REVIEW ARTICLE

Cell cycle regulation of homologous recombination in Saccharomyces cerevisiae David P. Mathiasen & Michael Lisby Department of Biology, University of Copenhagen, Copenhagen N, Denmark

Correspondence: Michael Lisby, Department of Biology, University of Copenhagen, Ole Maaloeesvej 5, DK-2200 Copenhagen N, Denmark. Tel.: +45 3532 2120; fax: +45 3532 2128; e-mail: [email protected] Received 5 July 2013; revised 20 January 2014; accepted 22 January 2014. Final version published online 24 February 2014. DOI: 10.1111/1574-6976.12066 Editor: Jure Piskur

MICROBIOLOGY REVIEWS

Keywords cell cycle; homologous recombination; Saccharomyces cerevisiae; DNA end resection; post-translational regulation; transcriptional regulation.

Abstract Homologous recombination (HR) contributes to maintaining genome integrity by facilitating error-free repair of DNA double-strand breaks (DSBs) primarily during the S and G2 phases of the mitotic cell cycle, while nonhomologous end joining (NHEJ) is the preferred pathway for DSB repair in G1 phase. The decision to repair a DSB by NHEJ or HR is made primarily at the level of DSB end resection, which is inhibited by the Ku complex in G1 and promoted by the Sae2 and Mre11 nucleases in S/G2. The cell cycle regulation of HR is accomplished both at the transcription level and at the protein level through post-translational modification, degradation and subcellular localization. Cyclin-dependent kinase Cdc28 plays an established key role in these events, while the role of transcriptional regulation and protein degradation are less well understood. Here, the cell cycle regulatory mechanisms for mitotic HR in Saccharomyces cerevisiae are reviewed, and evolutionarily conserved principles are highlighted.

Introduction Homologous recombination (HR) is defined as the exchange of genetic information between donor and recipient DNA molecules with similar or identical sequence. At least two criteria must be fulfilled in order for HR to take place. First, there must be an initiating event typically a DNA double-strand break (DSB) or a collapsed replication fork. Second, an intact homologous duplex must be available to serve as a sequence donor for the recombination event. The initiating event for HR is often accidental, but much of our understanding of the process comes from programmed events such as DSB-induced mating-type interconversion and meiotic recombination (Hunter, 2006; Haber, 2012). Accidental DSBs are rare – less than one per cell cycle in yeast – but they can arise as a consequence of environmental ionizing radiation or replication fork collapse. DSB repair can proceed via several distinct pathways of HR (Fig. 1, detailed below). Besides DSBs, potent inducers of HR in mitotically growing cells include alkylating agents such as methyl methanesulfonate (MMS), ultraviolet (UV) light and UV mimetics such as ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

4-nitroquinoline-1-oxide (4NQO), topoisomerase inhibitors such as camptothecin (Nitiss & Wang, 1988; Kaiser et al., 2011), inhibition of ribonucleotide reductase by hydroxyurea (Reichard, 1988; Galli & Schiestl, 1996), and DNA cross-linking agents such as cisplatin [reviewed in (Kupiec, 2000)]. Common for these agents is that their recombinogenic potential requires DNA replication indicating that blockage of replication fork progression may trigger HR, which allows for bypass of the lesion and therefore completion of genome duplication in the presence of DNA damage (Fig. 2). Although DNA replication stimulates HR (Kadyk & Hartwell, 1993; Cortes-Ledesma & Aguilera, 2006), recombination can also take place without passage through S phase (Aylon et al., 2004; Barlow & Rothstein, 2009). For repair of DSBs, nonhomologous end joining (NHEJ) serves as an alternative repair pathway in the G1 phase of the cell cycle (Karathanasis & Wilson, 2002). Availability of a homologous sequence for recombinational repair is mainly defined by ploidy and cell cycle phase. However, additional factors such as proximity of the donor and recipient sequences, chromatin structure, and nuclear compartmentalization also contribute to the availability of a sequence for HR. As a consequence of

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Fig. 1. Pathways of DNA DSBR. DNA DSBR can proceed by NHEJ, where the DNA ends are ligated after no or little end processing, or by HR. HR is initiated by resection in the 50 to 30 direction from the DSB. Following resection, DSBs that are flanked by repeat sequences (shown in orange) can be repaired by SSA, which is an error-prone process deleting the sequence between the repeats. The classical DSB repair pathway is initiated by invasion of the single-stranded 30 end into an intact homologous duplex (shown in red), leading to the formation of a D-loop. The invading 30 end primes DNA synthesis to restore the genetic information disrupted at the DSB. HR can now proceed via three alternative pathways: secondend capture resulting in the formation of a dHJ, SDSA or BIR in the case of a one-ended DSB. dHJs can be branch-migrated and resolved by dissolution to yield noncrossover products exclusively. In contrast, dHJ resolvases catalyze the formation of both crossover and noncrossover recombinants. In mitotic cells, SDSA causes the noncrossover products to prevail due to frequent displacement of the invading strand from the D-loop followed by its annealing to the other recessed end of the DSB and subsequent gap filling and ligation.

sister chromatid cohesion (Sjogren & Nasmyth, 2001), recombination between sister chromatids is believed to be 1–2 orders of magnitude more efficient than interhomologue or ectopic recombination (Fabre et al., 1984; Wu et al., 1997; Agmon et al., 2009; Jain et al., 2009), although direct evidence for this estimate remains to be established. Chromatin may serve as a barrier to recombi-

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nation, which is indicated by the requirement for chromatin-remodeling factors during HR (Chai et al., 2005; Shim et al., 2005; Tsukuda et al., 2005, 2009; Kent et al., 2007; Sinha & Peterson, 2009; Chen et al., 2012; Costelloe et al., 2012; Adkins et al., 2013). A special example is telomeres, which are essentially one-ended DSBs shielded against recombination by specialized telomere-binding ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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Cell cycle regulation in budding yeast

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which many recombination proteins are excluded to suppress rDNA recombination (Torres-Rosell et al., 2007); (2) the nuclear periphery, which is generally suppressive for HR except for the nuclear pore complexes, where gene conversion is stimulated at persisting DNA lesions (Nagai et al., 2008); and (3) the nucleoplasm, which is permissive for HR [reviewed in (Taddei & Gasser, 2012)]. In addition to the essential requirements for HR, the individual steps of the process are regulated to facilitate efficient recombination in S and G2 phase of the cell cycle and suppress recombination in the G1 phase. These mechanisms of regulation are the subject of this review.

5’

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Fig. 2. Homology-dependent DNA damage tolerance during replication. A replication fork stalled by a lesion (triangle) on the leading strand template can be rescued by template switching to an ectopic donor (left), the sister chromatid (middle), or through fork regression (right). After the initial template switch, DNA synthesis is re-primed from the 30 end that terminated at the DNA lesion. Next, the extended 30 end is thought to reanneal to the leading strand template by another switch or by fork reversal thereby effectively bypassing the lesion on this strand. The bypassed lesion remains in the DNA for excision repair at a later stage.

proteins [reviewed in (Wellinger & Zakian, 2012)]. Another example is the resistance of heterochromatin to the DNA damage-induced modification by phosphorylation of H2A(X) in both yeast and human cells (Kim et al., 2007), suggesting that DSB repair is suppressed in heterochromatin. The yeast nucleus can roughly be divided into three principal compartments, which behave differently with respect to HR: (1) the nucleolus from ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

Entry into and progression through the different phases of the cell cycle is tightly regulated by the cyclin-dependent protein kinases (CDKs). The genome of Saccharomyces cerevisiae encodes a total of five CDKs – Cdc28, Pho85, Kin28, Ssn3, and Ctk1 [reviewed in (Mendenhall & Hodge, 1998)]. The Cdc28 kinase is the master regulator of the cell cycle, while Pho85 is thought to play a minor role. The remaining 3 CDKs are mainly involved in transcription through the phosphorylation of RNA polymerase II. Regulation of the cell cycle occurs through tightly controlled fluctuations in Cdc28 activity. The level of Cdc28 protein is constant and in excess throughout the cell cycle. The catalytic activity of Cdc28 is regulated through its association with one of nine different cyclins, the G1 cyclins (Cln1-3) and the B-type cyclins (Clb1-6). Cyclins are unstable proteins whose protein and transcriptional levels vary periodically through the cell cycle. For example, the half-life of Cln1-3 is only 3–10 min (Barral et al., 1995). Importantly, conditional mutants of Cdc28 cause defects in mitotic chromosome stability and mitochondrial DNA transmission (Devin et al., 1990), failure to resect DSB ends and perform HR (Ira et al., 2004; Barlow et al., 2008; Huertas et al., 2008), radiation sensitivity (Koltovaya et al., 1998), and increased sensitivity to chronic DNA damage (Enserink et al., 2009). These data indicate a role for Cdc28 activity in cell cycle control of DNA repair. Transition from G1 to S phase, through the checkpoint called START, marks the beginning of the cell division cycle and is dependent on the activation of three classes of transcription factors; Mcm1, Swi6-Swi4 (SBF, SCBbinding factor)/Swi6-Mbp1 (MBF, MCB-binding factor), and Swi5 (Fig. 3). The first step toward progression past START is taken already in late telophase, where the transcription of genes essential for CDK regulation and transcriptional activation in G1 is induced. The first wave of transcriptional activation occurring at late telophase is FEMS Microbiol Rev 38 (2014) 172–184

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Transcription factor (DNA element) SBF (SCB) MBF (MCB) Swi5 (tGCTGGT) Mcm1 (ECB)

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POL3 PSY3 RAD5 RFA1 EXO1 ELG1 NSE4

SMC1 SMC6 TOP3 RFA2 SAE2 RAD51 RAD54 RDH54 PIF1 POL30

RFA3

Fig. 3. Transcriptional and proteolytic cell cycle regulation of HR. The transcriptional regulation of HR during the cell cycle in budding yeast. The inner circle illustrates the transcriptional levels of the cyclins Cln1-3 and Clb1-6 through the cell cycle and is adapted from (Richardson et al., 1992; Schwob & Nasmyth, 1993; Tyers et al., 1993; McInerny et al., 1997; Fitch et al., 2003). The cell-cycle-regulated genes involved in HR are derived from genome-wide microarray data set performed by Cho et al., 1998; Spellman et al., 1998; Pramila et al., 2002; and de Lichtenberg et al., 2003. Four transcription factors and corresponding upstream promoter elements are shown: SBF, MBF, Swi5, and Mcm1 (Spellman et al., 1998). A colored dot in front of each gene name indicates whether the gene expression is controlled by one of the four transcription factors. Protein degradation signals of the D-box, KEN-box or PEST region types are illustrated by the presence of a colored arrow following each gene. The annotation of degradation signals in known proteins related to HR was based on the consensus sequences: D-box, RXXLXXXXN; KEN-box, KENXXXN; and PEST region, rich in proline, glutamic acid, serine, and threonine (Jensen et al., 2006); (available at http://cyclebase.org).

orchestrated by the transcription factor Mcm1 at promoter elements called early cell cycle boxes, ECBs (McInerny et al., 1997). Mcm1 is normally inhibited at ECB sites due to its association with two repressors Yox1 and Yhp1, but at late telophase the bypass of C kinase, Bck2, outcompetes the two repressors for binding to Mcm1, leading to its activation (Bastajian et al., 2013). Mcm1Bck2 binds to ECB sites in the promoters of CLN3 and SWI4 and induces transcription that leads to the peak of CLN3 transcription observed at the M-G1 boundary with SWI4 transcription peaking shortly after (McInerny et al., 1997; MacKay et al., 2001; Mai et al., 2002). Cln3 is the first cyclin to be activated in the cell cycle. The catalytic activity of Cdc28-Cln3 is mainly directed toward activation of the SBF (Swi6-Swi4) and MBF (Swi6-Mbp1)-regulated genes through inhibition of the SBF/MBF repressor Whi5 (de Bruin et al., 2004; Ferrezuelo et al., 2009). Activation of SBF and MBF leads to the second wave of transcriptional activation that facilitates the progression into S phase (Koch et al., 1993, 1996). FEMS Microbiol Rev 38 (2014) 172–184

SBF and MBF activate the transcription of the cyclin genes CLN1,2 and CLB5,6. CLB5,6 are the only B-type cyclin genes to transcriptionally peak already in G1, but the gene products are inhibited by Sic1 until START. CLN1,2 transcription peaks at START to activate DNA replication through Cdc28-Cln-dependent proteolysis of the Cdc28Clb inhibitor Sic1 at START (Schwob et al., 1994). Clb5,6 are required for DNA replication. The remaining B-type cyclins function in S-M phase – induced in S phase and degraded in M phase due to mitotic D-boxes or the activity of protein kinase C, Pkc1. With the exception of Clb5, all cyclins contain a PEST sequence (Rechsteiner, 1990), which is a signal for rapid ubiquitin-mediated proteolysis via the Skp1/Cullin/F-box protein complex. The majority of cyclins also contain either a D- or KEN-box motif, which are recognized and leads to degradation via the anaphase promoting complex Cdc20-APC and Cdh1APC, respectively (Pfleger & Kirschner, 2000). Similar to the cyclins, many HR proteins also contain putative degradation signals (Fig. 3). ª 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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DSB repair pathways in S. cerevisiae DSBs are repaired by either NHEJ or HR (Fig. 1). The two pathways compete for DSB repair, and the choice of pathway is influenced by cell cycle phase and the nature and context of the DSB. Thus, NHEJ is the preferred pathway for DSBs with compatible ends in G1, whereas HR is the predominant pathway in S/G2 phase. NHEJ is generally considered to be an error-prone pathway because it rejoins ends from a DSB without the use of a repair template, potentially introducing deletions in the genome. NHEJ is, despite of this, essential for maintaining genome stability during G1 and consist of three phases: protection, bridging, and religation of the exposed DSB ends. Three core protein complexes, MRX (Mre11-Rad50-Xrs2), Ku (Yku70-Yku80), and DNA ligase IV (Dnl4-Lif1-Nej1), are required for DSB repair by NHEJ in S. cerevisiae. All three complexes physically interact at the DSB (Palmbos et al., 2005). The Ku complex binds DSB ends and protects against DNA end resection thereby suppressing HR (Zhang et al., 2007). Kumediated end protection is important for NHEJ as illustrated by a yku80 deletion causing a 50-fold reduction in total NHEJ and an increase in the efficiency of HR (Palmbos et al., 2005; Zhang et al., 2007). The MRX complex bridges DNA ends via the zinc-hook in Rad50 (de Jager et al., 2001; Hopfner et al., 2002; Lobachev et al., 2004). Dnl4 facilitates the last step of NHEJ through ligation of the two ends. For a detailed review of NHEJ, see Daley et al. (2005). At the DNA level, HR is initiated by the 50 to 30 resection of DSB ends, which commits repair to this pathway (Fig. 1). The single-stranded (ssDNA) tails are used to probe the genome for a homologous sequence in a process referred to as homology search. Recognition of homology is followed by strand invasion and formation of a displacement loop (D-loop). The invading 30 end serves to prime DNA synthesis using the intact duplex as a template. In the classical DSB repair model (Szostak et al., 1983), the other ssDNA end of the DSB is captured by the displaced strand of the D-loop, which also serves as a template for DNA synthesis. The 30 ends of the extended tails are ligated to the adjacent 50 ends to generate a double Holliday junction (dHJ), which can be resolved to produce either crossover or noncrossover recombination products. However, genetic data indicate that in mitotically growing cells, the D-loop is often disrupted before second-end capture by displacement of the extended invading strand, which can then anneal to the complementary strand of the other end of the DSB in a process referred to as synthesis-dependent strand annealing (SDSA). In SDSA, repair is completed by gap filling and ligation resulting in noncrossover products

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D.P. Mathiasen & M. Lisby

exclusively. In the special case of a one-ended DSB, which may arise during replication fork collapse, DNA synthesis within the D-loop may continue to the end of the chromosome in a process referred to as breakinduced replication (BIR). Another special case is the repair of a DSB flanked by repeat sequences, which can anneal upon resection of the DSB ends. The extruded 30 tails are removed by a flap endonuclease followed by gap filling and ligation in a process referred to as single-strand annealing (SSA). This repair pathway leads to deletion of one of the repeat sequences and the intervening DNA.

Cell cycle control of HR The cell cycle regulation of HR is accomplished at the transcription level and at the protein level through posttranslational modification, degradation, and subcellular localization. On top of the cell-cycle-regulated mechanisms, some genes and proteins are additionally regulated in response to DNA damage (Krejci et al., 2012; Tkach et al., 2012). The majority of genes involved in HR are transcribed throughout the cell cycle. A subset of genes, however, exhibits a cell-cycle-dependent pattern of expression. The collective output from several genomewide transcriptional data sets revealed 26 cell-cycle-regulated genes with a role closely linked to HR (Fig. 3) (Cho et al., 1998; Spellman et al., 1998; Pramila et al., 2002; de Lichtenberg et al., 2003). The NHEJ factor Yku70 transcript levels peak in the beginning of the G1 phase with lowest level of transcription during S phase, consistent with NHEJ being most active in G1. Presumably, to facilitate efficient HR during S and G2 phase, the transcription of 18 genes involved in HR is activated at the G1 to S boundary by the Cln/Clb controlled transcription factors SBF, MBF, and Swi5. In postreplicative cells, HR can be completely blocked by overexpression of the Clb-CDK inhibitor, Sic1 (Aylon et al., 2004). Sic1 is massively expressed from M phase but degraded through Cdc28-Cln-dependent proteolysis at START.

DNA end resection and pathway decision Within minutes following a DSB, the Ku heterodimer and the MRX complex are independently recruited to the DSB ends (Wu et al., 2008). The MRX complex is essential for keeping the broken DNA ends in close proximity (Lobachev et al., 2004), while the Ku complex inhibits DNA end resection to provide a window of opportunity for NHEJ (Mimitou & Symington, 2010).

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The control over 50 to 30 end resection of DSB ends, and hence over the choice between the HR and NHEJ pathways, is directed by the actions of Ku, MRX, and the endonuclease Sae2 (Lee et al., 1998; Barlow et al., 2008; Clerici et al., 2008). End resection is a two-step process where initial resection of 50–200 nt is catalyzed by the MRX complex and Sae2 (Mimitou & Symington, 2008; Zhu et al., 2008), and subsequent extensive resection of up to several kilobases is catalyzed by Exo1 or the Sgs1Top3-Rmi1 (STR) complex together with the Dna2 nucle(a) DSB end resection

ase (Fig. 4a) [reviewed in (Mimitou & Symington, 2011; Ferretti et al., 2013)]. Clb cyclins activated late in G1 and START exert strict control over DSB end resection, and strains lacking Clb-Cdc28 activity have been reported to fail to undergo end resection (Aylon et al., 2004). The capacity of the cell to perform resection is regulated by Clb/Cln-Cdc28 activity at multiple levels. First, the transcription of EXO1, SGS1, and SAE2 is cell cycle regulated. Cln3 regulates the expression of Exo1 through the transcription factors SBF/MBF, and the transcriptional levels (b) Strand invasion and HJ formation

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Fig. 4. Post-translational cell cycle regulation of HR. The schematic shows the repair of a DSB by the classical DSB repair model and its regulation by PTMs. The broken DNA is colored in blue and the intact donor duplex in red. MRX, Mre11-Rad50-Xrs2. STR, Sgs1-Top3-Rmi1. SHU, Shu1-Shu2-Csm2-Psy3. P, phosphorylation. A, acetylation. S, sumoylation. (a) DSB end resection. (b) Strand invasion and HJ formation. See text for details.

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of both Exo1 and Sae2 peak in late G1/START while Sgs1 peaks in early S phase. Second, a sae2D strain is defective in HR due to impaired DNA end resection, and Sae2 is a target of CDK-dependent phosphorylation in S/G2 (Huertas et al., 2008). In its inactive form, Sae2 forms large oligomeric structures, and upon CDK phosphorylation at Ser267, the oligomeric structure disrupts into monomeric and dimeric active forms (Fu et al., 2014). Mutation of the CDK-phosphorylated Ser267 residue in Sae2 causes hypersensitivity to CPT, impaired DNA end processing and faulty assembly of HR factors. Ku has low binding affinity toward ssDNA and likely dissociates from DSB ends upon Sae2 mediated resection (Foster et al., 2011). Third, extensive resection is promoted by CDK-dependent phosphorylation of Dna2, which facilitates its relocalization from the cytoplasm to the nucleus in S/G2 (Kosugi et al., 2009; Chen et al., 2011). Both Sae2 and Exo1 are negatively regulated by acetylation-mediated degradation (Robert et al., 2011). In human cells, CDK promotes DSB end resection through phosphorylation of NBS1 and CtIP (Huertas & Jackson, 2009; Falck et al., 2012), although the phosphorylation of NBS1 appears to be important for resection only in some cell lines (Wohlbold et al., 2012). Ku mainly inhibits initiation of DNA end resection in G1 (Barlow et al., 2008; Zierhut & Diffley, 2008). Yku70 transcript levels fluctuate during the cell cycle, peaking in the beginning of the G1 phase with the lowest expression during S phase, consistent with NHEJ being most active in G1. Ku association to DSB ends is inhibited in S and G2 (Clerici et al., 2008). However, removal of all the putative Cdc28 (CDK) phosphorylation sites (S/T-P) on Yku70 and 3 of 4 sites on Yku80 did not affect DSB repair, suggesting that the regulatory role of CDK on Ku is indirect (Zhang et al., 2009). MRX elicits control over Ku, being able to promote both its stabilization and dissociation from DSB ends (Zhang et al., 2007). The dissociation of Ku from unrepaired DSBs depended on the intact MRX complex, indicating that MRX can terminate an NHEJ repair phase (Wu et al., 2008). Ku is capable of preventing extensive resection at DSB ends in the absence of functional MRX and Sae2, whereas the requirement of the MRX complex to initiate HR is bypassed when Ku is absent and resection is instead executed by the exonuclease Exo1 (Mimitou & Symington, 2010). This proposes MRX as a cell-cycle-regulated switch between NHEJ in G1 and HR in S/G2. The precise mechanism of how this switch is cell cycle regulated is unclear. The Sae2 endonuclease, however, emerges as a likely candidate, as both Sae2 transcription and activity are strongly regulated throughout the cell cycle and Sae2 has been shown to control the initiation of end resection (Clerici et al., 2005).

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D.P. Mathiasen & M. Lisby

Strand invasion The single-strand tails generated during resection are bound by replication protein A (RPA) to protect the ends from degradation and formation of secondary structures (Alani et al., 1992; Sugiyama et al., 1997, 1998). However, RPA also acts as a barrier for loading the Rad51 recombinase onto single-stranded DNA (Sugiyama et al., 1997). Rad52 mediates the displacement of RPA from singlestranded DNA to facilitate the formation of a Rad51 nucleoprotein filament (Sung, 1997a, b; Shinohara & Ogawa, 1998; Krejci et al., 2002; Sugiyama & Kowalczykowski, 2002). The mediator function of Rad52 is further aided by the Rad51 paralogues Rad55-Rad57 (Sung, 1997b), which stabilize the Rad51 filament (Hays et al., 1995; Johnson & Symington, 1995; Fortin & Symington, 2002). Rad51 filaments are also protected by the SHU complex (Shu1-Shu2Csm2-Psy3) against the Srs2 helicase (Bernstein et al., 2011; Godin et al., 2013), which acts as an antirecombinase to disassemble Rad51 filaments. RPA is recruited to DSBs at all phases of the cell cycle although less efficiently in G1 phase reflecting the reduced resection activity in G1 (Barlow et al., 2008). Rad52 is recruited by RPA to DNA damage-induced foci preferentially during S and G2 phase except if cells are exposed to pathologically high doses of ionizing radiation (> 400 Gy equivalent to 20 DSBs per haploid cell) (Lisby et al., 2001). The cell-cycle-dependent recruitment of Rad52 to RPA foci is not understood, but it may be related to the phosphorylation of Rad52 during S/G2 or to a cell-cycle-dependent modification of RPA that controls its recruitment of Rad52 (Ant unez de Mayolo et al., 2006). Alternatively, the DNA damage dosedependent rescue of Rad52 focus formation could be interpreted to suggest that a G1-specific inhibitor of Rad52 exists but is depleted at excessive loads of DNA damage. The Rad51 nucleoprotein filament probes the genome for an intact homologous duplex until synapsis is accomplished (Fig. 4b). For interhomologue or ectopic recombination, both cytological and molecular assays indicate that homology search takes 20–60 min (Aylon et al., 2003; Bzymek et al., 2010; Mine-Hattab & Rothstein, 2012), but synapsis is likely to be significantly faster during sister chromatid recombination due to the proximity of the two DNA molecules imposed by cohesion. After synapsis is achieved, Rad51 catalyzes the formation of a D-loop through invasion of the single-stranded tail into and base pairing with one strand of an intact duplex. The D-loop is stabilized by binding of RPA to the displaced strand (Alani et al., 1992; Sugiyama et al., 1997; Eggler et al., 2002). Next, the invading strand primes DNA synthesis in a process that requires PCNA, Dpb11 and either Pold or Pole (Wang et al., 2004; Germann et al., 2011;

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Hicks et al., 2011). After extension of the 30 invading strand, HR can proceed by four alternative pathways: (1) the classical DSB repair (DSBR) pathway; (2) SDSA; (3) SSA; or (4) BIR. The classical DSBR pathway is suppressed in mitotic cells by the Mph1 helicase, which dissociates D-loops to facilitate annealing of the extended 30 tail to the other end of the DSB, leading to SDSA (Prakash et al., 2009). Gap-filling DNA synthesis and ligation subsequently completes repair of the DSB. DNA synthesis within the D-loop is promoted by Rad54 through displacement of Rad51 from the invading 3’-OH end (Solinger & Heyer, 2001; Sugawara et al., 2003; Li & Heyer, 2009). However, the cell cycle transcriptional regulation of RAD54 is apparently not crucial for HR, because disruption of the transcription factors Swi4 and Mbp1, which are believed to be responsible for the regulation, does not cause increased sensitivity to MMS (Johnston & Johnson, 1995). In contrast to SDSA, during DSBR, Rad52 and Rad59 promote capture of the second DSB end by the D-loop, and a dHJ is formed (Sugawara & Haber, 1992; Mortensen et al., 1996; Sugiyama et al., 1998; Petukhova et al., 1999; Wu et al., 2006; Lao et al., 2008). Second-end capture is favored in meiosis, but is suppressed in mitotic cells by the Srs2 helicase, which displaces Rad51 from single-stranded DNA, which is likely to prevent second-end capture. The antirecombinase activity of Srs2 is promoted by its CDK-dependent phosphorylation, which counteracts its inhibition by sumoylation (Saponaro et al., 2010).

dHJ dissolution/resolution The classical DSBR pathway leads to a dHJ that must be dissolved or resolved before the interlinked DNA molecules segregate during mitosis. Some dHJs are branchmigrated and dissolved by the Sgs1-Top3-Rmi1 (STR) complex in a process referred to as dissolution (Wu & Hickson, 2003; Cejka et al., 2010), which leads exclusively to noncrossover products. Any remaining dHJs in mitosis are targeted for resolution by the Mms4-Mus81 resolvase, which is activated by CDK and Cdc5 phosphorylation in M phase (Loog & Morgan, 2005; Matos et al., 2011). Resolution by Mms4-Mus81 produces both crossover and noncrossover products. The synthetic lethality between Sgs1 and Mus81 indicates that these two complexes are responsible for removal of the majority of dHJs (Mullen et al., 2001). However, another HJ resolvase, Yen1, is activated by dephosphorylation upon entry into anaphase and is thought to eliminate any remaining dHJs prior to nuclear division (Loog & Morgan, 2005; Matos et al., 2011). The human HJ resolvases MUS81-EME1 and GEN1 are regulated similarly by phosphorylation (Matos et al., 2011). FEMS Microbiol Rev 38 (2014) 172–184

Perspectives There is ample evidence for several roles of post-translational modifications in the cell cycle regulation of HR, but so far a functional role of the transcriptional upregulation of HR genes at the G1-S transition remains to be established. It is possible that phenotypes associated with the deregulation of the transcriptional program for HR genes will be subtle or that phenotypes can only be observed if multiple genes are affected as suggested by the MMS sensitivity of a swi6Δ mutant (Johnston & Johnson, 1995). Another open question is the purpose of residual resection in G1 phase of the cell cycle (Barlow et al., 2008; Clerici et al., 2008; Balogun et al., 2013). A minimal resection in G1 phase could be important for alternative end-joining pathways and for activation of the G1 checkpoint (Matsuzaki et al., 2012; Balogun et al., 2013). Finally, the functional importance of degradation motifs found in many HR proteins is indicative of a downregulation of HR during mitosis. The biological significance of these motifs remains to be studied, but their function may be related to recent studies from both human and yeast cells, indicating that sister chromatids are often linked by ultrafine DNA bridges upon entry into anaphase (Chan et al., 2007; Sofueva et al., 2011; Germann et al., 2014). In the latter study, it was shown that ultrafine DNA bridges form independently of HR (Germann et al., 2014), but it remains to be established if these DNA bridges are potential substrates for HR or other repair pathways. This study also showed that chromatinized anaphase bridges are less frequent and formed primarily through HR and accumulate in a dissolutiondefective sgs1Δ mutant, suggesting that they may represent chromatids connected by dHJs.

Acknowledgements We thank members of the Lisby and Holmberg laboratories for helpful discussions on this work. This work was supported by The Danish Agency for Science, Technology and Innovation, the Villum Kann Rasmussen Foundation, the Lundbeck Foundation and the European Research Council (ERC) under the European Union’s Seventh Framework Programme (FP7/2007-2013)/ERC Grant Agreement No. 242905 to M.L., and the ERC to D.P.M. The authors declare no conflict of interest.

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