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Biochimica et Biophysica Acta xxx (2011) xxx–xxx

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Biochimica et Biophysica Acta

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Review

Functions of the Snf2/Swi2 family Rad54 motor protein inhomologous recombination☆

Shannon J. Ceballos a, Wolf-Dietrich Heyer a,b,⁎a Department of Microbiology, University of California, Davis, Davis, CA 95616-8665, USAb Department of Molecular and Cellular Biology, University of California, Davis, Davis, CA 95616-8665, USA

☆ This article is part of a Special Issue entitled: Snfunction.⁎ Corresponding author at: Department of Molecular a

of California, Davis, Davis, CA 95616-8665, USA. Tel.: +13011.

E-mail address: [email protected] (W.-D. Heyer

1874-9399/$ – see front matter © 2011 Elsevier B.V. Aldoi:10.1016/j.bbagrm.2011.06.006

Please cite this article as: S.J. Ceballos, W.-DBiochim. Biophys. Acta (2011), doi:10.1016

a b s t r a c t
a r t i c l e i n f o
Article history:Received 26 April 2011Received in revised form 27 May 2011Accepted 6 June 2011Available online xxxx

Keywords:DNA repairDNA replicationGenome stabilityHomologous recombinationMeiosisMotor protein

Homologous recombination is a central pathway to maintain genomic stability and is involved in the repair ofDNA damage and replication fork support, as well as accurate chromosome segregation during meiosis. Rad54is a dsDNA-dependent ATPase of the Snf2/Swi2 family of SF2 helicases, although Rad54 lacks classical helicaseactivity and cannot carry out the strand displacement reactions typical for DNA helicases. Rad54 is a potentand processive motor protein that translocates on dsDNA, potentially executing several functions inrecombinational DNA repair. Rad54 acts in concert with Rad51, the central protein of recombination thatperforms the key reactions of homology search and DNA strand invasion. Here, we will review the role of theRad54 protein in homologous recombination with an emphasis on mechanistic studies with the yeast andhuman enzymes. We will discuss how these results relate to in vivo functions of Rad54 during homologousrecombination in somatic cells and during meiosis. This article is part of a Special Issue entitled: Snf2/Swi2ATPase structure and function.

f2/Swi2 ATPase structure and

nd Cellular Biology, University530 752 3001; fax:+1 530 752

).

l rights reserved.

. Heyer, Functions of the Snf2/Swi2 family R/j.bbagrm.2011.06.006

© 2011 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 02. Homologous recombination — a mechanistic overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 03. Genetic analysis of RAD54 in S. cerevisiae and vertebrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 04. Biochemical and cytological analysis of Rad54 protein in S. cerevisiae and vertebrates . . . . . . . . . . . . . . . . . . . . . . . . . . . . 05. Rad54 paralogs in S. cerevisiae and humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 06. Functions of Rad54 protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

6.1. Chromatin remodeling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 06.2. Rad51-ssDNA filament stability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 06.3. Homology search and DNA strand invasion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 06.4. D-loop dissolution and branch migration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 06.5. Dissociation of Rad51 from heteroduplex DNA to allow extension of the invading 3′-OH end by DNA polymerase . . . . . . . . . . . 06.6. Stimulation of Mus81–Mms4/EME1 nuclease activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 06.7. Turnover of Rad51-dsDNA dead-end complexes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 06.8. Regulation of Rad54 function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

7. Functions of the Rad54 paralogs, ScRdh54 and hRAD54B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 08. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 0

1. Introduction

Homologous recombination (HR) is a complex mechanism tomaintain genomic stability. This process uses template-dependentDNA synthesis to repair or tolerate DNA damage, such as DNA double-stranded breaks (DSB), single-stranded DNA gaps, and interstrand

ad54 motor protein in homologous recombination,

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crosslinks [1]. HR is intimately linked to DNA replication, as initiallyelaborated in phage T4 [2], and the function of HR in the recovery ofstalled and collapsed replication forks is critical to faithfully duplicateand segregate the genome to daughter cells [1–4]. Moreover, HR isessential for accurate chromosome segregation during the firstmeiotic division, also generating genetic diversity among the meioticproducts in the process [5].

RAD54 is a member of the RAD52 epistasis group and a corecomponent of the recombination machinery found in all eukaryotesand in some archaea, but not in bacteria [6–8]. The Rad54 proteinbelongs to a group of the Snf2/Swi2 family of SF2 helicases that share aprominent motor domain which powers ATP-dependent tracking ondsDNA but does not lead to strand separation like classical DNAhelicases [9]. The Snf2/Swi2 family of proteins is represented by well-known ATP-dependent chromatin remodelers, such as Snf2/Swi2 orISWI, but also other proteins that remodel non-chromatin protein–dsDNA complexes such as Mot1, which displaces the TATA-bindingprotein from DNA [10,11] (see other reviews in this issue of BBA). Thebudding yeast Saccharomyces cerevisiae contains 17 Snf2 familyproteins, of which 10 function in different aspects of the DNA damageresponse, including Rad54, Rdh54, Uls1, Rad5, Rad16, Rad26, Ino80,Snf2, Sth1, and Swr1 [7,12].

This review will focus on yeast and human Rad54 proteins, theirparalogs, and their function in HR. We will also discuss the yeast Uls1protein, which appears to have a partially overlapping function withRad54 andRdh54. Excellent reviews are availablewith comprehensivediscussions of the HR pathway [13–16], the Rad52 group proteins

dHJ

(1)

(2)

(3)

(4)

Crossover

dHJ

D-loop

Pre-synapsis

Synapsis

(5)

(6)

(7)

(8a)

Rad50-MDna2, Sg

Rad51, RRad55-RRad54, R

Rad51, R

Rad51, R

Sgs1-Top[BLM-TOYen1 [GE

Post-synapsisPolδ, RFC

Mus81-M

Fig. 1. Double-strand break repair by homologous recombination. Homologous recombinatiopost-synapsis (5–11). The Saccharomyces cerevisiae proteins identified to function at the indlisted in blue in square brackets and human proteins not found in yeast are listed in blue witproduct of DNA strand invasion by the Rad51-ssDNA filament. The dHJ (double Holliday juHolliday junction intermediate (dHJ), which can be resolved into crossover and non-crossovestrand after DNA synthesis on the target duplex to anneal the newly synthesized strand wconversion without crossover. BIR (break-induced replication; step 11) was proposed to assDSB site leading to long gene conversion events.

Please cite this article as: S.J. Ceballos, W.-D. Heyer, Functions of the SnBiochim. Biophys. Acta (2011), doi:10.1016/j.bbagrm.2011.06.006

[17,18], meiotic recombination [5,19], and the Snf2/Swi2 familychromatin remodelers [12,20–22]. We will emphasize the significantnovel developments since our last review on Rad54 in 2006 [7] andrefer the reader also to other reviews dedicated to the Rad54 protein[6,8]. To distinguish between the yeast and human proteins, we willadhere to the following nomenclature: S. cerevisiae Rad54 protein,ScRad54; human RAD54 protein, hRAD54; S. cerevisiae Rdh54 protein(also knownas Tid1), ScRdh54; and humanRAD54B protein hRAD54B.We will use the generic term Rad54 when we refer to both yeast andhuman Rad54.

2. Homologous recombination — a mechanistic overview

HR can be conceptually divided into three mechanistic stages: pre-synapsis, synapsis, and post-synapsis (Fig. 1) [13–18]. During pre-synapsis, the DNA damage is processed to generate ssDNA, onto whichRad51 can assemble the pre-synaptic filament. The Rad51-ssDNAfilament performs homology search and DNA strand invasion, the keysteps during HR that define synapsis. During post-synapsis, HR iscompleted by several pathways that entail DNA synthesis and differentmodes to resolve recombination-mediated junction intermediates. Thedouble Holliday Junction pathway (dHJ in Fig. 1) is of particular interest,because it can lead to crossovers, which are essential for meioticrecombination [5,19]. In somatic (vegetative) cells, the predominantrecombinational repair pathway appears to be Synthesis-DependentStrand Annealing (SDSA in Fig. 1), as it inherently avoids crossover andthus the possibility of deleterious genome rearrangements. Lastly,

SDSA

(9)

(10)

Non-crossover

Non-crossover

(8b)

re11-Xrs2 [NBS1], Sae2 [CtIP], Exo1, s1-Top3-Rmi1 [BLM-TOPOIIIα-RMI1/2], RPA

ad52,ad57, RAD51B/C/D, XRCC2/3, BRCA2-DSS1AD54B

ad54, RAD54B, RAD51AP1

ad54

3-Rmi1,POIIIα-RMI1/2]N1]

BIR(11)-PCNA

ms4 [EME1]

n can be conceptually divided into three stages: pre-synapsis (1–2), synapsis (3–4), andividual stages are listed in black, the human proteins with alternate nomenclature arehout brackets. Three different pathways emanate from the D-loop intermediate (4), thenction; steps 5–8) pathway engages both ends of the DSB to ultimately form a doubler products. SDSA (synthesis-dependent strand annealing; step 10) retracts the invadingith the single strand resulting from resection of the second end, leading to localizedemble a replication fork at the D-loop to copy the entire chromosome arm distal to the

f2/Swi2 family Rad54 motor protein in homologous recombination,


3S.J. Ceballos, W.-D. Heyer / Biochimica et Biophysica Acta xxx (2011) xxx–xxx

Break-Induced Replication (BIR in Fig. 1) appears restricted toconditions of one-sided DSBs, where the second DSB end is missing,for example after breakage of the replication fork.

Recent genetic and biochemical work defined two independent DSBresection pathways in budding yeast controlled by Exo1 and Sgs1-Top3-Rmi1withDna2, respectively,which function after the initial processingby Rad50-Mre11-Xrs2 in conjunction with Sae2 (for review [23]).Similar pathways exist in humans (see Fig. 1). The resulting 3′overhanging ssDNA is bound by the ssDNA binding protein, RPA,which removes potential secondary structure in the ssDNA. Since RPAhas exceptionally high affinity to ssDNA, it interferes with binding ofRad51. This impediment is overcome by so-called mediator proteins,Rad52 and Rad55–Rad57 in yeast, which allow the formation of Rad51-ssDNA filaments on RPA coated ssDNA. In humans, the breast andovarian cancer tumor suppressor BRCA2 and its associated factor DSS1performmediator function, and the RAD51 paralogs, RAD51B, RAD51C,RAD51D, XRCC2, and XRCC3 have been implicated as well [24–26]. TheRad51 filament performs homology search and DNA strand invasion.Extension of the invading 3-OH endmarks the onset of post-synapsis. Inyeast, the primary DNA polymerase for D-loop extension appears to beDNA polymerase delta with its cofactors PCNA/RFC [27]. In vertebrates,genetic and biochemical data suggest an involvement of the translesionpolymerase Pol eta [28,29].

In the dHJ pathway, the displaced strand of theD-loop anneals to thesecond DSB end through the action of Rad52 protein [30], to form ajunction intermediate that has the potential to mature into a dHJ uponligation of all strand interruptions. Such junction intermediates,including also nicked and partial dHJs, can be cleaved to crossover ornon-crossover products by specific endonucleases, such as Yen1(human GEN1) or Mus81–Mms4 (human MUS81–EME1) [31,32].Alternatively, the two junctions of a dHJ can be migrated towardseach other to result in a hemi-catenane that can be dissolved into non-crossover products by the combined action of Sgs1 helicase with thetype IA topoisomerase Topo3 and the specificity factor Rmi1 (humanBLM-TOPOIIIα-RMI1/RMI2 [33]). In the second post-synaptic pathway,the DNA synthesis-extended strand of the D-loop can be extricated toanneal with the second DSB end, in a process called Synthesis-Dependent Strand Annealing (Fig. 1). Lastly, in the absence of a secondDSB end, Break Induced Replication establishes a replication fork at theD-loop to initiate long-range DNA synthesis to copy the entire distalchromosome arm resulting in loss-of-heterozygosity [34]. In thisreview, wewill use thismechanistic framework to discuss the functionsof Rad54 and its paralogs during HR.

3. Genetic analysis of RAD54 in S. cerevisiae and vertebrates

In eukaryotes, RAD54 is a highly conserved gene. There is only asingle Snf2/Swi2-like protein in bacteria, RapA (HepA). However,RapA does not directly function in DNA repair but rather intranscription to release the nascent transcript from transcriptioncomplexes [35,36]. A Rad54 homolog has been identified in theArchaeon Sulfolobus solfataricus, but Rad54 is absent in many othergroups of Archaea [37,38]. Biochemical analysis of SsoRad54 suggeststhat the archaeal protein has a comparable function to its eukaryotichomolog in stimulating the recombination activity of the cognateRad51 homolog [37].

ScRAD54was first discovered in screens isolatingmutants that weresensitive to ionizing radiation (IR) [39–41]. In S. cerevisiae, rad54 is oneof the most IR-sensitive single mutants along with rad51 and rad52. Asingle DSB induces significant lethality in rad54 cells [42]. Consideringthe relatively normal growth of rad54 (and rad51 and rad52) mutantscompared towild-type cells, the data suggest that spontaneousDSBs arerelatively rare in yeast [43]. Similar to other mutants in the RAD52epistasis group, rad54mutants are hypersensitive not only to IR but alsoto crosslinking (e.g., mitomycin C, cis-platinum) or alkylating agents(e.g., methyl methanesulfonate), and a multitude of other agents that


are capable of causing DSBs [17,18]. Rad54-deficient S. cerevisiae cellsare defective in intrachromosomal gene conversion as well asinterchromosomal recombination [44]. In hetero-allele assays (HRbetween different alleles of one gene residing on two homologs indiploids cells) rad54 mutants displayed a similar defect as rad52mutants [45]. The effect of the rad54mutationonmeiotic recombinationis much milder than on mitotic recombination, which is likely due topartial redundancy in function between ScRad54 and ScRdh54 (seebelow) [44,46]. The analysis of the Walker A box mutations (Rad54-K341R/A) established that ATP hydrolysis is essential for proteinfunction in yeast and mice, as these catalytically defective mutantsdisplayed phenotypes in DNA repair assays that were quantitativelyidentical to null mutations [47–49]. In general, the defects of ScRad54-deficient yeast cells in DNA repair are highly similar in extent to thedefects in Rad51-deficient cells in yeast,whereas themeiotic phenotypeof ScRad54-deficient yeast cells is much less pronounced than that ofRad51-deficient cells [5,7,17,50].

Of particular importance regarding the in vivo function of ScRad54,are results from the analyses of double mutants, specifically thesynthetic lethality of the srs2 rad54 double mutant. Srs2 is an anti-recombinase that dissociates Rad51 from ssDNA, antagonizing theassembly of the presynaptic filament [51,52]. Importantly, theobserved synthetic lethality in srs2 rad54 cells is suppressed bymutations in the RAD51, RAD52, RAD55, and RAD57 genes [53–55].This implies that Rad51 filaments assembled in the srs2 rad54 doublemutants lead to recombination-dependent toxic intermediates thatcause the lethality. This strongly suggests that the critical function ofScRad54 is after the assembly of Rad51-ssDNA filaments.

Genetic analysis of RAD54 in mouse and chicken DT40 cellsestablished the importance of RAD54 and its essential ATPase activityfor recombination in vertebrates [49,56,57]. The function of hRAD54 inhuman cells is inferred from these results. The hRAD54 cDNA partiallysuppressed the MMS-sensitivity of rad54Δ yeast cells, suggestingremarkable conservation in protein function [58]. Mouse RAD54knockout embryonic stem (ES) cells display a reduction inHR(measuredas gene conversion and sister chromatid exchange), sensitivity to IR andinterstrand crosslinking agents, and defects in recombination-mediatedgene targeting [56,59]. At the organismal level, however, RAD54 is notessential for embryonic development and Rad54-deficient mice do notdisplay IR-sensitivity, but are hypersensitive to the interstrand cross-linking agent mitomycin C [56]. Double mutants of mouse RAD54 withdefects in nonhomologous endjoining (NHEJ), an alternative pathway ofDSB repair, causedbydeletionofDNAdependentproteinkinase (SCID) orDNA ligase 4 (LIG4), exhibit synergistic sensitivities to IR and interstrandcrosslinking agents, showing competition of HR and NHEJ for DSB repairin vertebrates [60,61]. RAD54-deficient mice do not display an obviousmeiotic defect [56]. It is interesting to note that inmice, unlikemutationsin RAD54, disruption of the RAD51 gene causes early embryonic lethality[62,63]. This represents an interesting contrast to budding yeast, whererad51 and rad54 mutants are both viable with nearly identicalphenotypes in terms of DNA damage sensitivities or mitotic recombina-tion defects. This difference might be related to functional differencesbetween the yeast and human Rad51 and Rad54 proteins, or possiblyindicates the existence of an additional proteinwith a function similar toRAD54 in vertebrates. As the RAD54RAD54B doublemutantmouse is alsoviable [64], hRAD54B is not that candidate, but yet another third genewould have to be postulated. The genetic data, largely obtained in yeastand mouse, are critical to guide the interpretation of the biochemicalresults discussed below.

4. Biochemical and cytological analysis of Rad54 protein inS. cerevisiae and vertebrates

ScRad54 and hRAD54 are highly conserved proteins with 68%similarity and 48% identity [58,65]. Sequence analysis identifiedRad54 as amember of the SF2 superfamily of helicases with the classic



Table 1Rad54 and its paralogs in budding yeast and humans.

Protein Organism Mr, length ATPase min−1 Protein interactions

ScRad54 Saccharomycescerevisiae

101,753 Da898 aa

1,200a

[75,114]Mus81–Mms4 [145]Rad51 [76,78]Histone H3 [106]Mek1 [161]

hRAD54 Homo sapiens 84,352 Da747 aa

800a

[67,125]RAD51 [77]MUS81-EME1 [147]

ScRdh54 Saccharomycescerevisiae

107,946 Da958 aa

2200[165]

Dmc1 [88]Rad51 [165]

hRAD54B Homo sapiens 102,836 Da910 aa

69[99]

DMC1 [169,170]RAD51 [95,99]

a The ATPase of ScRad54 and hRAD54 is given for linear dsDNA, the activity isenhanced on junction DNA substrates or substrates partially covered with the cognateRad51 protein.


seven “helicase” motifs: I, Ia, II, III, IV, V, and VI that define the ATP-dependent motor domain essential for translocation on DNA (Fig. 2)[9,66]. More precisely, Rad54 falls in the Swi2/Snf2 group of ATP-dependent motor proteins that track along dsDNA but are unable tocatalyze the strand displacement reactions typical for a DNA helicase[12]. The ATPase activity requires dsDNA and is not supported byssDNA [67] (Table 1), whereas classical DNA helicases translocate onssDNA and ssDNA stimulates their ATPase activity [66]. dsDNAtracking by Rad54 is associated with topological changes in the DNA(positive and negative supercoils in topologically constrained DNAmolecules) that were documented biochemically and through the useof atomic force microscopy [68–72]. Translocation on dsDNA wasdirectly visualized in single-molecule experiments with ScRad54,measuring a rate of ~300 bp/s with an average travel distance of11.5 kbp [73]. This high level of processivity suggests that Rad54forms a multimeric assembly on the DNA, possibly a ring encirclingthe DNA. Indeed, there is biochemical and electron microscopicevidence for Rad54 multimerization on DNA [47,68,74], but the exactarchitecture of this assembly remains to be determined. Thefundamental biochemical activity of Rad54 is its dsDNA-dependentATPase which powers translocation on duplex DNA, and the ATPaseactivity is significantly stimulated by the cognate Rad51-DNAcomplex [69,70,75]. As discussed below, this biochemical findingstrongly supports that the physical interaction between Rad54 andRad51 [76–78] is of biological significance and that both proteinscooperate during HR.

Two X-ray crystallographic structures for Rad54 proteins areavailable: the core (motor) domain of the zebra fish protein [79](Fig. 2) and of the Sulfolobus enzyme, which has also been crystallizedwith duplex DNA [38]. Both proteins fold into two lobes (Fig. 2), eachconsisting of a RecA-like domain found in helicases [66] with cleartopological and structural similarity to other SF2 helicases [80]. Thetwo lobes contain specific insertions, only found in the Snf2/Swi2group. The overall structural homology to other SF2 helicasessupports an inchworm mechanism of translocation, tracking along

A

B

Fig. 2. Structure and architecture of Rad54 proteins and Rad54 paralogs. (A) Schematic aligmotor domains are outlined in green (DEXDc) and red (HELICc) [172], the 7 conserved ATPasRad54 protein, an N-terminal truncation representing amino acids 91–738, is shown with t


the minor groove of the DNA [81]. The relative orientation of the twolobes in the two crystal structures of the zebra fish and Sulfolobusenzymes is significantly different [38,79]. The lobe orientation of theDNA-bound form of the Sulfolobus Rad54 protein was very similar tothe free form of the protein [38]. The significance of this difference ispresently unclear, but may be related to the mechano-chemical cycleof the enzyme [82]. The specificity of Rad54 function is likelymediated by its protein interactions, in particular with Rad51. It willbe of interest to obtain structural information for the Rad51interaction domain of Rad54, which maps to the N-terminus ofRad54 protein [76,83]. Unfortunately, this region is lacking in thepresent structures.

In vivo cytological analysis provides significant insights into thebiological functions of Rad54. A comprehensive analysis of HRproteins in mitotically growing yeast cells after IR-induced DNAdamage revealed that Rad54 forms DNA damage-induced foci,dependent on the presence of Rad51 protein [84]. Conversely,formation of DNA damage-induced Rad51 foci was independent of

nment of Rad54 proteins and Rad54 paralogs from Homo sapiens and S. cerevisiae. Theemotifs are highlighted in yellow. (B) X-ray crystal structure of the zebra fishDanio reriohe DEXDc and HELICc domains colored as in (A) (PDB code: 1Z3I) [79].




Rad54 [84], suggesting that Rad54 acts downstream of the formationof the Rad51 presynaptic filament. Analogous experiments in micediscovered that DNA damage-induced RAD51 foci are not as robust inRAD54-deficient cells compared to wild type [64,71]. Further workwith chromosomally integrated ATPase-deficient RAD54 mutants inmice showed that this function of RAD54 protein was independent ofits ATPase activity [49]. Elegant real-time imaging and photo-bleaching experiments revealed additional facets of RAD54 proteinfunction and dynamics in living cells [49]. Mouse cells expressing theRAD54 ATPase-deficient mutants (RAD54-K189A/R) show an increasein spontaneous RAD54 foci and RAD51 foci, whereas RAD54 nullmutant cells have normal levels of spontaneous RAD51 foci.Interestingly, this increase was not accompanied by increasedendogenous DNA damage, as indicated by normal levels of γ-H2AX,a sensitive cellular marker for DNA damage that can be repaired byHR. Although foci of DNA repair proteins appear as relatively stablestructures, the residence time of individual proteins can vary greatly,from a few seconds for RAD54 to severalminutes for RAD51 [85]. FRAP(fluorescence recovery after photobleaching) experiments revealedthat the RAD54 ATPase activity is required for its dynamic associationwith DNA repair foci inmammalian cells [49]. Surprisingly, the ATPaseactivity of RAD54 was also required to release DNA repair foci afterrelocalization to the nuclear periphery [49]. The mechanisms andsignificance of intranuclear redistribution of DNA repair foci are notunderstood, but this phenomenon has also been observed in yeast[86]. The release of DNA repair foci from the periphery may reflect thecompletion of the repair event. These important in vivo observationsare invaluable to interpret the biochemical data and establish thebiological significance of the in vitro results.

5. Rad54 paralogs in S. cerevisiae and humans

There are 17 Snf2/Swi2-like proteins in budding yeast [12], butScRdh54 is the paralog that is evolutionarily most similar to Rad54 byfunctional and phylogenetic criteria, and we therefore treat ScRdh54 asthe sole Rad54 paralog in the stricter sense. Both proteins appear toperform partially overlapping roles in HR as discussed in more detailbelow (Fig. 2). Similarly in humans, where several dozen Snf2/Swi2-likeproteins exist, hRAD54B represents the only hRAD54 paralog clearlyinvolved in the coremechanismofHR (Fig. 2) [12]. Other Snf2/Swi2-likeproteins, such as Rad5 (human HLTF and SHPRH), Rad16, and Rad26(human CS-B) are involved in different DNA repair pathways. Inaddition, the classical ATP-dependent chromatin remodeling factors andother Snf2/Swi2-like factors function in other aspects of the DNAdamage response and transcriptional control (for review see [7]).

Budding yeast cells deficient for the Rad54 paralog ScRdh54 displayvery mild phenotypes compared to mutants in RAD54. Mutants inRDH54 exhibit no MMS or IR sensitivity, somewhat increased levels ofintrachromosomal recombination compared towild-type, and only veryslightly reduced interchromosomal recombination [44]. Mitotic recom-bination is reduced in the diploid mutant strains but no reduction isobserved in haploids [87]. The meiotic phenotype of rdh54 cells is alsorather mild with 82% spore viability compared to 53% for rad54 cells.However, the rad54 rdh54 double mutant is completely defective inmeiosis, suggesting that both proteins perform partially overlappingfunctions in meiosis [44]. It has been suggested that ScRdh54preferentially functions in HR between homologs, possibly through itspreferential interaction with Dmc1 (see Table 1), the meiosis-specificRad51 paralog that is required for meiotic interhomolog recombination[5,88], whereas Rad54 is envisioned to primarily function in DNA repairinvolving the sister chromatid through its association with Rad51[44,87,89]. Further insights into the differential roles of ScRad54 andScRdh54 will likely shed light on the mechanisms governing inter-homolog events during meiotic recombination.

In somatic yeast cells, Rdh54 is recruited to aDSB byRad51 andRad52,as demonstrated in ChIP experiments [90]. This result is rather surprising,


given that ScRdh54-deficient cells are not sensitive to this type of DNAdamage. However, ScRdh54 has also been implicated in signaling fromDSBs, specifically during adaptation from DSB-induced cell cycle arrest incells with a non-repairable DSB [91]. While wild type cells eventuallyadapt after prolonged arrest and resume the cell cycle even in thepresenceof aDSB, rdh54 cells remain terminally arrested. It is possible thatthe localization of ScRdh54 and its recruitment by Rad51 and Rad52 isrelated to this signaling function [92]. Themechanisms involved remain tobe determined. In vivo, ScRdh54 localizes to kinetochores, but thefunctional significance is unclear [84]. In cells lacking ScRdh54, ScRad54appears to take the place of ScRdh54 at kinetochores [84], suggesting thatthis localization is not related to the ScRdh54 signaling function inadaption evident in the single mutant.

Biochemically, ScRdh54 is exceedingly similar to ScRad54, and itappears that their specificity is mediated by their differential affinitiesfor Rad51, Dmc1, and possibly other proteins (Table 1). Like ScRad54,ScRdh54 is a dsDNA-specific ATPase, albeit with apparently lowerspecific activity (Table 1). ScRdh54 translocates on dsDNA, with lowervelocity (84 bp/min) and processivity than ScRad54, inducing thetopological changes identified to be associated with ScRad54 translo-cation [93,94]. In summary, the genetic, biochemical, and cytologicaldata suggest some functional overlap between ScRdh54 and ScRad54,particularly in meiosis, but there are clearly unique functions for bothproteins, e.g. of ScRad54 in DNA repair in somatic cells and ScRdh54 inadaptation from DNA damage checkpoint-mediated cell cycle arrest.

Unlike the yeast Rad54 paralog, ScRdh54, the mammalian RAD54paralog, hRAD54B, has a clear function in DNA repair. In human cellshRad54B colocalizes with hRad51 and hRad54 in the nucleus [95]. Theavailability of a human cell line (HCT116) knocked out for both copies ofRAD54B directly demonstrated the relevance of hRad54B to HR, as thesecells show reduced gene targeting frequencies and genomic instability[96,97]. Importantly, hRAD54B-deficiency is synthetically lethal withdepletion of the FEN1 nuclease, which is critical for lagging strandreplication [97]. This mimics the synthetic lethality of rad27 mutants(RAD27 encodes yeast FEN1)with any HRmutant in budding yeast [98].The conclusions from the genetic studies in human cells are corrobo-rated by results with RAD54B-deficient mouse ES cells, which displaysensitivity to IR and interstrand crosslinking agents (mitomycin C) [64].Interestingly, RAD54 RAD54B double mutant ES cells do not exhibitincreased mitomycin C sensitivity, whereas the double mutant animaldoes, suggesting possible tissue-specific expression and functionof bothproteins [64]. Likewise, the yeast rad54 rdh54 double mutant iscompletely deficient in meiosis, whereas the RAD54 RAD54B doublemutant mouse is fertile [44,64]. The overall biochemical properties ofhRAD54B are highly similar to hRAD54, although the ATPase activityappears to be surprisingly low (Table 1, see below) [64,99,100].

While it is tempting to draw the analogy that both yeast andvertebrates (human) contain one Rad54 protein (ScRad54, hRAD54)and one principal Rad54 paralog (ScRdh54, hRAD54B), the geneticdata in yeast and mouse strongly suggest that hRAD54B is not theequivalent of ScRdh54. The phenotypes of the respective singlemutants are too different, and the genetic analysis in mice documentsclear differences to yeast.

6. Functions of Rad54 protein

Rad54 protein has been implicated in nearly all mechanistic stagesof HR, except DNA end resection [49]. Here, we discuss the functionsof Rad54 protein in the consecutive stages of HR (see Fig. 1) and itsregulation, followed by a brief summary of the roles of Rad54 paralogsin HR.

6.1. Chromatin remodeling

Snf2/Swi2 is the founding member of ATP-dependent chromatinremodeling factors that can slide or eject nucleosomes, as well as




exchange constituent histones ([101]; also see other contributions inthis issue of BBA). The homology between Rad54 and Snf2/Swi2provided an immediate impetus to test whether Rad54 functions torelieve the repressive context of chromatin on HR, which could berelevant during DSB resection, Rad51 filament assembly, homologysearch, DNA strand invasion or later stages (Fig. 3). Biochemicalexperiments using purified S. cerevisiae, Drosophila melanogaster, andhuman Rad54 proteins on reconstituted chromatin templates demon-strated that Rad54 remodels nucleosomes in anATP-dependentmannerin vitro [72,102–104]. The chromatin remodeling activity of Rad54 wasstimulated by the Rad51 presynaptic filament [72,102–104], which isknown to stimulate the Rad54 ATPase activity from studies involvingnon-chromatinized substrates [6–8]. Rad54 enhanced DNA strandinvasion by Rad51 on chromatin substrates [102,104], but ScRad54was not required for Rad51-mediated homology search on suchsubstrates [105]. Rad54 is a potent and processive dsDNAmotor protein[73] and may remodel nucleosome in a non-specific manner as ittranslocates on duplex DNA. Rad54-mediated nucleosome remodelingis relatively inefficient compared to established remodelers such as theSnf2 complex. Rad54 chromatin remodeling appeared limited tomono-nucleosomes and Rad54 did not affect nucleosome positioning in anucleosomal array [72]. While the Rad54 ATPase activity is highly

Fig. 3. Chromatin remodeling during HR. Schematic representation of possible stages,where nucleosomes on chromosomal DNA (1) may exert an effect on HR, potentiallyrequiring chromatin remodeling, including resection (2), homology search (3), DNAstrand invasion (4), branch migration (5), and recombination-associated DNA synthesis(6). The questionmark in (2) indicates the possibility that nucleosomesmay be associatedwith ssDNA after resection. The architecture of theRad54 protein (monomer,multimer) inassociation with the Rad51-ssDNA filament or duplex DNA is unknown. For convenience,we represent here and in subsequent figures Rad54 associated with the Rad51-ssDNAfilament as amonomer. Theactive translocatingmotor onduplexDNA isdepictedhereandin subsequent figures as a hexameric ring encircling the DNA in an interpretation of thesingle molecule and electron microscopic studies discussed in the text based on theanalogy of processive, homo-hexameric DNA helicases.


stimulated by the Rad51-dsDNA filament [75], a similar stimulation bynucleosomes has not been reported. Moreover, there is a fundamentaldifference between the architecture of Rad54, whichmay assemble in ahomo-polymeric ring on DNA [47,73,74], and that of established ATP-dependent chromatin remodelers that occur in large multi-subunitassemblies [101]. However, a direct protein interaction has beendescribed between ScRad54 and histone H3 [106], which is consistentwith a specific function of Rad54 in chromatin remodeling.

Does ScRad54 remodel chromatin during HR in vivo? At themoment this question cannot be answered definitively, but it can benarrowed down. In yeast, Rad51 filaments form in vivo and canperform successful homology search in vivo and in vitro withchromatinized substrates in the absence of ScRad54 [105,107]. Thissuggests that Rad54 and its ATP-dependent chromatin remodelingactivity are not essential for DSB resection, Rad51 presynapticfilament formation or homology search. Rad54 chromatin remodelingmay still be relevant in later stages of HR, such as DNA strand invasion,branch migration, or during recombination-associated DNA synthesis.During in vivo recombinational repair in the MAT system using theHML locus as a donor site, changes in a positioned nucleosome and theaccessibility by HO endonuclease dependent on ScRad54 have beennoticed [108,109]. However, it is presently not possible to distinguisha direct chromatin remodeling action by Rad54 from an indirect effectby DNA strand invasion, branch migration, or DNA synthesis. Theoccurrence of Rad54 in thermophilic Crenarchaea, such as Sulfolobus,shows that the appearance of Rad54 is not linked to the appearance ofhistones in evolution, as many members of this group of Archaea lackhistones [110]. In summary, more work is needed to establish thespecificity and biological significance of chromatin remodeling byRad54.

6.2. Rad51-ssDNA filament stability

The Rad51 presynaptic filament is the central HR intermediate.During its formation, mediator proteins help in the assembly of Rad51filaments on RPA-covered ssDNA. These mediators may act either inthe nucleation or stability of the Rad51-ssDNA filament (Fig. 4).Biochemical experiments uncovered a role of ScRad54 in stabilizingRad51-ssDNA filaments [111]. This function was independent ofATPase activity, as also the ATPase-deficient ScRad54-K341R proteinstabilized Rad51 filaments. The increase in stability is likelycontributed by the additional protein–protein contacts promoted bythe specific binding of Rad54 to Rad51. Stabilization of the Rad51-ssDNA filaments provides a satisfying mechanism for the observedmediator function of Rad54 in reconstituted in vitro recombinationreactions [112].

Fig. 4. Rad51 presynaptic filament stability. Schematic representation of ATPase-independent stabilization of the Rad51-ssDNA filament by Rad54. The exact interactionof Rad54 with the Rad51-ssDNA filament, laterally or terminally, monomer or multimeris unknown.




Multi-faceted evidence from yeast and mammalian cells stronglysuggests that Rad54 stabilizes Rad51-presynaptic filaments as well invivo in an ATP-independent fashion. Cytological data show that Rad51filament formation at IR-induced DSBs is independent of Rad54 [84].However, closer examination by chromatin-immunoprecipitationrevealed that Rad51 filament assembled at the MAT locus after HO-mediated DSB formation in ScRad54-deficient cells were not as robustas in wild type cells [107,113]. Stabilization of Rad51 filaments in vivowas independent of ATP-hydrolysis and was also observed in ATPase-deficient ScRad54-K341R mutant cells, congruent with the biochem-ical observations [108,111]. Also in mammalian cells it was observedthat Rad51 filaments formed in RAD54 deficient cells are less robustand sensitive to fixation conditions [64,71]. In fact, an elaborate studyusing the mouse knock-in of the RAD54 ATPase-deficient mutantdemonstrated slow accumulation of RAD51 foci at break sites,consistent with a stability defect. Importantly, this RAD54 functionwas independent of its ability to hydrolyze ATP, as the ATP-deficientmutant cells showed normal RAD51 focus formation [49]. In yeast,Rad51 filaments do form in the absence of ScRad54 and are competentfor homology search, possibly with diminished efficiency [107,109].Results from yeast with the ScRad54 ATPase-deficient mutants andthe recombination-dependent lethality of the srs2 rad54 doublemutant discussed earlier suggest that the ATP-independent functionsmay be important, but not essential for HR, and that the criticalATPase-dependent function of ScRad54 is after assembly of theRad51-ssDNA filament. In summary, stabilization of the Rad51presynaptic filament appears to be a conserved, ATPase-independentfunction of Rad54 proteins, which may be important but is notessential for HR.

A

B

C

D

E

Fig. 5. Homology search and DNA strand invasion. A. Scheme of the three-stranded DNA stB. Scheme of the D-loop reaction between supercoiled, circular duplex DNA and a single-strandsDNA sliding model envisions that the Rad54 motor protein slides duplex DNA along the Rmodel suggests that Rad54 translocation on the duplex target DNA results in strand openingneeds to transition from an ATPase inactive form when associated with the Rad51-ssDNA filE. The Rad51-dsDNA complex dissociation model maintains that in the in vitro reaction somdissociated from duplex DNA by Rad54 and entirely redistributed to bind to the ssDNA.


6.3. Homology search and DNA strand invasion

The Rad51-ssDNA filament performs homology search and DNAstrand invasion, the key steps in HR. Through its association with thepresynaptic filament, Rad54 is targeted to the pairing site, where itmay bind duplex DNA, which support the Rad54 ATPase activity andability to translocate on dsDNA [69,70]. In fact, the seminal discoverythat ScRad54 stimulates the in vitro recombination activity of Rad51protein opened the field of mechanistic studies with Rad54 protein[114]. Sub-stoichiometric amounts of Rad54 stimulate Rad51 in thethree-strand DNA strand exchange reaction (circular ssDNA andlinear dsDNA; Fig. 5A) and the D-loop reaction (linear ssDNA andsupercoiled duplex DNA; Fig. 5B) in all systems examined[37,102,114–116]. The stimulation depends on the Rad54 ATPaseactivity and requires species-specific protein interactions between theRad51 and Rad54 proteins, suggesting that the observed in vitro effectis of biological importance [6–8].

The mechanism of how Rad54 stimulates Rad51-mediated recom-bination remains to be determined. Since the Rad54-mediatedstimulation is well recognized on non-chromatin substrates, chromatinremodeling by Rad54 cannot be the primary mechanism. Severalmodels can be entertained: first, the dsDNA translocation activity ofRad54 may help by sliding the target duplex DNA along the Rad51filament for improved homology search [114] (Fig. 5C). In vivochromatin-immunoprecipitation experiments, however, show thatRad51 filaments formed at theMAT locus can perform homology searchand target Rad51 to the HML donor locus in the absence of ScRad54[107], arguing against such a homology search model. However, acontribution to homology search by Rad54 cannot be entirely excluded

Rad51Rad54

rand exchange reaction between linear duplex DNA and circular single-stranded DNA.ded DNA oligonucleotide, which is depicted with a heterologous duplex DNA tail. C. Thead51 presynaptic filament to enhance homology search. D. The dsDNA strand-openingthat facilitates the conversion to stable joint molecules. In both models, Rad54 protein

ament to an active ATPase on duplex DNA, a process which is not depicted in the figure.e Rad51 will bind to the duplex DNA, which inhibits D-loop formation, unless Rad51 is



A

B

C

D

E

Fig. 6. Branch migration, D-loop dissociation, migrating D-loop during DNA synthesis,and fork regression. Schematic representation of the potential roles of Rad54 in branchmigration: A) enlarging D-loops, B) moving nicked (not shown) or closed Hollidayjunctions, C) migrating D-loops during recombination-associated DNA synthesis, D)dissociating D-loops, and E) regressing replication forks. For details see text.


by these experiments, because the low kinetic resolution in theseexperiments (1 h time points) may have missed a subtle effect. Second,the Rad54 translocation activitymay lead to transient strand openingontheduplexDNA to facilitate joint formation [68–70] (Fig. 5D). Consistentwith this proposal is the ability of Rad51-ssDNA filaments to performhomology search in vivo and in vitro without Rad54 [105,107].Importantly, in the biochemical experiments, the ScRad54 ATPaseactivity was required to convert protein-stabilized, homology-depen-dent joints mediated by Rad51 in a D-loop assay into stable DNA jointsthat could be analyzed by gel electrophoresis after deproteinization.This transition may be a reflection of duplex opening by Rad54 to allowintertwining of the invading strand with the target template DNA.Alternatively, it is possible that Rad54 is required to dissociate Rad51from the duplex target DNA in the in vitro reaction, as Rad51 bound tothe target DNA blocks DNA strand invasion [117] (Fig. 5E). It isinteresting to observe that yeast Rad51 is completely dependent onScRad54 for D-loop formation [114], whereas human RAD51 can formD-loops in the absence of hRAD54 [115,116]. This suggests that Rad54function may relate to the DNA binding properties of its cognate Rad51protein [118–120]. At present, it is difficult to distinguish between thelatter two models. The strand-opening model predicts that Rad54protein is required for D-loop formation in vivo, whereas the Rad51dissociation model predicts that in vivo D-loops can be formed in theabsence of Rad54. There are presently no tools to demonstrate D-loopintermediates in vivo and indirect measures, such as chromatin-immunoprecipitation, cannot distinguish between successful homologysearch and DNA strand invasion.

6.4. D-loop dissolution and branch migration

Branch migration describes the movement of recombination-mediated joints, which appears to be isoenergetic, as it involvesbreaking and forming the same number of Watson–Crick base pairs(Fig. 1). To achieve directionality, however, ATP-dependent branchmigrationmotor proteins are required. The paradigm for such amotoris the RuvB protein which is targeted to Holliday junctions through itspartner protein RuvA [121]. Branch migration can occur in severaldifferent contexts: first, branch migration could enlarge the D-loopand generate longer heteroduplex DNA (Fig. 6A) or second, branchmigration could move nicked junctions or classical Holliday junctionscontaining four uninterrupted strands (Fig. 6B). Both processes affectthe length of heteroduplex DNA. However, the initial resection of theDSB also determines heteroduplex DNA length [5]. In bacteria, branchmigration moves junctions to the preferred cleavage site of the RuvCHolliday junction resolvase [121]. The recombination junctionnucleases known in eukaryotes, Mus81–Mms4 (human MUS81–EME1), Slx1–Slx4, Rad1–Rad10 (human XPF-ERCC1), and Yen1(human GEN1), have not been reported to display any sequencepreference. Therefore, there is no compelling need for branchmigration. It is currently unclear whether branch migration occursduring HR in eukaryotes [5,18,19], but genetic data suggest thatheteroduplex length is defined by more than just end resection [122].Rad54 has also been suggested to be involved in other processes thatresemble branch migration, such as D-loop migration duringrecombination-associated DNA synthesis (Fig. 6C) or D-loop dissolu-tion (Fig. 6D). The interested reader is also referred to a previousdetailed review on the role of Rad54 in branch migration [8].

In reconstituted recombination reactions and using specificallydesigned synthetic substrates, Rad54 exhibits ATP-dependent branchmigration activity in vitro. The first indication came from experimentsusing the three-strand DNA strand exchange reaction (Fig. 5A) [123].Rad54 protein enhanced branch migration in this assay by about five-fold with defined directionality and in a species-specific manner, asyeast Rad54has noeffect on the RecA-mediated reaction. Thesefindingswere confirmedand extended to the related four-strand reaction,wheremigration of a four-stranded DNA junction is measured, for the yeast


andhumanRad54proteins [124]. hRAD54 (and ScRad54)were found tobindwith preference to partial X or X junctions (see Fig. 8) compared tolinear duplex DNA and were able to migrate junction substratesreconstituted from oligonucleotides [124,125]. Binding to its preferredsubstrate also stimulated the ATPase activity of hRAD54 about three tofive-fold over dsDNA [124,125]. Inclusion of hRAD51 protein alsostimulated hRAD54 branch migration activity [126], likely through thestimulation of the Rad54ATPase activity [75,116]. hRAD54was also ableto drive branch migration through regions of heterology when usingjunction substrates reconstituted from oligonucleotides or in recon-stituted recombination reactions with hRAD51 [124,127]. While thebiochemical data clearly support a role of Rad54 in branch migration,there are presently no specific in vivo data to demonstrate biologicalsignificance.

D-loopmigration during recombination-associated DNA synthesis ismediated by theDdahelicase in the phage T4 recombination/replicationsystem,where the processwas termedbubblemigration (Fig. 6C) [128].The role of Rad54 in recombination-associatedDNAsynthesis, extensionof the invading strand in the D-loop, has been directly tested inreconstituted reactions using yeast proteins (RPA, Rad51, ScRad54, DNA




polymerase delta, PCNA, and RFC), but the Rad54 motor protein wasunable to migrate the D-loop during DNA synthesis [27].

ScRad54 is required for D-loop formation in vitro catalyzed by theS. cerevisiae Rad51 protein [114], and hRAD54 stimulates D-loopformation by hRAD51 [115]. During a D-loop time course in reactionsincluding Rad54, a decrease in initial product formation can be noted,which is due to ATP-dependent disruption of the D-loop by Rad54[129]. It has been proposed that this D-loop-dissociation activity(Fig. 6D) is biologically significant [129], because overexpression ofATPase-deficient ScRad54 (Rad54-K342R/A) leads to an increase inconversion tract length [130]. It was interpreted that the absence ofRad54motor activity leads to longer heteroduplex length because of adefect in D-loop dissociation [129]. However, this result could alsoreflect other mechanisms, for example the failure to dissociate Rad51from heteroduplex DNA after DNA strand invasion (see Section 6.5).D-loop dissolution occurs during SDSA (Fig. 1), but there is no directevidence suggesting that Rad54 protein is required specifically forSDSA, and other motor proteins such as Mph1/Fml1/FANCM, Srs2 andBLM/Sgs1 have been implicated [131–133].

hRAD54 protein has also been implicated in the process ofreplication fork regression, a mechanism to provide lesion bypasswhen the replication fork encounters a blocking lesion (Fig. 6E) [134]. Inreactions utilizing oligonucleotide-based replication fork substrates,Rad54 was shown to migrate such substrates in an ATP-dependentfashion. This activity is reminiscent of the branchmigrationmediated byRad54 on similar substrates, but there is presently no evidence tosupport the biological significance of this activity.

In conclusion, Rad54 is a potent motor protein that tracks ondsDNA and is capable of moving junctions that are encountered as ittranslocates on duplex DNA in vitro. Although branch migration is astandard feature of HR models, there is actually no direct evidencethat it occurs in eukaryotes. At present, the biological significance ofthe branchmigration, D-loop dissociation, or fork regression activitiesof the Rad54 protein are unclear.

6.5. Dissociation of Rad51 from heteroduplex DNA to allow extension ofthe invading 3′-OH end by DNA polymerase

After DNA strand exchange Rad51 remains bound to theheteroduplex (double-stranded) DNA [135], much like the bacterialRecA protein, which serves as a paradigm for Rad51 [136] (Fig. 7).RecA requires ATP-dependent turnover and release of the productduplex DNA before DNA polymerase III holoenzyme can access theinvading 3′-OH end for recombination-associated DNA synthesis[137]. Rad51, however, displays about 200-fold lower ATPase activityon dsDNA than RecA [138,139], resulting in very low dynamics ofRad51 cycling off duplex DNA [140]. ScRad54 specifically dissociatesRad51 from dsDNA in a time- and ATP-dependent manner [117,140].This reaction displays strong species preference, as human RAD51 isvery poorly displaced by ScRad54 [117]. Furthermore, the ScRad54ATPase activity was enhanced up to 6-fold on dsDNA substratescovered with sub-saturating amounts of Rad51 protein, again

Fig. 7. Rad51 dissociation from heteroduplex DNA. Schematic representation of Rad54dissociating Rad51 from heteroduplex DNA after DNA strand invasion. The stippledblue arrow indicates new DNA synthesis by a DNA polymerase that gains access afterRad51 dissociation.


displaying significant preference for the cognate Rad51 protein[75,140]. Hence, the absence of a Rad54-like HR protein in bacteriais consistent with bacterial RecA being capable of autonomousturnover driven by its intrinsic nucleotide cofactor cycle of ATP-binding and hydrolysis, whereas eukaryotic Rad51 may have come torely on an extrinsic turnover factor, namely Rad54, to release duplexDNA. Electron microscopic (EM) analysis of the interaction betweenRad54 and Rad51-dsDNA filaments suggests that Rad54 is eitherdirectly recruited to the ends of Rad51-dsDNA filaments or trans-locates along dsDNA to dock at the terminus of a Rad51-dsDNAfilaments before dissociating the filament [74]. Efficient dissociationrequired the Rad54 ATPase activity, likely to power Rad54 transloca-tion on dsDNA, but also the intrinsic Rad51 ATPase activity. Whilefilaments of the ATPase-deficient Rad51-K191R mutant protein ondsDNA stimulated the Rad54 ATPase activity like wild type Rad51,their dissociation became very inefficient [140]. Furthermore, Rad54dissociated Rad51-dsDNA filaments containing Rad51-ADP protomersmuch more efficiently than ATP-bound Rad51 protomers, suggestingthat Rad54 may target the Rad51-filament end with a terminal ADP-bound subunit [140]. These conclusions are consistent with extensivesingle molecule studies of human RAD51 that demonstrate slowrelease of RAD51 from dsDNA even under conditions allowing ATPhydrolysis [141–143]. In summary, the results from the biochemicaland EM analysis demonstrate that the Rad51-dsDNA filament is aspecific substrate for the Rad54 motor protein.

UsingKlenowpolymerase as a tool to probe access to the invading3′-OH end, Rad54 and its ATPase activity were shown to be essential forDNA synthesis from the invading strand in reconstituted recombinationreactions using yeast proteins [135]. Although human RAD51 isproficient in D-loop formation, no DNA synthesis was observed in thepresence of ScRad54, suggesting that species-specific protein interac-tions are necessary. These reactions used D-loop formed between linearDNA molecules to circumvent the need for Rad54 in D-loop formation(as is the case when circular dsDNA is used), allowing analysis of Rad54function in steps that occur after synapsis [135]. Examination ofreconstituted recombination-associatedDNA synthesiswith the cognateDNA polymerase delta and its co-factors PCNA and RFC also revealed adependence on Rad54 and Rad54 ATPase activity [27]. However, theseexperiments used the classic D-loop assay with supercoiled dsDNA, sothat Rad54was first required for D-loop formation and, as inferred fromthe studies with linear D-loops, later for D-loop extension.

What is the evidence in vivo that Rad54 is specifically required toallow extension of the invading 3′-OH end in the D-loop? Chromatin-immunoprecipitation experiments monitoring protein occupancy at asingle DSB site in yeast (theMAT locus) and its recombination target site(HML) showed that Rad51filaments can form in the absence of ScRad54in budding yeast [107], although these filaments show diminishedstability as discussed above under Section 6.2. Live-cell imaging datawith fluorescently tagged proteins also supports the conclusion that IR-induced Rad51 filaments form in the absence of ScRad54 [84].Importantly and as noted earlier, Rad51 can be immunoprecipitated atthe HML target sequence in ScRad54-deficient cells, suggesting thatScRad54 is not essential for homology search [107]. ScRad54-deficientcells are unable to perform recombination-associated DNA synthesis,measured bya PCR-assay that requires less than100 ntofDNAsynthesis[107]. Recent work showed that ScRad54 is required for the mobility ofthe positioned nucleosome L6 at the HML target region duringrecombination [109]. However, these results cannot distinguishwhether the Rad54-dependent step is DNA strand invasion (D-loopformation) or D-loop extension or both. The question is whether Rad51can form D-loops in vivo in the absence of ScRad54. However, the toolsto detect D-loops in vivo are lacking. Consistent with the notion thatRad51 displacement from heteroduplex DNA is required for DNAsynthesis is the observation that recombination-associated DNAsynthesis in meiotic recombination, measured as BrdU incorporation,is never associated with Rad51 foci, suggesting that Rad51 dissociation




is required before recombination-associated DNA synthesis can takeplace [144]. This important result supports the notion that Rad51 mustbe displaced from heteroduplex DNA, likely by Rad54, before recom-bination-associated DNA synthesis can commence from the D-loop. Insummary, the present data are consistent with an ATP-dependent invivo role of ScRad54 in D-loop formation and/or D-loop extension andfurther work is needed to clarify the Rad54-dependent step(s).

6.6. Stimulation of Mus81–Mms4/EME1 nuclease activity

A two-hybrid screen using ScRad54 protein as a bait led to theoriginal identification of Mus81, the catalytic subunit of the Mus81–Mms4 structure-selective endonuclease [145]. Binding of Mus81 toScRad54 potentially recruits the nuclease to junction intermediatesduring HR, specifically the D-loop during synapsis or later junctionintermediates that mature from second-end capture and branchmigration (Figs. 1 and 8). Consistent with this model, the activity oflow and limiting amounts of yeast Mus81–Mms4 (0.25 nM) wasstimulated by ScRad54 achieving amaximum at 20 ormore foldmolarexcess of ScRad54 protein [146]. Similar results were found with thehuman proteins, using 2.5 nMMUS81–EME1with amaximal nucleasestimulation at 40-fold molar excess of hRAD54 [147]. Whether thiseffect depends on species-specific protein interaction is unclear,because hRAD54 stimulated the yeast nuclease Mus81–Mms4 [146],whereas ScRad54 did not stimulate, rather inhibited, human MUS81–EME1 [147]. In both studies, ScRad54 or hRAD54 did not change thesubstrate preference for the cognate endonuclease [146,147]. Inparticular, there was no significant cleavage of the intact Hollidayjunction in the presence of ScRad54/hRAD54 [146,147], suggestingthat even when recruited by the Rad54 motor protein, Mus81–Mms4/hMUS81–EME1 maintains its preference for junctions contain-ing at least one strand interruption [32,148–152]. The ScRad54

A

Fig. 8. Recruitment of the Mus81–Mms4 structure-selective endonuclease. A) Recruitmentmediated junctions by Rad54, including a DNA synthesis extended D-loop, a partial double Hoall three junction intermediates depicted are excellent substrates for Mus81–Mms4 cleavastrands has not been reported to be cleaved by Mus81–Mms4 and a single Holliday junctio


Walker-A box mutant proteins (ScRad54-K341R/A) stimulatedMus81–Mms4 cleavage like wild type ScRad54, suggesting thatMus81–Mms4 stimulation occurred independent of Rad54 ATPbinding or hydrolysis [146]. Using non-hydrolysable ATP analogs, itwas concluded instead that hRAD54 requires ATP binding but nothydrolysis to stimulate human MUS81–EME1 [147]. It is unclearwhether this is a difference between the yeast and human enzymes orwhether nucleotide cofactor analogs affect theMUS81–EME1 catalyticactivity. In vivo, Mus81 forms foci in about 5% of yeast cells undernormal growth conditions, which is increased to 9% after IR (40 Gy)[146]. Importantly, deletion of RAD54, but not the Walker A-boxmutants, abolished the IR-induced Mus81 foci. The RAD54 deletionhad no effect on spontaneous Mus81 foci, whereas the Walker A-boxmutants increased the frequency of spontaneousMus81 foci to 8–13%.This suggests that ScRad54 recruits Mus81–Mms4 to sites of DNAdamage in an ATPase-independent fashion [146]. Recruitment ofMus81–Mms4 is the second ATPase-independent function of Rad54,but it is unclear when this recruitment occurs during the HR processin wild type cells: during Rad51 presynaptic filament formation,homology search, DNA strand invasion, or even subsequent steps?

6.7. Turnover of Rad51-dsDNA dead-end complexes

Unlike bacterial RecA protein, yeast or human Rad51 readily binddsDNA [118,153]. This biochemical property poses a significantproblem for the cell, as Rad51 may form dead-end complexes onchromosomal DNA, which exists in vast excess over sites of DNAdamage, where Rad51 is destined to assemble on ssDNA [117] (Fig. 9).This problem was first experimentally demonstrated with Dmc1, themeiosis-specific Rad51 paralog (see Section 7) [154]. As discussedabove, Rad54 protein dissociates Rad51 bound to dsDNA in a time-and ATP-dependent manner [117], but how much Rad54 contributes

B

of the Mus81–Mms4 endonuclease by Rad54 to a D-loop and B) other recombination-lliday junction after second end capture, or a nicked double Holliday junction. Note thatge in vitro, whereas the mature double Holliday junction with four full-length ligatedn is either not cleaved at all or very inefficiently [32,152].



Fig. 10. Regulation of the Rad54–Rad51 protein interaction. Inhibition of the Rad54–Rad51 interaction by Mek1-mediated phosphorylation of Rad54-T132 and Hed1binding to Rad51.


to the turnover of Rad51-dsDNA dead-end complexes in vivo isuncertain. Genetic and cytological analysis of yeast strains withdefects in ScRad54, Rdh54, and Uls1 suggested that ScRad54 primarilyturns over Rad51 from DNA damage-induced foci, whereas Rdh54,and to lesser degree Uls1, turns over spontaneous Rad51 foci [155].This may be interpreted that ScRad54 primarily removes Rad51 fromdsDNA in the context of DNA strand invasion, i.e. from theheteroduplex DNA, whereas Rdh54 (and Uls1) dissociate Rad51-dsDNA dead-end complexes. However, the nature of spontaneousRad51 foci in vegetative (somatic) yeast cells remains unclear — theymay represent dead-end complexes or sites of spontaneous DNAdamage with Rad51 in the presynaptic filament, D-loop, or on theheteroduplex DNA. An analysis of Rad51 foci in meiotic cells mayclarify the role of ScRad54 protein in the turnover of dead-end Rad51complexes (see [154]). The turnover of Rad51 from duplex DNA byRad54, either in dead-end complexes or heteroduplex DNA (seeSection 6.5), is very different from the anti-recombinationmechanismmediated by the Srs2 helicase, which translocates on ssDNA todissociate Rad51 from the presynaptic filament [51,52].

6.8. Regulation of Rad54 function

HR is a key pathway to maintain genomic stability; however,uncontrolled HR can lead to loss-of-heterozygosity, genomic rearran-gements, and toxic recombination intermediates that lead to celldeath [14]. Hence the cell must achieve a careful balance in regulatingHR, and Rad54 appears to be a target of HR regulation (Fig. 10). HRfavors the use of the sister chromatid as a template in DNA repair insomatic (vegetative) cells, and in S. cerevisiaeHR-mediated DSB repairis down-regulated in the G1 phase of the cell cycle by limiting endresection [156–158]. This problem is exacerbated in haplonticorganisms, such as the fission yeast Schizosaccharomyces pombe,where accurate DSB repair by HR is impossible in the G1 phase for lackof an appropriate template. One mechanism to downregulate HR inthe G1 phase of fission yeast is targeting Rad54 (termed Rhp54 in S.pombe) for ubiquitin-mediated proteolysis [159]. This mechanism isalso operational in meiosis. Rad54 is primarily involved in sisterchromatid recombination during meiosis, and failure to downregulateRad54 (Rhp54) using a non-degradable Rhp54 mutant shiftedrecombination from interhomolog to intersister events [159].

Rad54 protein is also targeted for regulation during meiosis inbudding yeast, involving two distinct mechanisms to inhibit thefunctionally critical interaction between Rad51 and ScRad54 (Fig. 10).The meiosis-specific kinase Mek1 is required to bias meiotic recombi-nation towards interhomolog instead of intersister interactions [160].Mek1 kinase phosphorylates ScRad54 in vitro and T132 is foundphosphorylated on ScRad54 isolated from cells arrested in meioticprophase [161]. The meiotic phenotype of the non-phosphorylatableallele rad54-T132A is consistentwith being a directMek1 target site and

Fig. 9. Turnover of Rad51-dsDNA dead-end complexes. Schematic representation ofRad54 dissociating Rad51 dead-end complex on chromosomal duplex DNA.


mimics themek1 phenotype in derepressing inter-sister recombinationduring meiosis. ScRad54-T132D mimics the phosphorylated form ofScRad54 and provided a valuable tool to identify the mechanismsinvolved. The T132D mutation significantly weakened the ScRad54–Rad51 protein interaction, reducing the stimulation of the ScRad54ATPase activity by Rad51 and, importantly, the formation of D-loop inreconstituted recombination reactions with Rad51 compared to thewild type ScRad54 protein. These effects were specific to the Rad51interaction, as the ScRad54 ATPase activity in the absence of ScRad51was the same between the wild type and ScRad54-T132D mutantproteins. The ScRad54-T132D mutant protein also exhibited a DNArepair defect when expressed in vegetative cells [161]. Interestingly,Rad53, a DNA damage checkpoint kinase that is related to Mek1, wasalso found to target ScRad54-T132 in vegetative yeast cells [162],suggesting that ScRad54may also be subject to negative control outsidethe meiotic context.

The Rad51–ScRad54 interaction is also the target of an indepen-dent regulatory mechanism during budding yeast meiosis involvingHed1, a meiosis-specific Rad51 binding protein that prevents bindingof ScRad54 to Rad51 [163,164]. Analogous to ScRad54-T132 phos-phorylation, Hed1 inhibits the protein interaction between Rad51 andScRad54, the Rad51-stimulation of the ScRad54 ATPase activity, andthe functional interaction between Rad51 and ScRad54 in D-loopformation in vitro [163]. The interaction defect is also evident inchromatin-immunoprecipitation experiments monitoring the recruit-ment of ScRad54 to a DSB at the MAT locus. Hed1 also inhibits theinteraction of Rad51 with ScRdh54, but to a lesser degree than theRad51–ScRad54 interaction [163]. When ectopically expressed invegetative cells, Hed1 inhibits recombinational DNA repair, suggest-ing that no other meiosis-specific protein is needed for the inhibitoryeffect [164]. The inhibition of ScRad54 function during meiosis islikely to be only temporary, as ScRad54 is required for successfulmeiosis. It is possible that ScRad54 is transiently inhibited to bias HRevents towards homologs, and that after the establishment of inter-homolog events that lead to the essential meiotic crossovers,repression by Hed1 is relieved and ScRad54 functions in the repairof residual meiotically induced DSBs by sister chromatid repair [5].

Hence, in budding yeast two mechanisms, Mek1-mediatedScRad54-T132 phosphorylation and Hed1, target the Rad51–ScRad54interaction during meiosis, and genetic results suggest that theyoperate independently of each other [161]. Based on the biochemicaldata, these inhibitory mechanisms will prevent Rad51-mediatedrecombination at the level of D-loop formation or D-loop extension,and direct chromatin-immunoprecipitation experiments suggest thatabsence of Hed1 does not affect Rad51 filament formation/stability




[163]. Currently, the effect of ScRad54-T132 phosphorylation onRad51-ssDNA filament formation or stability is unknown.

7. Functions of the Rad54 paralogs, ScRdh54 and hRAD54B

The biochemical properties of ScRad54 and ScRdh54 are, wherecomparable, highly similar, as discussed above. It appears that bothproteins derive a degree of specificity through differential proteininteractions. ATP-dependent chromatin remodeling activity has beendirectly demonstrated for ScRdh54 using mono-nucleosomal sub-strates, showing nucleosome sliding and enhancement of restrictionenzyme accessibility to chromatinized substrates [90]. The specificityof this activity remains to be established, and it is unknown whetherScRdh54 directly interacts with nucleosomal histones or whether itsATPase activity is stimulated by nucleosomal substrates. Similar toScRad54, ScRdh54 enables yeast Rad51 to form D-loops, both onprotein-free and chromatinized substrates [90,165]. Stimulation ofDmc1-mediated D-loop formation by Rdh54 on protein-free sub-strates was also observed [166], suggesting that ScRdh54 could be aco-factor for Rad51 and Dmc1. Chromatin-immunoprecipitationexperiments show that ScRdh54 is recruited to DSBs during HR-mediated repair in a Rad51- and Rad52-dependent manner [90].However, the absence of an obvious DNA repair defect in ScRdh54-deficient cells demonstrates that Rad54 is the predominant Rad51-cofactor in vegetative yeast cells. ScRdh54 efficiently dissociates Dmc1bound to duplex DNA in vitro [166], and cytological and immunopre-cipitation experiments in meiotic cells demonstrated that Dmc1accumulates on chromatin in ScRdh54-deficient cells [154]. Impor-tantly, Dmc1 accumulated on chromatin in a DSB-independentfashion, strongly supporting the model that ScRdh54 turns overdead-end complexes of Dmc1 on undamaged chromosomal DNA[154]. ScRdh54 has a similar function in vegetative cells, turning overspontaneous Rad51 foci [155]. It is not entirely clear whether theseRad51 foci represent sites of spontaneous DNA damage or dead-endcomplexes, but the absence of colocalizing RPA favors the interpre-tation that they represent dead-end complexes [155]. Adding amutation in RAD54 did not enhance this phenotype, but a triplemutant with a deletion in the ULS1 gene, encoding another yeastSnf2/Swi2-like protein, further increased spontaneous Rad51 focusformation [154]. IR-induced Rad51/RPA foci require ScRad54 and Uls1for their turnover, and to a far lesser degree Rdh54 [154]. These dataled to the model that ScRdh54 primarily targets Dmc1 and Rad51dead-end complexes, whereas ScRad54 targets Rad51 bound toheteroduplex DNA after DNA strand invasion [154]. Although Uls1 isa member of the Snf2/Swi2 family, it shows no similarity to Rad54outside the motor domain. Uls1 is a SUMO-targeted ubiquitin ligase(StUbL) that is involved in proteolytic control of sumoylatedsubstrates and functions in transcriptional silencing of the silentmating type locus HMR [167,168]. Uls1 was also identified as Tid4 in atwo-hybrid screen with Dmc1, but the significance of this interactionstill needs to be tested [88]. The specific function of the Uls1 protein inthese processes remains to be established, whether it acts as a motorprotein on DNA or via its StUbL activity. It will be interesting tounderstand how ScRad54 and ScRdh54 can distinguish between thedifferent functional contexts of the Rad51-dsDNA complexes. Insummary, the in vivo phenotypes of ScRad54- and ScRdh54-deficientcells demonstrate functional separation with evidence for partialoverlap, but the underlying biochemical mechanisms that distinguishboth proteins still need to be established.

Much less is known about the biochemical mechanisms of thehuman Rad54 paralog, hRAD54B. hRAD54B interacts with humanRAD51 andDMC1, and enhances theD-loop formation of both proteins[64,169,170]. Further work is needed to understand the functionalspecification between hRAD54 and hRAD54B in vertebrates. WhileScRad54 and hRAD54 are clearly functional homologs, this does notextend to ScRdh54 and hRAD54B. Genetic analysis in mouse ES cells


and animals of single and double RAD54 and RAD54B mutants arguesstrongly against this possibility, because single RAD54B-deficientmouse ES cells display DNA repair phenotypes, unlike yeast singleRDH54mutants [64]. It is possible that vertebrates contain yet anotherRAD54 paralog among their cohort of Snf2/Swi2-like proteins. Such aprotein is expected to exert a meiotic function, because the mouseRAD54 RAD54B double knockout displays no obvious meiotic pheno-type [64], whereas the RAD54 RDH54 double mutant in budding yeastdisplays a strong meiotic defect [44].

8. Summary

Biochemical analysis generated a multitude of potential functions forRad54 protein during HR, including mediator function in Rad51 filamentassembly, chromatin remodeling, plectonemic joint formation, branchmigration, D-loop dissolution, turnover of Rad51-dsDNA filaments afterDNA strand invasion, dissociation of dead-end Rad51-dsDNA complexes,and recruitment of the Mus81–Mms4 endonuclease. Multi-facettedevidence from the yeast and vertebrate systems supports a role ofRad54 in Rad51 filament assembly/stability as a first function of Rad54 invivo. However, this function is independent of the Rad54 ATPase activity,leaving open the critical question of what is the essential, ATP-dependentfunctionof Rad54protein inHR in vivo. A keyquestion iswhetherRad54 isrequired in vivo for D-loop formation, D-loop extension or both, but wepresently lack the tools to measure D-loop formation in vivo. Recentprogress to detect physical HR intermediates duringDSB repair in somaticyeast cells provides hope that this limitationwill be overcome [171]. Suchnovel in vivo approaches will be needed to help establish the biologicalsignificance of the biochemical results. We are also still lacking a high-resolution structure of the full-length Rad54 protein or its paralogs, inparticular in conjunctionwith its binding partner Rad51. The combinationof structural and single-molecule studiesmay also answer the question ofwhether the active form of Rad54 is a hexameric or double-hexamericring, as suggested by the initial single molecule experiments [73]. Lastly,morework is needed to understand the functional overlap and separationbetween Rad54 and its paralogs ScRdh54 and hRAD54B in yeast andvertebrates. Since at least Rad54 is forming multimeric assemblies, it ispossible that Rad54 forms a mixed multimer with its paralog. We lookforward to the resolutionof these problems and the identification of novelfacets of Rad54 function in the future.

Acknowledgments

We thank members of the Heyer (Clare Fasching, Damon Meyer,Erin Schwartz, Jie Liu, Kirk Ehmsen, Rinti Mukherjee, Ryan Janke,WilliamWright, Xiao-Ping Zhang) and the Nicolas labs (Aurèle Piazza,Jean-Baptiste Boulé, Gael Millot) for critical comments on themanuscript and John Ceballos for help on the artwork. This workwas supported by grants from the US National Institutes of Health(GM58015, CA92276) and US Department of Defense (W81XWH-09-1-0116). SJC was supported by NIH training grant (T32GM007377).

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