rad59 and rad50 mutants (Malkova et al. 1996; Klein1997; Shinohara et al. 1997; Signon et al. 2001). Incontrast, spontaneous recombination is reduced 20- to1,000-fold in rad52 and more than 20-fold in rad51 cells(Bai and Symington 1996), but is increased only 6-fold inrad59 cells (Bai and Symington 1996). Therefore, allelicrecombination leads preferentially to gene conversionsunassociated with crossovers and occurs by a majormechanism that requires the strand-exchange Rad51protein in cooperation with Rad55, Rad57 and Rad54.

Fig. 3 Replication-associated recombination. A lesion in the DNAmay block the progression of the replication fork and lead to agapped replication structure. Annealing of the newly synthesizedDNA generates a cruciform junction (HJ). DNA synthesis and HJunwinding overcomes the obstacle. Alternatively, HJ resolution bya resolvase generates a free end to invade the homologous DNAmolecule and restart replication. Each line represents a single-strand DNA molecule

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These data fit with SDSA as an important mechanismof allelic recombination, with a prominent role forRad51. Nevertheless, the 10–20% of associated cross-overs may reflect a minor proportion of DSBR events.In addition, the high level of repaired molecules detectedin rad51 cells upon HO-induced DSBs suggests thatother mechanisms take place when Rad51 is absent. Ithas been shown that a HO-induced DSB in one chro-mosome can be efficiently repaired in rad51 cells by amechanism which copies the information from the ho-mologous chromosome (from the 3¢ end of the DSB untilthe end of the chromosome). These events require Rad52and have been proposed to occur by BIR (Malkova et al.1996). Further characterization has provided evidencethat BIR requires Rad59 and Rad50, but not Rad51,Rad54, Rad55 and Rad57 (Signon et al. 2001).

Ectopic recombination

Ectopic recombination refers to those events occurringbetween any homologous pairs of sequences located atnon-allelic positions, whether or not in the same chro-mosome. Since intramolecular recombination displaysparticular genetic and molecular features, it will be dis-cussed below in a separate section. Ectopic recombina-tion between naturally spread Ty elements or artificiallycloned leu2 or ura3 alleles placed in heterologous chro-mosomes occurs at the same frequency (Kupiec andPetes 1988; Lichten and Haber 1989) and requires thesame gene products (Steele et al. 1991; Liefshitz et al.1995; Jablonovich et al. 1999) as when located at allelicpositions. These results suggest that homologousand heterologous chromosomes interact with thesame probability during mitosis, in contrast to meioticrecombination, in which allelic recombination occurs ata much higher frequency than non-allelic recombination(Kupiec and Petes 1988). As previously mentioned forallelic recombination, ectopic recombination betweensequences located in heterologous chromosomes exhibitsfrequencies of crossovers associated with gene conver-sion of 10–20% (Lichten and Haber 1989). Therefore, itseems that both allelic and ectopic recombination areunder the same genetic control.

Similar genetic requirements are observed for DSB orgap repair of a plasmid by a recombination event thatuses information from a homologous sequence locatedeither in a chromosome or in a second plasmid. There arestudies reporting that crossover (integration of the pla-smid) is associated with gene conversion at a frequency of50%, consistent with the DSBR model (Orr-Weaver andSzostak 1983), or lower than 25%, consistent with theSDSA model (Plessis and Dujon 1993; Nassif et al. 1994;Ferguson and Holloman 1996; Bartsch et al. 2000). Asexpected, all events are dependent onRad52 (Orr-Weaveret al. 1981) and are strongly reduced in rad51 mutants(Bartsch et al. 2000). Also, gap repair is negativelyaffected by some alleles of the RFA1 gene (Elias-Arnanzet al. 1996). Plasmid gap repair displays a high frequency

of associated crossovers in the absence of Rad51. As forRAD51-independent allelic recombination, plasmid gaprepair has been proposed to take place via two BIR eventsinitiated at the plasmid ends (Bartsch et al. 2000).

Ectopic recombination seems to be an importantsource of telomere propagation in yeast cells lackingtelomerase. Such mutants are able to survive by stabi-lizing telomere, using RAD52-dependent mechanisms.The major group of survivors also needs Rad51, whilea minor group is able to grow without Rad51 butwith Rad59 (Lundblad and Blackburn 1993; Teng andZakian 1999; Teng et al. 2000).

Direct-repeat recombination

Recombination between direct repeats can lead to geneconversion and/or to the deletion of the intervening re-gion and one of the repeats. Deletions can occur eitherby intramolecular recombination or by sister chromatidexchange (SCE; see SCE section below). In any case,deletions can result from a crossover between the repeatsoccurring via DSBR. However, different genetic andmolecular data suggest that single-strand annealing(SSA; Lin et al. 1984) is an important mechanism lead-ing to deletions (Fig. 4). In SSA, upon DNA resection ofthe 5¢-ended tails, the exposed homologous sequences

Fig. 4 Single-strand annealing (SSA) as a mechanism responsiblefor deletions. A DSB at a direct repeat is followed by 5¢ endresection, leading to complementary single strands. Strand anneal-ing followed by removal of the 3¢ overhanging strands leads to thedeletion of both the intervening sequence and one of the repeats.Each line represents a single-strand DNA molecule

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are annealed and the overhanging 3¢-ended tails areremoved.

Deletions have been shown to be kinetically separablefrom gene conversions. DSB-induced deletions are de-tected earlier than gene conversions in tandem repeatshaving no DNA between them and are detected laterwhen the repeats are 4.4 kb apart (Fishman-Lobell et al.1992). This uncoupling between gene conversion andcrossover rules out DSBR as the major mechanismleading to deletions, as DSBR implies a physical con-nection between both recombination events. Besides,only 5% of spontaneous deletions have been shown tobe associated with the formation of circles, the recipro-cal product of a crossover between repeats (Schiestl et al.1988; Santos-Rosa and Aguilera 1994).

In contrast to other HR events, deletions are effi-ciently initiated by a DSB at a heterologous DNAregion: the intervening sequence located between therepeats (Mezard and Nicolas 1994; Prado and Aguilera1995). Indeed, DSBs in direct repeats are preferentiallyrepaired by a deletion rather than by recombination witha third copy located either far away in the same chro-mosome or in a different chromosome (Rudin andHaber 1988; Sugawara and Haber 1992).

Much of the genetic data is consistent with SSA asone of the most important mechanisms leading to dele-tions. Deletions are slightly stimulated in rad51, rad54,rad55 and rad57 (McDonald and Rothstein 1994;Aguilera 1995; Rattray and Symington 1995; Ivanovet al. 1996; Klein 1997), suggesting that they occur in theabsence of strand exchange. Also, deletions require theproducts of the nucleotide-excision repair (NER) genes,RAD1 and RAD10, to clip off the overhanging 3¢ endsformed after annealing of the homologous single-stranded DNAs (Fig. 4; Fishman-Lobell and Haber1992; Ivanov and Haber 1995). In contrast to allelicrecombination, the mismatch repair proteins Msh2/Msh3 stimulate deletions between short DNA repeats,most likely by stabilizing the annealed sequences(Saparbaev et al. 1996; Sugawara et al. 1997; Evans et al.2000). Finally, mutations in the Mre11/Rad50/Xrs2complex do not prevent, but markedly retard, the for-mation of DSB-induced deletions (Ivanov et al. 1996).

Deletions are reduced 10- to 100-fold in rad52mutants (Jackson and Fink 1981; Ronne and Rothstein1988; Schiestl and Prakash 1988; Dornfeld and Living-ston 1992; Prado and Aguilera 1995). However, thisdependency on Rad52 decreases with the length of thehomology between the repeats (Paques and Haber 1999).These results are consistent with Rad52 providing thesingle-strand annealing activity. One of the roles pro-posed for Rad52 is to overcome the inhibitory effect ofthe DNA-binding RPA complex, since some rfa1 allelesstimulate Rad52-independent deletions (Smith andRothstein 1995, 1999). Other annealing activities mayparticipate. Rad59 is required for deletion formation,especially with short homologous sequences. This neg-ative effect of rad59 on deletions is synergistic with thatof rad52 and of msh3 (Jablonovich et al. 1999; Sugawara

et al. 2000; Davis and Symington 2001). However, de-letions are in some cases unaffected by rad52, as is thecase of the natural rDNA and CUP1 arrays (Ozenbergerand Roeder 1991). This observation suggests that, incontrast to other recombination events, deletions canalso occur via a Rad52-independent pathway.

Inverted-repeat recombination

Recombination between inverted repeats may lead togene conversion of one of the repeats and/or the inver-sion of the intervening sequence. Spontaneous inver-sions between particular inverted repeats are ten timesless abundant than deletions occurring between the samerepeats in direct orientation (Prado and Aguilera 1995).This is likely because inversions cannot be initiated by aDSB at the intervening region (Prado and Aguilera1995).

Inversions can be generated by reciprocal exchangevia DSBR. Accordingly, they are strongly dependenton Rad52 (Aguilera and Klein 1989a; Rattray andSymington 1994; Aguilera 1995; Prado and Aguilera1995; Bai and Symington 1996; Bai et al. 1999; Malagonand Aguilera 2001). Interestingly, the reduction in bothspontaneous and DSB-induced inversions in the rad51mutant is about 5-fold (Rattray and Symington 1994;Aguilera 1995; Prado and Aguilera 1995; Bai andSymington 1996; Kang and Symington 2000; Malagonand Aguilera 2001; Gonzalez-Barrera et al., unpublisheddata). To explain these observations, it has beenhypothesized that there is a recombination processleading to inversions that can function in the absence ofstrand exchange (Fig. 5). A BIR event occurringbetween inverted repeats could lead to the duplication ofthe inverted repeats. As a consequence, internal directrepeats would be generated; and such direct repeatswould be processed via SSA. Depending on the directrepeat to be annealed, the recombination event will orwill not be associated with the inversion (Fig. 5; Bartschet al. 2000; Kang and Symington 2000; Malagon andAguilera 2001). Interestingly, analysis of HO-inducedDSB repair at inverted repeats in sae2 and rad50 mu-tants has provided some evidence for this mechanism ofinversions. In these mutants, 54% of the repair eventslead to the duplication of the inverted repeat that couldreflect a BIR event (Rattray et al. 2001). Indeed, similarduplications of the inverted repeats have been observedin the repair of HO-induced DSBs in which only one endis homologous to the donor (Colaiacovo et al. 1999).

As previously mentioned, both BIR and SSA requireRad59; and at least SSA requires Rad1. Accordingly,inversions are reduced 5- to 10-fold in rad59 mutantsand up to 1,000-fold in rad51 rad59 double-mutants (Baiand Symington 1996; Malagon and Aguilera 2001;Gonzalez-Barrera et al. 2002). Also, the frequency ofRad51-independent inversions is reduced 5-fold in rad51rad1 strains (Rattray and Symington 1995; Kangand Symington 2000; Gonzalez-Barrera et al. 2002).

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However, inversions are poorly affected in rad1 mutants(Aguilera and Klein 1989b; Rattray and Symington1995; Gonzalez-Barrera et al. 2002), suggesting thatBIR/SSA is not the major mechanism leading to inver-sions in wild-type cells (Kang and Symington 2000;Gonzalez-Barrera et al. 2002). Consistent with this idea,HO-induced gene conversions are associated withinversions in less than 5% in wild-type and rad1mutants, which is consistent with the SDSA mechanismof recombination (Kang and Symington 2000).

DSBR and BIR/SSA are not the only mechanismsleading to inversions. As mentioned for deletions, in-versions have also been proposed to occur by unequalsister gene conversion (Fig. 6B; Chen and Jinks-Robertson 1998). This is discussed in the next section.

Sister chromatid exchange

The study of SCE has been hampered by the impossi-bility of genetically detecting the recombination product.

Despite this, several results support the importance ofSCE in DSB repair. First, diploid G2 cells are moreresistant to gamma irradiation than G1 cells (Brunborgand Williamson 1978). Also, Rad52/Rad51 foci forma-tion after gamma irradiation in diploid cells is observedduring S and G2 but not in G1, unless irradiation dosesare increased 16-fold (Gasior et al. 2001; Lisby et al.2001). Further, post-replicative DSB repair requires thecohesin complex, which holds sister chromatids togetherduring G2 and S phases, and the proteins needed to loadcohesins onto chromosomes (Sjogren and Nasmyth2001).

Most of our knowledge on SCE comes from the studyof unequal SCE events between repeated sequences, ineither direct or inverted orientation (Fig. 6). In theseevents, repeats located in sister chromatids may even-tually be misaligned, leading to deletions or inversionsby HR. Different genetic approaches have allowed thedetermination of the levels of DSB-induced allelicrecombination and SCE in diploid cells (Paques andHaber 1999). Whereas X-ray-irradiated G1 cells are

Fig. 5 BIR followed by SSA asa mechanism responsible forinversions. A DSB at one of therepeats generates the substratefor a BIR event that duplicatesthe repeats. As a consequence,two direct repeats (A/B¢, A¢/B)are formed, which are processedvia SSA to produce either theinversion of the interveningsequence or the original orien-tation. Each line represents asingle-strand DNA molecule.The way that the BIR productis represented does not neces-sarily imply that BIR occurs bya conservative mechanism. Thisis yet unknown

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preferentially repaired by allelic recombination, SCE isthe prominent recombination event in cells irradiatedduring G2. These data suggest that sister chromatids arepreferred over homologous chromosomes as substratesfor recombination repair (Kadyk and Hartwell 1992).

SCE can occur by different mechanisms in yeast,which are dependent on Rad52 (Jackson and Fink 1981;Kadyk and Hartwell 1993), Rad50 (Bressan et al. 1999)and Rad54 (Dronkert et al. 2000). Firstly, SCE canoccur by SDSA or DSBR. Accordingly, DNA damage-induced unequal SCE requires Rad51 (Fasullo et al.2001) and leads mostly to gene conversion events(Kadyk and Hartwell 1993). Unequal SCE can lead todeletions between direct repeats either by gene conver-sion or crossover (Fig. 6A). Since deletions are stimu-lated by rad51 (see above), these events are not expectedto be the most abundant. In contrast, unequal SCE geneconversion could be an important source of inversions,as proposed to explain the role of mismatch repair geneson recombination between homeologous inverted se-quences (Chen and Jinks-Robertson 1998; Fig. 6B). Inaddition, SCE can occur by BIR. In misaligned directrepeats located in sister chromatids, unequal sisterchromatid BIR generates deletions (Kadyk and Hartwell1993; Fig. 6A). Indeed, spontaneous SCE is independentof Rad51 (Fasullo et al. 2001). Therefore, it seems thatthe recombination mechanism leading to SCE dependson the nature of the initiation event and presumably thephase of the cell cycle in which it is initiated.

Regardless of the mechanism used, SCE has beenproposed to control genome stability by reducing rear-rangements generated by other types of recombinationevents. Consistent with this, the decrease in DNAdamage-induced SCE observed in rad51 correlates withan increase in the frequency of non-reciprocal translo-cations (Fasullo et al. 2001). In this context, it may wellbe that SCE has become the putative mechanism of DSBrepair.

Elements influencing the choiceof recombination mechanism

A lesion in DNA can be repaired by different HRmechanisms, using a wide spectrum of homologoussequences. The choice of partner can determine both themechanism by which the lesion is repaired and the re-sulting recombination product. As we have seen, DSBR/SDSA and BIR can efficiently lead to viable recombi-nation products in most types of HR, whereas SSA isvalid only for deletions and BIR plus SSA for theformation of inversions and plasmid gap repair.

The genomic location of the substrates seems to playan important role in the choice of partner. Sister chro-matid sequences seem to be the preferred and to be moreefficient donors (Brunborg and Williamson 1978; Kadykand Hartwell 1992). Whereas homologous and heterol-ogous chromosomes most likely interact with the sameprobability in mitosis, as deduced from the same fre-quencies of allelic and ectopic recombination (Kupiecand Petes 1988; Lichten and Haber 1989), SCE might befavored by the proximity of the substrates (Kadyk andHartwell 1992).

The chromatin structure is likely a relevant element inthe choice of partner and the type of recombinationmechanism used in DSB repair. We have seen that thesame general mechanisms can operate in recombinationregardless of the location of the homologous sequences.However, the efficiency of inter- versus intramolecularrecombination might in part be determined by thechromatin structure of the partner. As mentioned above,intra- but not intermolecular events are efficient in rad51cells. Rad51-independent deletions between directrepeats can be explained by the high efficiency of SSA.However, inversions and plasmid gap repair can alsooccur by similar RAD51-independent mechanisms, thatis, BIR followed by SSA (Bartsch et al. 2000; Kang andSymington 2000; Malagon and Aguilera 2001). DSBrepair has been shown to be involved in chromatinmodifications that make DNA more accessible toDNaseI (Downs et al. 2000). In intramolecular recom-bination, a DSB at one of the repeats may lead to a moreaccessible chromatin structure or less topologicallyconstrained DNA structure at the adjacent repeat that

Fig. 6A,B Unequal sister chromatid exchange (USCE) as amechanism responsible for deletions and inversions. Deletionsand inversions can be formed via USCE between misaligned DNArepeats. A Deletions can be formed either by gene conversion orcrossover via DSBR or SDSA or by BIR. B Inversions can beformed by gene conversion via DSBR or SDSA. Each linerepresents a double-strand DNA molecule

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might facilitate strand invasion in the absence of Rad51.As mentioned earlier, Rad51 and Rad59 seem to play apivotal function in recombination. It is plausible thatthey help Rad52 to carry out the invasion step, probablystabilizing the DNA pairing. However, observationsthat mutations affecting chromatin structure, such asspt6 and spt12, increase recombination in the absence ofRad51 but not in the absence of Rad59 (Malagon andAguilera 2001) and that Rad51 is not required whenthe donor is actively transcribed (Sugawara et al. 1995)suggest an additional role for Rad51 in overcoming non-permissive chromatin structures in recombination.

The mechanism of recombination used for the repairof a DSB is affected by other elements besides the lo-cation of the partner. Thus, the choice of SDSA versusDSBR may be determined, at least in part, by the lengthof homology. Thus, while gene conversion can occurbetween sequences that share a short stretch of homol-ogy, crossovers require longer homologous partners(Klar and Strathern 1984; Ahn et al. 1988; Jinks-Robertson et al. 1993; Inbar et al. 2000; Sugawara et al.2000) or are frequently associated with long gene con-version tracts (Klein 1984; Ahn and Livingston 1986;Aguilera and Klein 1989b). In the case of DSB-inducedrecombination, physical detection of crossovers has beenshown to require a minimal homology length of about1.7 kb, whereas gene conversions are detectable withsequences as short as 250 bp (Inbar et al. 2000). This hasbeen interpreted in terms of stability of the heteroduplexintermediate (Jinks-Robertson et al. 1993; Inbar et al.2000; Sugawara et al. 2000) or as a minimal length re-quirement to allow either the HJ to be formed or thewhole recombination machinery to be loaded (Klar andStrathern 1984; Aguilera and Klein 1989b).

The choice of BIR as the DSB repair mechanismmay have a simple explanation, as we proposed recently(Aguilera 2001). BIR and DSBR/SDSA mechanisms ofDSB repair may occur as different processing alterna-tives of the same initial recombination event (Fig. 1).The main difference between BIR and SDSA/DSBR isthat newly replicated DNA is not captured by a secondend and may proceed for long regions (up to severalhundred kilobases). Although BIR can occur in rad51cells, it is likely that Rad51 facilitates DNA invasion inwild-type cells. BIR may be favored in one-ended inva-sion reactions, such as those predicted during the repairof replication-associated lesions (Fig. 2). Alternatively,in a two-ended invasion reaction, BIR might be impededif the polymerase initiating DNA synthesis at one 3¢invading end is blocked by the Rad51–DNA plectone-mic joints formed at the other 3¢ invading end. As theplectonemic joints would not form in rad51 cells, BIRcould efficiently occur in this mutant (Aguilera 2001).

Concluding remarks and future perspectives

It is becoming evident that mitotic recombination is aprocess intimately linked with replication. However, HR

is both a general mechanism of DSB repair in vegeta-tively growing cells and a putatively important source ofgenetic instability. Even though SCE may be a frequentmechanism of recombinational repair, most geneticallydetectable HR events occur either between homologousDNA sequences located at allelic positions in homol-ogous chromosomes or between DNA repeats locatedat ectopic positions in either the same, homologousor heterologous chromosomes. As we have explored,mitotic recombination may occur by multiple mecha-nisms. These include DSBR, SDSA, BIR and SSA. Theoccurrence of one recombination mechanism versusanother depends on different elements, including theposition of the homologous partner (whether in thesame or a different molecule), the initiation event(whether a replication fork blockage or an inducedDSB) and the length of homology of the recombinantmolecules.

The genetic and physical analysis of mitotic recom-bination in yeast, which takes advantage of the ease ofgenetic and molecular manipulation of its genome,provides an important amount of data for understand-ing the mechanisms of mitotic recombination and the invivo role of the recombinational repair genes. However,as we know more about the molecular mechanisms ofmitotic recombination, new questions arise. How doesrecombinogenic DNA damage other than DSBs, becomerepaired? How is a stalled replication fork processed intoa recombination event? Do chromatin and DNA struc-ture influence the mechanism of recombination? Howdoes recombination occur in DNA substrates simulta-neously undergoing transcription? What is the func-tional role of Rad52 as a central protein of mitoticrecombination? Does mitotic recombination involve theformation of HJs? The answers to these and futurequestions require further classic genetic and molecularanalyses. The use of new methodologies for DNA mi-croarrays, genome-wide location analysis or cell biologyin vivo and the reconstitution of minimal HR systems invitro should in the near future become valuable tools todecipher the mechanisms of HR.

Acknowledgements We thank D. Haun for style supervision. Thiswork has been funded by grants from the Ministry of Educationand Culture of Spain (BMC2000-0437), the Human Frontier Sci-ence Program (RG0075/1999-M) and Junta de Andalucıa (CVI-102). F.C.-L. and P.H. are recipients of pre-doctoral training grantsfrom the Ministry of Education and Culture and the Ministry ofScience and Technology, respectively.

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