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Mechanism of EukaryoticHomologousRecombinationJoseph San
Filippo,1 Patrick Sung,1
and Hannah Klein21Department of Molecular Biophysics and
Biochemistry, Yale University Schoolof Medicine, New Haven,
Connecticut 06520; email: [email protected] of
Biochemistry and Kaplan Comprehensive Cancer Institute,New York
University School of Medicine, New York, New York 10016;email:
[email protected]
Annu. Rev. Biochem. 2008. 77:22957
First published online as a Review in Advance onFebruary 14,
2008
The Annual Review of Biochemistry is online
atbiochem.annualreviews.org
This articles doi:10.1146/annurev.biochem.77.061306.125255
Copyright c 2008 by Annual Reviews.All rights reserved
0066-4154/08/0707-0229$20.00
Key Words
DNA motor proteins, genome maintenance, meiosis,recombinases,
recombination mediators
AbstractHomologous recombination (HR) serves to eliminate
deleteriouslesions, such as double-stranded breaks and interstrand
crosslinks,from chromosomes. HR is also critical for the
preservation of repli-cation forks, for telomere maintenance, and
chromosome segrega-tion in meiosis I. As such, HR is indispensable
for the maintenanceof genome integrity and the avoidance of cancers
in humans. TheHR reaction is mediated by a conserved class of
enzymes termedrecombinases. Two recombinases, Rad51 and Dmc1,
catalyze thepairing and shufing of homologous DNA sequences in
eukaryoticcells via a lamentous intermediate on ssDNA called the
presynapticlament. The assembly of the presynaptic lament is a
rate-limitingprocess that is enhanced by recombination mediators,
such as thebreast tumor suppressor BRCA2. HR accessory factors that
facil-itate other stages of the Rad51- and Dmc1-catalyzed
homologousDNA pairing and strand exchange reaction have also been
identied.Recent progress on elucidating the mechanisms of action of
Rad51and Dmc1 and their cohorts of ancillary factors is reviewed
here.
229
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FurtherANNUALREVIEWS
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Contents
BIOLOGICAL FUNCTIONSOF HOMOLOGOUSRECOMBINATION. . . . . . . . .
. . . . 230
HOMOLOGOUSRECOMBINATIONPATHWAYS ANDBIOLOGICAL RELEVANCE . . .
231
HR GENES AND PROTEINS . . . . . 233THE RAD51 RECOMBINASE
AND PRESYNAPTICFILAMENT FORMATION . . . . . 233
THE MEIOSIS-SPECIFICRECOMBINASE DMC1 . . . . . . . . 236
ROLE OF ATP BINDING ANDHYDROLYSIS INPRESYNAPTIC
FILAMENTDYNAMICS. . . . . . . . . . . . . . . . . . . . . 236
OPPOSING EFFECTS OF RPA INPRESYNAPTIC FILAMENTASSEMBLY. . . . .
. . . . . . . . . . . . . . . . . 236
CONSERVED FUNCTIONALATTRIBUTES OF THERECOMBINATIONMEDIATORS . .
. . . . . . . . . . . . . . . . . 237
THE S. CEREVISIAE RAD52PROTEIN AND ITSRECOMBINATIONMEDIATOR
ACTIVITY . . . . . . . . 237
OTHER FUNCTIONS OF THES. CEREVISIAE RAD52PROTEIN. . . . . . . .
. . . . . . . . . . . . . . . 238
THE HUMAN RAD52 PROTEINAND ITS HR FUNCTION. . . . . . 239
THE HR ROLE OF THE TUMORSUPPRESSOR BRCA2 . . . . . . . . . .
239
SALIENT FEATURES OF BRCA2ORTHOLOGUES. . . . . . . . . . . . . .
. 240
RECOMBINATION MEDIATOR
ACTIVITY OF U. MAYDISBRH2 AND HUMAN BRCA2PROTEINS . . . . . . .
. . . . . . . . . . . . . . 242
A MODEL FOR THE BRCA2RECOMBINATIONMEDIATOR ACTIVITYAND SOME
QUESTIONS . . . . . . 243
THE BRCA2-ASSOCIATEDPROTEINS DSS1 ANDPALB2 (FANCN) . . . . . . .
. . . . . . . . . 243
THE S. CEREVISIAERAD55-RAD57 COMPLEXAND ITS
RECOMBINATIONMEDIATOR ACTIVITY . . . . . . . . 244
THE HUMAN RAD51B-RAD51CCOMPLEX AND ITSRECOMBINATIONMEDIATOR
ACTIVITY . . . . . . . . 245
THE S. POMBE SWI5-SFR1COMPLEX AND ITSRECOMBINATIONMEDIATOR
ACTIVITY . . . . . . . . 245
RELATIONSHIP OF THES. CEREVISIAE MEI5-SAE3COMPLEX TO THE S.
POMBESWI5-SFR1 COMPLEX . . . . . . . . . 246
BIPARTITE ACTION OF THEHOP2-MND1 COMPLEXIN
RECOMBINASEENHANCEMENT . . . . . . . . . . . . . . 246
THE MULTIFUNCTIONALROLE OF THE DNA MOTORPROTEIN RAD54 IN HR . .
. . . . . 247
RAD54-RELATED DNA MOTORPROTEINS: S. CEREVISIAERDH54 AND
HUMANRAD54B . . . . . . . . . . . . . . . . . . . . . . . . .
249
CONCLUSIONS. . . . . . . . . . . . . . . . . . . 250
BIOLOGICAL FUNCTIONSOF HOMOLOGOUSRECOMBINATION
Homologous recombination (HR), theexchange of genetic
information between
allelic sequences, has essential roles in meio-sis and mitosis.
In meiosis, HR mediates theexchange of information between the
mater-nal and paternal alleles within the gamete
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precursor cells and thus generates diversityamong the progeny
derived from commonparents. HR has a second critical functionin
meiosis; it ensures proper segregation ofhomologous chromosome
pairs at the rstmeiotic division through the formation
ofcrossovers, resulting in gametes with the cor-rect number of
chromosomes. These func-tions of HR ensure stability of the
organismkaryotype. Meiotic HR is initiated by Spo11-mediated DNA
double-strand breaks (DSBs)(1). HR maintains somatic genomic
stabil-ity by promoting accurate repair of DSBs in-duced by
ionizing radiation and other agents,repair of incomplete telomeres
that arise whenthe enzyme telomerase is nonfunctional, re-pair of
DNA interstrand crosslinks, and therepair of damaged replication
forks. Althoughcells have alternate DNA repair pathways suchas
nonhomologous DNA end-joining, thesemay not be operative at all
phases of the cellcycle, they do not always act on injured
repli-cation forks, nor are they as precise as HR inthe repair of
broken chromosomes.
Mutations in genes encoding the enzy-matic steps of HR result in
extreme sensitiv-ity to DNA-damaging agents such as
ionizingradiation in model organisms such as Saccha-romyces
cerevisiae (2). Additionally, these mu-tant strains are defective
in processes that in-volve the repair of naturally occurring
DSBssuch as those breaks made during mating-typeswitching and
during meiosis (3, 4). In ver-tebrate organisms, the equivalent
mutationsare very often lethal, most likely reectingthe higher
occurrence of spontaneous DSBsduring somatic growth and the
essential rolethat HR plays in the repair of damaged DNAreplication
forks (5, 6). HR in higher eukary-otes involves additional factors
not found inall model organisms. Heritable mutations inthe
cancer-prone disease Fanconi anemia andfamilial breast cancer have
turned out to bein some of these factors (7). These are gener-ally
hypomorphic mutations as the genes arefrequently essential.
The focus of this review is on the factorsthat promote HR
through their action on the
Crossovers:recombinationproducts that entailan exchange of
thearms of theparticipantchromosomes
Recombinase: anenzyme thatmediates the pairingand exchange ofDNA
strands duringhomologousrecombination
Gene conversion:nonreciprocalhomologousrecombinationbetween an
intactdonor duplex and agapped or brokenrecipient molecule
recombinases Rad51 and Dmc1. The Rad51recombinase mediates the
formation of DNAjoints that link homologous DNA molecules.It is
active in somatic and meiotic cells. Asecond recombinase, Dmc1,
promotes similarassociations of homologous DNA molecules,but is
active only in meiosis and acts in concertwith Rad51. Several DNA
helicases have beenfound to either negatively regulate HR
initia-tion or specically suppress crossover forma-tion. The
biological roles of these DNA he-licases and their mechanism of
action are thesubject of recent reviews (8, 9) and are notcovered
here.
HOMOLOGOUSRECOMBINATION PATHWAYSAND BIOLOGICAL RELEVANCE
Many HR genes were rst identied bymutants that are
hypersensitive to DNA-damaging agents that cause DSBs, and by
afailure to give viable meiotic products (seebelow for a more
detailed description). Fromstudies of these mutants using
recombinationreporters, models of HR and classication ofHR pathways
have emerged. These modelsare based on the repair of a DSB usinga
homologous DNA sequence. The rstHR model for repair of a DSB was
basedon observations of transformation in yeastusing linear
plasmids that carried sequenceshomologous to yeast chromosomal DNA
(10,11). This model, called the double-strandbreak-repair (DSBR)
model, can explainmuch of the meiotic segregation in the fungiand
linked crossing over and gene conversionas different outcomes of
DSB repair (12).Although this model has been modiedfrom its
original conception, subsequentmodels retain several key features.
The mostimportant are (a) initiation of HR by a DSB,(b) processing
of the DSB by nucleolytic re-section to give single-strand tails
with 3-OHends, (c) formation of a recombinase lamenton the ssDNA
ends, (d ) strand invasion intoa homologous sequence to form a
D-loopintermediate, (e) DNA polymerase extension
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Holliday junction(HJ): a cruciformintermediategenerated late
inhomologousrecombination.Resolution of theHolliday junctioncan
result incrossover products
Replicationcheckpoints: signaltransductioncascades triggered
bydamaged replicationforks and that lead tocell cycle arrest
ordelay
from the 3 end of the invading strand, ( f )capture of the
second DSB end by annealingto the extended D loop, ( g) formation
of twocrossed strand or Holliday junctions (HJ)s,and lastly (h)
resolution of the HJs to givecrossover or noncrossover
products.
Although the DSBR model explains manyobservations related to
meiotic recombina-tion products in the fungi, one of its
maintenets, the linking of gene conversion andcrossovers through
the resolution of a com-mon intermediate, is not supported by
mitoticrecombination data where most DSB repair ismost frequently
unassociated with crossovers.To keep the original DSBR model for
mi-totic HR would necessitate the imposition ofstrict rules on HJ
resolution in a noncrossovermode only. A second model that avoids
thisrestriction and is based on mitotic DSB re-pair data in model
organisms has been de-veloped (1315). The essence of this modelis a
migrating D loop that never leads tocapture of the second DSB end.
Instead, af-ter the initial steps of DSB resection, DNAstrand
invasion, and repair DNA synthesis,the invading strand is displaced
and annealswith the second resected DSB end. Becauseno HJ is
formed, only noncrossover prod-ucts are made. Since the model
involves DNAsynthesis followed by strand annealing, it iscalled
synthesis-dependent strand annealing(SDSA). Although the SDSA model
was ini-tially developed to explain mitotic DSB repair,there is now
substantial evidence to suggestthat SDSA is also important during
meioticHR. Not all meiotic DSBs result in crossoverproducts: only a
small fraction of these breaksdo. The existing data suggest that
there aretwo waves of meiotic DSB-promoted HR.The rst wave proceeds
by SDSA and is onlynoncrossover, whereas the second wave pro-ceeds
by DSBR, forms double HJs, and ismainly, if not solely,
crossover.
Sometimes a DSB is closely anked bydirect repeats. This DNA
organization pro-vides the opportunity to repair the DSB by
adeletion process using the repeated DNA se-quences, called
single-strand annealing (SSA)
(1618). In the SSA process, the DSB ends areresected, but then
instead of engaging a ho-mologous DNA sequence for strand
invasion,the resected ends anneal to each other. Theprocess is
nished by nucleolytic removal ofthe protruding single-strand tails,
and resultsin deletion of the sequences between the di-rect repeats
and also one of the repeats. Sincestrand invasion is not involved,
SSA is inde-pendent of strand invasion and HJ resolutionfactors
(19).
Some DSBs, such as those that can occurat telomeres or at broken
replication forks,are single-ended (2022). These too can
par-ticipate in HR, through a single-ended in-vasion process called
break-induced replica-tion (BIR) (2329). In BIR, the DSB end
isnucleolytically processed similar to the re-section that occurs
in other DSB HR repairevents. The single-strand tail then invades
ahomologous DNA sequence, often the sis-ter chromatid or homolog
chromosome butsometimes a repeated sequence on a differ-ent
chromosome. The invading end is usedto copy information from the
invaded donorchromosome by DNA synthesis. When thesister chromatid
or homolog chromosome isused, the repair is accurate. When a
repeatedsequence on a nonhomologous chromosomeis engaged to
initiate repair, the result is a non-reciprocal translocation. Most
BIR events aredependent on the HR factors used in DSBRand SDSA, but
a small fraction can occur inde-pendently of these factors that
include Rad51.BIR is often used to repair broken or short-ened
telomeres (26, 29).
The requirement for HR in DNA repli-cation is highlighted by the
nding thatmany replication mutants and mutants infactors required
for checkpoint activationwhen replication is stalled are dependent
onHR genes for viability (3032). This ndingsuggests that
replication checkpoints preventHR at stalled or damaged forks by
stabilizingthe replication complex at the fork, thusavoiding the
occurrence of HR-promotingor HR-like intermediates. The nding
alsosuggests that defective replication can result
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in HR-provoking intermediates, e.g., gaps ator behind the
replication fork. Because HR atstalled replication forks can lead
to genomicrearrangements, it might be expected that itwould be
tightly controlled. In the case ofcollapsed replication forks, HR
is used forone-ended strand invasion events using thesister
chromatid to reconstruct the fork. Thisprocess may be promoted by
sister chromatidcohesion complexes.
HR GENES AND PROTEINS
A large proportion of the genes needed forHR were initially
identied in the buddingyeast S. cerevisiae by the classical
procedure ofmutant isolation (typically based on sensitiv-ity of
mutant cells to DNA-damaging agentssuch as ionizing radiation),
in-depth epistasisanalyses of the available mutants, and cloningof
the corresponding genes by complementa-tion of the mutant
phenotype. These genesare collectively known as the RAD52
epistasisgroup. The structure and function of the pro-teins encoded
by genes of the RAD52 groupare highly conserved among eukaryotes,
fromyeast to humans. Table 1 lists the membersof the RAD52 gene
group as rst dened inS. cerevisiae and their human equivalent.
Generally speaking, in addition to me-diating mitotic HR events,
members of theRAD52 group are needed for meiotic recom-bination as
well. Aside from the RAD52 coregroup, a whole host of genes that
uniquely af-fect meiotic recombination have been uncov-ered in
screens designed to search for them(1). For instance, the DMC1
gene, which en-codes one of the two recombinase enzymes,was
identied as a cDNA species that isstrongly upregulated when S.
cerevisiae cellsenter into meiosis. Overall, more is knownabout the
properties of the mitotic HR factorsthan of meiosis-specic factors.
We discussrecent progress in understanding the mecha-nism of the HR
machinery without providingan exhaustive account on the properties
of allthe known HR factors (reviewed in 4, 8, 19,33).
Epistasis group: agroup of genes thatfunction in the
samebiological pathway.Epistasis isestablished by doublemutant
analyses
Presynapticfilament: theright-handed helicalrecombinaselament
that isassembled on ssDNA
THE RAD51 RECOMBINASEAND PRESYNAPTICFILAMENT FORMATION
The enzymes that mediate the pairing andshufing of DNA sequences
during HR arecalled recombinases, and the reaction medi-ated by
these enzymes is termed homologousDNA pairing and strand exchange.
Two re-combinases, Rad51 and Dmc1, exist in eu-karyotes. Rad51 is
needed for mitotic HRevents such as DSB repair and also for
mei-otic HR, whereas Dmc1 is only expressed inmeiosis so its
function is restricted therein.The salient attributes of the DMC1
gene andencoded protein are discussed in a separatesection.
Much of our knowledge on the RAD51gene and its encoded protein
has been de-rived from genetic and biochemical stud-ies done in S.
cerevisiae. The S. cerevisiaerad51 mutants are highly sensitive to
DNA-damaging agents and show defects in mitoticand meiotic
recombination. Analysis of theS. cerevisiae RAD51 gene, which was
clonedindependently by three different groups, re-vealed signicant
homology of its encodedprotein to the bacterial recombinase
RecA,with particular conservation of those RecAresidues that are
critical for its recombi-nase function, including DNA binding
andATP hydrolysis (3436). The structure of theRad51 protein has
been conserved amongeukaryotes. Whereas S. cerevisiae rad51
mu-tants are viable mitotically, ablation of theRAD51 gene in
vertebrates engenders mitoticlethality (19), which likely reects an
essen-tial role of Rad51-mediated HR in the repairof damaged DNA
replication forks and hencethe successful navigation through S
phase.
Rad51 and its prokaryotic counterpartRecA exists as a
homo-oligomer in solution,being heptameric and hexameric,
respectively(33, 37). Just as in the case of RecA, with ATP(or an
analogue of ATP) available, S. cerevisiaeRad51 protein assembles
onto ssDNA or ds-DNA to form a right-handed helical poly-mer that
can span thousands of bases or base
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Table 1 Homologous recombination factors
Human S. cerevisiae Biochemical function Additional
featuresProteins that function with Rad51
MRN complex:Mre11-Rad50-Nbs1
MRX complex:Mre11-Rad50-Xrs2
DNA bindingNuclease activities
Involved in DNA-damage checkpointsAssociated with DSB end
resection
BRCA2 (none) ssDNA bindingRecombination mediator
Interacts with RPA, Rad51, Dmc1,PALB2, DSS1
Member of the Fanconi anemia groupRad52a Rad52 ssDNA binding and
annealing
Recombination mediatorInteracts with Rad51 and RPA
?b Rad59 ssDNA binding and annealing Interacts with
Rad52Homology to Rad52
Rad54Rad54B
Rad54Rdh54
ATP-dependent dsDNA translocaseInduces superhelical stress in
dsDNAStimulates the D-loop reaction
Member of the Swi2/Snf2 proteinfamily
Chromatin remodelerInteracts with Rad51The yeast proteins remove
Rad51 from
dsDNARad51B-Rad51CRad51D-XRCC2Rad51C-XRCC3
Rad55-Rad57 ssDNA bindingRecombination mediator activity
(Rad55-Rad57 & Rad51B-Rad51C)
Rad51B-Rad51C and Rad51D-XRCC2 form a tetrameric complex
Rad51C associates with aHolliday-junction resolvase activity
Hop2-Mnd1 Hop2-Mnd1 Stimulates the D-loop reactionStabilizes the
presynaptic lamentPromotes duplex capture
Interacts with Rad51 and Dmc1
Proteins that function with Dmc1Hop2-Mnd1 Hop2-Mnd1 Stimulates
the D-loop reaction
Stabilizes the presynaptic lamentPromotes duplex capture
Interacts with Dmc1 and Rad51
?b Mei5-Sae3 Predicted recombination mediatoractivity
Interacts with Dmc1Likely functional equivalent ofS. pombe
Sfr1-Swi5
Rad54B Rdh54 Stimulates the D-loop reaction Interacts with Dmc1
and Rad51
aRecombination mediator activity has been found in the yeast
protein only.bNo human equivalent has been identied.
pairs. The Rad51-DNA nucleoprotein la-ment harbors 1819 bases or
base pairs ofDNA and 6 protein monomers per helicalturn. It has a
pitch of close to 100 A, corre-sponding to an axial rise of 5.2 to
5.5 A perbase or base pair (38, 39). The DNA in thislamentous
structure is therefore held in ahighly extended conformation, i.e.,
stretchedabout 50% when compared to a naked B formduplex molecule.
Human Rad51 forms heli-cal laments on both ssDNA and dsDNA that
exhibit biophysical attributes similar to thosedescribed for S.
cerevisiae Rad51 (40). Bio-chemical studies have provided clear
evidencethat only the Rad51-ssDNA nucleoprotein l-ament species is
able to catalyze DNA jointformation (39), which supports the
notionthat HR in cells is initiated via recruitment ofRad51 to the
ssDNA generated via nucleolyticprocessing of DSBs (Figure 1) or
ssDNAthat is associated with stalled or damagedDNA replication
forks. The Rad51-ssDNA
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nucleoprotein lament is often referred to asthe presynaptic
lament, and the biochem-ical steps that lead to the assembly of
theRad51 lament are collectively known as thepresynaptic stage. The
formation of Rad51-dsDNA laments with bulk chromatin coulddiminish
the pool of Rad51 available for HRreactions. As discussed below,
the S. cere-visiae Rad54 and Rdh54 proteins dissociateRad51-dsDNA
laments, an activity that islikely critical for the intracellular
recycling ofRad51.
Once assembled, the presynaptic lamentcaptures a duplex DNA
molecule and searchesfor homology in the latter. From studies
donewith RecA (33), it is expected that the homol-ogy search
process occurs by way of randomcollisions between the presynaptic
lamentand the duplex molecule. Thus, segments ofthe duplex are
bound and tested in a reiterativefashion until homology is found.
Upon the lo-cation of homology in the duplex molecule,the
presynaptic lament is able to form DNAjoints that are either
paranemic or plec-tonemic in nature. In the paranemic joint,
aninternal region of the ssDNA is paired withthe duplex molecule
via canonical Watson-Crick hydrogen bonds, but the paired
DNAstrands are not topologically linked. Studiesdone in the Radding
laboratory have shownthat the paranemic linkage mostly involves
theformation of AT base pairs between the re-combining DNA
molecules (41). The three-stranded, paranemically paired
nucleoproteinintermediate is referred to as the synapticcomplex.
Recently published work has pro-vided evidence for a role of the
Hop2-Mnd1protein complex in functionally synergizingwith the
presynaptic lament in the capture ofduplex DNA and the assembly of
the synapticcomplex (42, 43) (see below). Although rela-tively
short-lived, the paranemic joint facili-tates the location of a
free DNA end to ini-tiate the formation of a plectonemic joint,
inwhich the participant DNA strands are boundby Watson-Crick
hydrogen bonds and topo-logically intertwined (44, 45). The
nascent
Double-strand break
End resection
Strand invasionDNA synthesis
D-loop dissociationAnnealing
DNA synthesisLigation
Noncrossover
SDSA Second end captureDNA synthesisLigation
Noncrossoveror
Crossover
HJ resolution
DSBR
c
3'
DNA damagea
b
3'
Figure 1Pathways of DNA double-strand break repair by
homologousrecombination. Double-strand breaks (DSBs) can be
repaired by distinctivehomologous recombination (HR) pathways, such
as synthesis-dependentstrand annealing (SDSA) and double-strand
break repair (DSBR). (a) AfterDSB formation, the DNA ends are
resected to yield 3 single-strand DNA(ssDNA) overhangs, which
become the substrate for the HR proteinmachinery to execute strand
invasion of a partner chromosome. After asuccessful homology
search, strand invasion occurs to form a nascentD-loop structure.
DNA synthesis then ensues. (b) In the SDSA pathway, theD loop is
unwound and the freed ssDNA strand anneals with thecomplementary
ssDNA strand that is associated with the other DSB end.The reaction
is completed by gap-lling DNA synthesis and ligation.
Onlynoncrossover products are formed. (c) Alternatively, the second
DSB endcan be captured to form an intermediate that harbors two
Hollidayjunctions (HJ)s, accompanied by gap-lling DNA synthesis and
ligation.The resolution of HJs by a specialized endonuclease can
result in eithernoncrossover (black triangles) or crossover
products (gray triangles).
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Synaptic complex:the ternary complexof the recombinaselament,
ssDNA,and dsDNA in whichthe DNA moleculesare held inhomologous
registry
Recombinationmediator: a proteinthat facilitates theassembly of
therecombinasepresynaptic lamentvia RPAdisplacement fromssDNA
plectonemic joint can be extended by DNAstrand exchange being
catalyzed by the presy-naptic lament. The DNA strand
exchangereaction is facilitated by the Rad54 protein(46). Moreover,
Rad54 also promotes a spe-cialized form of DNA strand exchange
thatinvolves the formation of a HJ and migrationof the branch point
in the HJ (47).
Nucleation of Rad51 onto ssDNA is a slowprocess, which renders
presynaptic lamentassembly prone to interference by the
ssDNAbinding protein RPA. Certain recombinaseaccessory factors,
which have been termedrecombination mediators and include the
tu-mor suppressor BRCA2, can overcome the in-hibitory effect of RPA
on the assembly of theRad51 presynaptic lament. As such, these
re-combination mediators are critical for the ef-ciency of HR in
vivo. We expand on the mech-anism of action of the known
recombinationmediators below.
THE MEIOSIS-SPECIFICRECOMBINASE DMC1
The DMC1 gene was isolated by Bishop et al.(48) in a screen for
cDNA species specicfor S. cerevisiae meiosis. The
DMC1-encodedprotein is present in almost all eukaryotes in-cluding
humans and is structurally related toRecA and Rad51 (48, 49).
Ablation of DMC1in S. cerevisiae, Arabidopsis thaliana, and
miceproduces a constellation of meiotic abnor-malities that reect
an indispensable role ofthe Dmc1 protein in meiotic
recombinationand chromosome segregation (1, 48, 50, 51).Dmc1 exists
as an octamer in solution (52),and recent biochemical studies have
providedcompelling evidence that it too forms right-handed, helical
laments on ssDNA in anATP-dependent manner and catalyzes the
ho-mologous DNA pairing and strand exchangereaction within the
context of these nucle-oprotein laments (5355). Thus, in its
ac-tion as a recombinase, Dmc1 possesses thesame functional
attributes as have been docu-mented for RecA and Rad51.
ROLE OF ATP BINDING ANDHYDROLYSIS IN PRESYNAPTICFILAMENT
DYNAMICS
Even though Rad51 and Dmc1 hydrolyzeATP, especially when DNA
bound (5659),ATP hydrolysis is not needed for the assem-bly of the
presynaptic lament. In fact, bio-chemical studies have provided
evidence thatATP hydrolysis within the microenvironmentof the
presynaptic lament leads to the disso-ciation of recombinase
molecules from DNA(53, 6062). As a consequence, the use of
anonhydrolyzable nucleotide analogue (such asAMP-PNP) (61, 62),
calcium ion (53, 60, 62),or a Rad51 variant that binds ATP but
lacksATPase activity (61) leads to the stabilizationof the
presynaptic lament. That ATP hydrol-ysis promotes the turnover of
the presynapticlament is also a well-known property of RecA(33,
63). The ATP hydrolysis-linked turnoverof the presynaptic lament
could promotethe intracellular recycling of the recombinases(i.e.,
preventing the nonproductive associa-tion of the recombinase
protein with DNA)and to make available the primer end in thenewly
made D loop to initiate the repair DNAsynthesis reaction.
OPPOSING EFFECTS OFRPA IN PRESYNAPTICFILAMENT ASSEMBLY
The heterotrimeric RPA (replication proteinA) is an abundant
nuclear protein that bindsssDNA with high afnity and can remove
sec-ondary structure in ssDNA. Depending onthe circumstances, RPA
can exert a stimu-latory or an inhibitory effect on the assem-bly
of the presynaptic lament (64, 65). Thestimulatory action of RPA
was noted in 1994when the recombinase activity of S.
cerevisiaeRad51 was rst reported (59). Subsequentstudies have
provided evidence that RPA facil-itates the assembly of the
presynaptic lamentvia the removal of secondary structure in
thessDNA (66) and also by sequestering ssDNAgenerated during the
homologous DNA
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pairing and strand exchange reaction (67, 68).However, an amount
of RPA that is sufcientto saturate the available ssDNA (the
ssDNAbinding site size of RPA is 25 nucleotides perheterotrimeric
molecule) strongly suppressesthe ssDNA-dependent ATPase and
recombi-nase activities of Rad51 and Dmc1 (64, 65, 6971). That RPA
can exclude the recombinasesfrom the HR substrate has been
validated instudies that employed chromatin immuno-precipitation
(ChIP) and cytological methods.Several recombination mediators have
beenshown to counteract the inhibitory action ofRPA (see
below).
CONSERVED FUNCTIONALATTRIBUTES OF THERECOMBINATION MEDIATORS
As mentioned above, the assembly of thepresynaptic lament can be
severely impededby RPA. Studies in several laboratories haveled to
the identication of recombination me-diators capable of overcoming
this inhibitoryaction of RPA. Specically, the addition ofthese
recombination mediators with the re-combinase protein to an
RPA-coated ssDNAtemplate permits the efcient assembly of
thepresynaptic lament (19, 64, 6972). The re-combination mediator
activity of a variety ofHR factors has also been demonstrated
usingthe restoration of the recombinases ssDNA-dependent ATPase as
the readout (70, 71) orby electron microscopy to directly
visualizetheir effect on presynaptic lament formation(72).
Mutations in the recombination medi-ators invariably impair the
delivery of theircognate recombinase to the HR substrate incells,
as revealed in cytological and ChIP ex-periments (19, 7377). Since
the K45E muta-tion in the largest RPA subunit is associatedwith a
synapsis defect, RPA likely also plays arole in DNA strand invasion
during HR (78).
The recombination mediators share com-mon features in that they
are all capableof physically interacting with their
cognaterecombinase and bind ssDNA preferentiallyover dsDNA (19, 71,
72, 79). In some in-
Chromatin im-munoprecipitation(ChIP): a powerfultechnique
fordetermining whethera protein associateswith a specic regionof
the genome
stances, an interaction of the recombinationmediators with RPA
has also been noted (8083). Only a catalytic quantity of the
recom-bination mediators is needed to see reversalof RPA
inhibition, which is very likely dueto the fact that the addition
of recombinasemolecules to a nascent presynaptic lament(i.e.,
lament growth) is sufcient to displaceRPA from ssDNA (84). The
genetic char-acteristics and salient features of the variousknown
recombination mediators are reviewedbelow.
THE S. CEREVISIAE RAD52PROTEIN AND ITSRECOMBINATIONMEDIATOR
ACTIVITY
The S. cerevisiae Rad52 protein has been themost intensely
studied recombination media-tor to date. Genetically speaking, S.
cerevisiaerad52 mutants are extremely sensitive to a va-riety of
DNA-damaging agents and engendera general defect in all the known
pathwaysof HR, including Rad51-independent reac-tions such as SSA
(see below). Rad52 is a ring-shaped oligomer (81, 85), and
oligomerizationof monomers to form the protein ring is medi-ated by
the N-terminal portion of the protein(86). Higher-order multimeric
structures ofRad52 have also been documented (86). Theinclusion of
a catalytic quantity of Rad52 leadsto highly efcient reversal of
RPA-imposedinhibition of the ssDNA-dependent ATPaseand recombinase
activities of Rad51 (65, 69,70). In both mitotic and meiotic cells,
the re-cruitment of Rad51 to DSBs is strongly de-pendent on Rad52,
but the DSB recruitmentof Rad52 shows no dependence on Rad51
(73,7577). Taken together, the genetic and bio-chemical studies on
S. cerevisiae Rad52 pro-vide compelling evidence that it helps
deliverRad51 to the ssDNA substrate during HR.
That Rad52 physically associates withthe Rad51 protein was rst
noted in ayeast two-hybrid protein-protein interactionanalysis
conducted by Milne et al. (87),and this was subsequently conrmed
by
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coimmunoprecipitation of the two proteinsfrom cell extract (65)
and also using pu-ried proteins in afnity pulldown assays(36). The
Rad51 interaction domain resideswithin the carboxyl-terminal
portion of theRad52 protein (87), and truncation mutations(rad52
327 and rad52 409412) that abol-ish Rad51 binding attenuate the
recombina-tion mediator activity of the latter (70, 88)
andcompromise HR deciency in both mitoticand meiotic cells (88,
89). These observationssupport the premise that complex
formationwith Rad51 is indispensable for the recombi-nation
mediator activity of Rad52. The HRdeciency of the rad52 409412
mutant canbe largely overcome by the overexpression ofRad51 (88),
indicating that the recombina-tion mediator function of ScRad52 can
be by-passed when the intracellular concentration ofRad51 is
elevated.
Mortensen et al. (90) rst reported thatScRad52 has a DNA binding
activity thatis specic for ssDNA. These authors foundthat the
N-terminal one third of Rad52 har-bors a DNA binding function, and
exten-sive subsequent studies have focused on howthe N-terminal
portion of human Rad52 en-gages ssDNA (9194). The structure of
theconserved N-terminal protein oligomeriza-tion/DNA binding domain
of the humanRad52 protein has been analyzed by X-raycrystallography
(91, 94). The crystallographicdata reveal an undecameric
(11-subunit) ringstructure with a deep groove on the outersurface,
with an abundance of basic and aro-matic residues lining this
groove (91, 94). De-tailed mutational analyses have provided
ev-idence for the involvement of these residuesin DNA engagement
(91, 92). Mutations ofthe equivalent residues in the ScRad52
pro-tein compromise DNA repair and HR ef-ciency, attesting to the
importance of theN-terminal DNA binding domain in Rad52protein
function (95). It remains to be deter-mined how these N-terminal
mutations af-fect the recombination mediator activity ofScRad52.
Recent studies in the laboratory ofthe authors have led to the
identication of a
second DNA binding domain in the carboxylterminal portion of
ScRad52 (L. Krejci, C.Seong & P. Sung, unpublished
observation).How this second DNA binding domain con-tributes to the
known functions of ScRad52 isthe subject of ongoing
investigations.
Rad52 protein also associates with RPAin S. cerevisiae cells
(80), and it appears thatboth the largest and middle subunits of
RPAare able to directly bind Rad52 (81, 96).The ability to interact
with RPA is con-served in the human Rad52 protein (96). Be-cause
Rad52 is unable to overcome inhibitionposed by the Escherichia coli
SSB protein onRad51-mediated homologous DNA pairingand strand
exchange (69), specic associationof Rad52 with RPA is very likely
necessary forthe recombination mediator activity of Rad52.
A model for the recombination mediatorfunction of ScRad52 is
presented in Figure 2.Among the most pertinent questions regard-ing
the recombination mediator function ofRad52 are the relative
contributions of the N-terminal and C-terminal DNA binding do-mains
of this protein and the relevance ofprotein oligomerization. In
this regard, itshould be noted that the N-terminal domainof ScRad52
is important for DNA annealingreactions during yeast meiosis
(97).
OTHER FUNCTIONS OF THES. CEREVISIAE RAD52 PROTEIN
Even though the rad52 327 and 409412alleles encode proteins that
are defective inRad51 interaction and consequently lack
re-combination mediator activity (70, 88), theyare not as decient
in mitotic and meiotic HRas is the rad52 null mutant (88, 89).
Moreover,the rad52 327 protein retains residual abilityto mediate
the recruitment of Rad51 to DSBs(C. Seong & P. Sung,
unpublished results).Clearly then, in HR events that are Rad51
de-pendent, Rad52 protein serves an undenedrole that is distinct
from its well-characterizedrecombination mediator activity. Aside
fromparticipating in Rad51-dependent HR events,Rad52 is also
required for Rad51-independent
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SSA and BIR reactions (19, 26). Consistentwith its involvement
in SSA, Rad52 pro-tein anneals DNA strands that are nakedor coated
with RPA (81, 82). It has beeninferred that Rad52-mediated
annealing ofRPA-coated DNA strands is relevant for thecapture of
the second DNA end in the DSBRpathway of HR (98). Rad52 is absent
in iesand worms but present in other eukaryotes,including
humans.
THE HUMAN RAD52 PROTEINAND ITS HR FUNCTION
Even though the human Rad52 protein re-sembles its S. cerevisiae
counterpart in struc-ture and biochemical attributes (i.e., it
isoligomeric and able to bind ssDNA and pro-mote ssDNA annealing),
and can under somespecialized circumstances enhance Rad51-mediated
homologous DNA pairing (99), arecombination mediator activity has
not yetbeen demonstrated for it. In fact, extensive ef-forts in the
laboratory of the authors (P. Sung,unpublished observations) have
failed to re-veal such an activity. The apparent lack of
arecombination mediator activity in hRad52may explain why it plays
only a subsidiaryrole in HR in vertebrates (19, 100).
Alterna-tively, hRad52 may augment the recombina-tion mediator
activity of other factors such asBRCA2 or complexes of the Rad51
paralogues(see below). In this regard, synthetic lethalityhas been
noted for a double mutant of Rad52and XRCC3 (a Rad51 paralogue; see
below)(100). It of course also remains possible that arecombination
mediator activity will only berevealed upon the posttranslational
modica-tion (such as phosphorylation) of hRad52 orthe inclusion of
a partner protein.
THE HR ROLE OF THE TUMORSUPPRESSOR BRCA2
Mutations in the BRCA2 gene predisposethe affected individuals
to breast, ovarian,and other cancers (101). Biallelic
inactivationof BRCA2 can cause the cancer-prone dis-
Rad51
Rad51 & RPA
Rad51 alone
Rad52
RPA Rad51DNA binding
DNA bindingOligomerization
Functionaldomain:
a
b
c
Rad52 RPA
Key:
Figure 2Rad52 and its role as a recombination mediator. (a) In
the presence of ATP,Rad51 forms the presynaptic lament on ssDNA,
but is unable to do so inthe presence of replication protein A
(RPA). (b) The functional domains inS. cerevisiae Rad52 are
indicated. (c) In its recombination mediator role,Rad52 forms a
complex with Rad51 and delivers it to RPA-coated ssDNAto seed the
assembly of the presynaptic lament. The polymerization ofadditional
Rad51 molecules results in the further displacement of RPA fromthe
DNA.
Paralogue: aprotein that is relatedto another protein inprimary
structurebut not necessarily infunction and thatarises through
geneduplication
ease Fanconi anemia (102) (see sidebar: Con-nection between
Fanconi Anemia and Ho-mologous Recombination). Cells from
ver-tebrate, plant, nematode, and fungal speciesdecient in BRCA2 or
its orthologue aresensitive to DNA-damaging agents and im-paired
for homology-directed elimination ofchromosome damage, including
interstrandcrosslinks and DSBs (51, 101, 103109). Thetransport of
RAD51 to the nucleus is im-paired in cells that harbor a
cancer-associated
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Orthologue: thestructural andfunctional equivalentof a protein
in adifferent species
CONNECTION BETWEEN FANCONIANEMIA AND HOMOLOGOUSRECOMBINATION
Fanconi anemia (FA) is an autosomal recessive
chromosomefragility syndrome characterized by progressive bone
marrowfailure, short stature, cancer predisposition, and a severe
de-ciency in the removal of lesions, interstrand crosslinks
inparticular, from DNA (209, 210). FA is a complex
multigenicdisorder comprised of 13 complementation groups (FA- A,
B,C, D1, D2, E, F, G, I, J, L, M, and N), and each of the
corre-sponding genes has been identied. FA-D1 and FA-N
patientsharbor biallelic mutations in BRCA2 and its associated
pro-tein PALB2, respectively, thus revealing a functional linkage
ofBRCA2-/PALB2-dependent homologous recombination tothe FA pathway
of DNA-damage repair and response (102,209, 210). Upon DNA damage,
a complex of several FANCproteins helps mediate the
monoubiquitination of FANCD2and FANCI, resulting in the chromatin
localization of the lat-ter two proteins. It is believed that the
chromatin-bound ubiq-uitinated FANCD2-FANCI complex recruits
BRCA2/PALB2and associated proteins, such as Rad51, to initiate DNA
repairby a homology-directed mechanism (102, 209212). BecauseFA
cells are frequently impaired for HR (209, 210), some ofthe FA
proteins may possibly inuence the homologous re-combination process
via BRCA2/PALB2.
BRCA2 truncation (110). Furthermore, ham-ster Brca2 mutant cells
exhibit an abnormalS phase DNA-damage checkpoint response(104).
Brca2-decient mice suffer embry-onic lethality, an observation that
highlightsthe importance of BRCA2-dependent DNA-damage repair and
response (107). There isalso good evidence that BRCA2 is impor-tant
for meiotic HR (51, 103, 105, 111). Alarge body of results point to
an importantrole of BRCA2 in the assembly of presynap-tic laments
of Rad51. Specically, and asexpounded below, BRCA2 and its
ortholo-gous proteins bind DNA (71, 72, 79, 105),physically
interact with Rad51 (101, 107, 112,113), and are needed for the
formation ofDNA damageinduced nuclear Rad51 foci(101, 114).
Importantly, studies on the Usti-lago maydis Brh2 protein (the
BRCA2 ortho-
logue in that organism) and a polypeptidethat harbors critical
functional domains ofthe human BRCA2 protein have provided di-rect
biochemical evidence for a recombina-tion mediator activity (71,
72) (see below). Anability of human BRCA2 and its
Arabidopsisthaliana orthologue to interact with Dmc1 hasalso been
demonstrated (115, 116), althoughwhether BRCA2 serves a
recombination me-diator role in Dmc1-mediated HR reactionsremains
to be explored. Notably, S. cerevisiaeand Schizosaccharomyces pombe
do not possess aBRCA2-like molecule.
SALIENT FEATURES OF BRCA2ORTHOLOGUES
The BRCA2 orthologues vary greatly in size,ranging from 3418
amino acid residues ofthe human protein to only 383 residues ofthe
Caenorhabditis elegans counterpart (calledBRC-2). Human BRCA2
utilizes two sepa-rate means to interact with Rad51 protein,through
a reiterated motif called the BRC re-peat (101, 112, 113, 117) and
also via a struc-turally distinct motif located at its
extremecarboxyl terminus (107, 118); the carboxylterminal Rad51
binding domain will be re-ferred to as the CTRB domain
henceforth.The BRC repeat, which comprises about30 amino acid
residues, is highly conservedamong the BRCA2 orthologues (117,
119), al-though there is extreme variance in the num-ber of copies
of this motif among the ortho-logues. For example, human BRCA2
harborseight BRC repeats, whereas Ustilago maydisBrh2 protein and
A. thaliana Brca2 proteinpossess a single BRC repeat and four such
re-peats, respectively. While most of the BRCrepeats from different
BRCA2 orthologuesbind Rad51 with varying afnity, the BRC5and BRC6
repeats of human BRCA2 do notseem to be functional in this regard
(112, 113).The second BRC repeat of the A. thalianaBrca2 protein
appears to interact with Dmc1but not Rad51 (115). Biochemical and
crys-tallographic approaches and also electron mi-croscopy have
been employed to dene the
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nature of the BRC-Rad51 complex. The re-sults from these studies
indicate that the BRCrepeat interacts with the monomeric form
ofRad51 [Rad51 by itself is heptameric in solu-tion (37)] in the
absence of DNA (120). Theatomic structure of a complex between
BRC4of human BRCA2 and the RecA-homologydomain of Rad51 has been
solved by X-raycrystallography (121). The structure revealsthat
BRC4 mimics the motif in Rad51 thatmediates protein
homo-oligomerization, thusproviding a structural basis as to why
BRCbinds the monomeric form of Rad51 (121).Yang et al. (71) have
suggested that bindingof the BRC repeat to Rad51 leaves one ofthe
Rad51 oligomerization interfaces avail-able for the recruitment of
another Rad51molecule, which could provide a means for
thenucleation of Rad51 presynaptic lament as-sembly in the
recombination mediator role ofBRCA2. Electron microscopy with
accompa-nying three-dimensional reconstruction anal-yses have
provided evidence that the BRCrepeat can interact with a preformed
Rad51presynaptic lament without disruption of thelament, and that
BRC3 and BRC4 of hu-man BRCA2 interact with different surfacesof
Rad51 in the context of the presynaptic l-ament (122). The sole BRC
repeat of the C.elegans BRC-2 protein is bipartite in struc-ture;
one region is specic for monomericRad51 and the other interacts
specically withand stabilizes the Rad51 presynaptic
lament(123).
As mentioned above, BRCA2 also bindsRad51 through the CTRB
domain (107, 118).An allele of mouse Brca2 lacking the CTRBdomain
is associated with reduced viabil-ity, hypersensitivity to the DNA
interstrandcrosslinking agent mitomycin C, chromoso-mal instability
(124), and a modest impair-ment of the HR-directed repair of a
site-specic DSB (106). Thus, the CTRB domainappears to make a
signicant contribution to-ward the HR function of BRCA2 but is
notessential for this function. Unlike the BRCrepeat, the CTRB
domain interacts with theoligomeric form of Rad51 in the absence
of
DNA (125, 126), and appears to bind theinterface of two adjacent
Rad51 monomersin the context of the presynaptic lament(126). The
CTRB domain harbors a serineresidue (serine 3291) that becomes
phospho-rylated by cyclin-dependent kinases (CDKs)in a cell
cycle-dependent fashion, a modica-tion that blocks the interaction
with Rad51. Ithas been suggested that the
CDK-mediatedphosphorylation of the CTRB domain servesas a molecular
switch in regulating Rad51-mediated recombination (127). Note that
theU. maydis Brh2 protein also harbors a Rad51binding domain in its
carboxyl terminus. ThisC-terminal Rad51 binding domain, which
iscrucial for the HR and DNA repair func-tions of Brh2 in vivo, is
unrelated to the BRCrepeat (128). Whether this Brh2 domain
isstructurally related to the CTRB domain ofBRCA2 is unclear. Dmc1
also interacts withthe CTRB domain in human BRCA2, albeitmore
weakly than Rad51 (116).
Although human BRCA2 and its A.thaliana equivalent both interact
with Dmc1,the primary Dmc1 binding domain appears tobe different in
the two proteins. None of theBRC repeats in the human BRCA2
proteinhas any afnity for Dmc1, and the main Dmc1interaction site
in human BRCA2 has beenmapped to a 26-amino-acid region
(residues23862411) termed the PhePP motif. Thismotif is highly
conserved in mammalianBRCA2 species but absent in the BRCA2
or-thologue in A. thaliana and other eukaryotes(116). In contrast,
A. thaliana Brca2 associateswith Dmc1 through BRC2, the second of
fourBRC repeats in this protein (115).
Aside from Rad51 interaction, BRCA2 hasalso been found to
associate with RPA (83) byseveral biochemical criteria. RPA binding
ismediated by the extreme N-terminal regionof BRCA2, and a
cancer-associated mutation,Y42C, in BRCA2 attenuates or abolishes
theability to bind RPA (83).
The DNA binding activity of BRCA2was rst revealed in a combined
biochemi-cal and structural examination of the mouseBrca2 (mBrca2)
protein by Yang et al. (79).
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The BRCA2 DNA binding domain (BRCA2DBD) is thought to harbor ve
subdomains:three oligonucleotide/oligosaccharide-bind-ing (OB)
folds (OB1, OB2, and OB3), a helix-turn-helix motif that is
appended to OB2and called the Tower domain, and a proxi-mal
alpha-helical region termed the helicaldomain (79). The OB folds of
BRCA2 arestructurally similar to the OB folds presentin RPA.
Embedded within the helical domainand OB1 is a surface that
mediates complexformation with a small, highly acidic pro-tein DSS1
(see below for a more detailed de-scription of DSS1). The mBrca2
DBD-Dss1complex binds ssDNA but not dsDNA andappears to recognize
the duplex-ssDNA tran-sition in various DNA substrates (79). In
thisregard, the U. maydis Brh2-Dss1 complex hasa high afnity for a
duplex-ssDNA junctionthat bears the 3 overhang polarity of a
re-sected DSB (71). Crystallographic and bio-chemical evidence has
implicated all threeOB folds of BRCA2 in DNA engagement(79).
RECOMBINATION MEDIATORACTIVITY OF U. MAYDIS BRH2AND HUMAN BRCA2
PROTEINS
The U. maydis Brh2 protein in complex withDss1 was found to
possess a recombinationmediator activity (71). Using several
biochem-ical approaches, Yang et al. demonstratedthat an amount of
Brh2-Dss1 substoichio-metric to that of the U. maydis Rad51
pro-tein is sufcient to seed the assembly ofthe presynaptic lament
on DNA precoatedwith RPA. The Brh2-Dss1 pair is particu-larly adept
at mediating Rad51 lament as-sembly at a duplex-ssDNA junction that
has a3 overhang polarity, which is in congruencewith the ability of
this protein complex tospecically recognize such a DNA
junction.These results suggest that Brh2-Dss1 pref-erentially seeds
Rad51 presynaptic lamentassembly at the duplex-ssDNA junction of
aresected DSB or another DNA lesion in vivo.
The sole BRC repeat of Brh2 can interactwith the human Rad51
protein and, accord-ingly, the Brh2-Dss1 complex is functionalas a
recombination mediator with humanRad51 (71).
Owing to its uncommonly large size, full-length human BRCA2
protein has not yetbeen puried. To circumvent this problem,San
Filippo et al. (72) fused the BRC3 andBRC4 repeats of BRCA2 to the
DNA bindingdomain of this protein and devised a methodto purify the
BRCA2-derived polypeptide(termed BRC3/4-DBD) to near homogene-ity.
BRC3/4-DBD binds both ssDNA anddsDNA but with a distinct preference
forthe former. As expected, BRC3/4-DBD in-teracts with Rad51 but
not RecA. A quantityof BRC3/4-DBD substoichiometric to
Rad51promotes the assembly of the Rad51 presy-naptic lament on
RPA-coated ssDNA ef-ciently, but this BRCA2-derived polypep-tide is
completely inactive toward RecA (72).Aside from its ability to
restore presynapticlament assembly, BRC3/4-DBD also specif-ically
targets Rad51 to ssDNA when an excessof dsDNA is present.
Polypeptides that har-bor just the BRC3 and BRC4 repeats or theDBD,
when used alone or in combination,do not exhibit recombination
mediator orssDNA targeting activity, indicating that boththe BRC
repeats and the DBD are neededfor biological efcacy and they act in
cis (i.e.,both entities have to be present on the samepolypeptide).
Thus, BRC3/4-DBD, and byinference BRCA2 protein, possesses two
dis-tinct functional properties, i.e., an ability totarget Rad51 to
ssDNA and a recombinationmediator activity, that are germane for
thepromotion of Rad51-mediated HR reactions.That BRC3/4-DBD
possesses recombinationmediator activity is congruent with a
recentnding that fusion proteins comprising RPAand either a single
BRC repeat or multiplesuch repeats can improve the HR prociencyof
Brca2-decient hamster cells and enablethese cells to form Rad51
foci upon DNAdamaging treatment (129).
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A MODEL FOR THE BRCA2RECOMBINATION MEDIATORACTIVITY AND
SOMEQUESTIONS
We present in Figure 3a model that incorpo-rates all the
biochemical attributes believed tobe germane for the recombination
mediatoractivity of BRCA2. Of special note is the coop-erative
action of the BRC repeats and CTRBdomain in the assembly of the
nascent Rad51lament, with the former acting as the initialdepositor
of monomeric Rad51 onto the HRsubstrate and the latter being a
recruiter of anintact Rad51 oligomer to seed the assembly ofthe
nascent presynaptic lament.
As reviewed above, recent studies haveprovided evidence for
functional differencesamong the BRC repeats of BRCA2 and possi-bly
a presynaptic lament stabilizing activityin some (or all) of these
repeats (122, 123).Future studies will address the contributionsof
the individual BRC repeats toward presy-naptic lament assembly and
preservation andthe mechanistic basis for their action. BRCA2is
also able to interact with Dmc1 (115, 116),and studies in A.
thaliana have provided di-rect evidence for functional interactions
be-tween Brca2 and Dmc1 in meiotic HR (51).It seems reasonable to
suggest that BRCA2also serves to nucleate Dmc1 presynaptic la-ment
assembly on resected DSBs during mei-otic HR. Even though the CTRB
domainof BRCA2 interacts with Rad51 more avidlythan Dmc1, the
phosphorylation of S3291 inthis domain attenuates Rad51 binding
with-out signicantly affecting the association withDmc1 (116). It
will be interesting to testwhether CDK-mediated phosphorylation
ofS3291 in BRCA2 provides a means for thedifferential regulation of
BRCA2-Rad51 andBRCA2-Dmc1 interactions.
THE BRCA2-ASSOCIATEDPROTEINS DSS1 ANDPALB2 (FANCN)
BRCA2 forms a complex with the small acidicprotein DSS1 (130),
which is needed for HR
+
b
Rad51 RPA
BRC-Rad51
CTRB-Rad51
BRCA2
Key:
or
Rad51 Rad51+ BRCA2
c
BRCA2
PALB2RPA
Rad51 Rad51Dmc1
BRC repeats DBD
Dmc1 DSS1
Interactiondomain:
a
HD1 2 3 4 5 6 7 8
OB
1
OB
2
OB
3
CTRB
Figure 3BRCA2 and its role in homologous recombination. (a) The
functionaldomains in human BRCA2 are indicated. (b) In this model
for the BRCA2recombination mediator activity, the BRC repeats each
bind a Rad51monomer (for simplicity, only two of the six functional
BRC repeats aredepicted as being associated with Rad51), while the
CTRB is charged withan intact Rad51 heptamer. One of the BRC
repeats and the CTRB thencooperate to assemble a nascent Rad51
lament, followed by the furtherdisplacement of RPA by the growing
Rad51 lament. (c) Recent results (72)suggest a function of BRCA2 in
targeting Rad51 specically to the ssDNAregion of a resected
DSB.
efciency in vivo (131, 132). In U. maydis, theDss1 orthologue is
thought to maintain Brh2in an active state by preventing the
forma-tion of Brh2 homo-oligomers (128). Whethermammalian DSS1
serves a similar role has not
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yet been explored. Since the DSS1 interactionsurface resides
within the DNA binding do-main of BRCA2 (79), DSS1 may well
modu-late the DNA binding properties of the latter.Even though a
homologue of DSS1 (calledSem1) is present in the budding yeast, it
ap-pears to affect diverse processes such as exocy-tosis (133) and
DSB repair by HR and NHEJ(134) via an association with the
proteasome(134, 135).
A novel BRCA2-associated protein calledPALB2 was recently
identied by Xia et al.(136). PALB2 promotes the proper
localiza-tion and maintains the stability of BRCA2 inchromatin and
nuclear matrix and appears tobe critical for the DNA repair and
checkpointfunctions of BRCA2 (136). The PALB2 in-teraction domains
lies within the amino ter-minus of BRCA2, and the
cancer-associatedY42C mutation that affects the interaction ofBRCA2
with RPA (83) also ablates PALB2binding (136). PALB2 possesses a
series ofWD40 repeats at its carboxyl terminus (136,137) that are
indispensable for complex for-mation with BRCA2 and the biological
ac-tivity of PALB2 (137). Importantly, muta-tions in PALB2 are
associated with familialbreast cancer (138, 139) and biallelic
inactiva-tion of PALB2 can cause Fanconi anemia (FA)of
complementation group N (140, 141) (seesidebar). Owing to its FA
connection, PALB2is also known as FANCN. How the HR func-tion and
specically how the recombinationmediator activity of BRCA2 may be
inu-enced by PALB2 will undoubtedly be a hotlypursued area of
research in the near future.
THE S. CEREVISIAERAD55-RAD57 COMPLEXAND ITS
RECOMBINATIONMEDIATOR ACTIVITY
Mutants of RAD55 and RAD57 genes sharethe uncommon property of
being coldsensitive for HR and DNA repair (19, 142),and both genes
are required for the deliveryof Rad51 to HR substrates in cells
(73, 76,77). Overexpression of Rad51 or Rad52
partially overcomes the DNA repair and HRdecits of rad55 and
rad57 mutant cells, andsimultaneous overexpression of Rad51
andRad52 leads to further suppression of themutant phenotype (143,
144). The RAD55-and RAD57-encoded proteins are regardedas
paralogues of Rad51 as they exhibitsignicant homology to Rad51,
includingsequence motifs that are thought to conferthe ability to
bind and hydrolyze ATP (19,145). Rad55 protein is phosphorylated in
acheckpoint-dependent manner when DNAdamage occurs, and this
modication appearsto be important for Rad55 function
upongenome-wide genotoxic stress (146, 147).Interactions between
Rad55 and Rad57 andof Rad55 with Rad51 have been noted inthe yeast
two-hybrid system (143, 144). Themajority or all of the Rad55 and
Rad57proteins in yeast cell extract can be coim-munoprecipitated,
indicating that they areassociated as a stable complex in cells
(148).When coexpressed in yeast cells, the Rad55and Rad57 proteins
assemble into a het-erodimeric complex that has ssDNA
bindingactivity and the ability to interact with Rad51(148; P.
Sung, unpublished results). Eventhough results from a genetic study
suggestthat biological activity of the Rad55 proteinis inuenced by
ATP (144), the Rad55-Rad57complex does not seem to have a
signi-cant ATPase activity and its DNA bindingfunction appears to
be refractory to ATP(P. Sung, unpublished results). Despite
thesimilarity of both Rad55 and Rad57 toRad51, the Rad55-Rad57
complex has norecombinase activity (148). Addition of anamount of
the puried Rad55-Rad57 complexsubstoichiometric to Rad51 overcomes
theinhibitory effect of RPA on Rad51-mediatedhomologous DNA pairing
and strand ex-change (148), indicative of a recombinationmediator
activity. As deduced from the anal-ysis of a RAD51 allele (RAD51
I345T ) thataffords partial suppression of the rad55 andrad57
mutant phenotype, the Rad55-Rad57complex possibly also stabilizes
the alreadyassembled Rad51 presynaptic lament (149).
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Much remains to be learned about the HRfunction of the
Rad55-Rad57 complex. Mostnotably, whether the recombination
mediatoractivity of Rad55-Rad57 involves a specic in-teraction with
RPA, as has been demonstratedfor Rad52, is not yet known. The role,
if any,of Rad55 phosphorylation and of ATP in themodulation of the
recombination mediatorfunction of Rad55-Rad57 are also
importantquestions that need to be answered.
THE HUMAN RAD51B-RAD51CCOMPLEX AND ITSRECOMBINATIONMEDIATOR
ACTIVITY
The RAD51B and RAD51C genes code fortwo of the ve Rad51
paralogues (the remain-ing three Rad51 paralogue-encoding genesare
RAD51D, XRCC2, and XRCC3) in verte-brate cells (19). There is good
evidence thatall the Rad51 paralogues participate in HR byinuencing
the assembly and/or maintenanceof the Rad51 presynaptic lament (19,
150),and the overexpression of human Rad51 pro-tein suppresses the
defects of chicken DT40cells that lack any one of the ve Rad51
par-alogues (151). The Rad51B and Rad51C pro-teins interact in the
yeast two-hybrid system(152) and form a stable complex as
evidencedby coimmunoprecipitation and other means(153, 154). The
Rad51B-Rad51C complexhas been puried to near homogeneity andfound
to possess ssDNA binding activity anda modest ATPase activity that
is stimulatedby DNA, ssDNA in particular, but the DNAbinding
activity of this complex seems to berefractory to ATP (154). The
DNA bindingand ATPase activities of the Rad51B-Rad51Ccomplex are
derived from both Rad51B andRad51C proteins (155). Biochemical
experi-ments (154) revealed a recombination media-tor activity in
the Rad51B-Rad51C complex.Like other recombination mediators that
havebeen characterized to date, Rad51B-Rad51Cacts in a catalytic
fashion in that optimal re-combination mediator activity requires a
rel-atively small amount of the complex (154).
Rad51B-Rad51C enhances the homologousDNA pairing activity of
Rad51 (154), aneffect that could stem from the ability ofRad51C to
promote the melting of duplexDNA (155). Whether Rad51B-Rad51C
inter-acts with RPA in nucleating Rad51 presynap-tic lament
assembly and if ATP binding andhydrolysis by the protein complex
inuenceits recombination mediator function are stillunanswered.
Yeast two-hybrid and biochemical studieshave found a
higher-order complex consist-ing of the Rad51B, Rad51C, Rad51D,
andXRCC2 proteins, termed the BCDX2 com-plex (153). The BCDX2
complex has thehighest afnity for branched DNA (156) andcan
recognize nicks in DNA (153). Rad51Dand XRCC2 on their own form a
het-erodimeric complex that has DNA bindingactivity (157) but
apparently no recombina-tion mediator activity (W. Bussen & P.
Sung,unpublished results). It will be particularly in-teresting to
test whether the BCDX2 complexhas enhanced recombination mediator
activ-ity compared to the Rad51B-Rad51C com-plex. Rad51C also
combines with the XRCC3protein to form a distinct protein complex
thatseems to be associated with a nuclease activitycapable of
resolving the HJ (158). This lat-ter observation is consistent with
an apparentlate role of the XRCC3 protein in HR (159)and the fact
that, in chicken DT40 cells, si-multaneously ablating the Xrcc3 and
Rad51Dgenes engenders a mutant phenotype more se-vere than that of
the respective single mutants(150).
THE S. POMBE SWI5-SFR1COMPLEX AND ITSRECOMBINATIONMEDIATOR
ACTIVITY
As determined by a combination of yeast two-hybrid,
coimmunoprecipitation, and geneticanalyses, S. pombe Swi5 protein
combines witheither Swi6 or Sfr1 to form two separate com-plexes
that have distinct functions. Specif-ically, the Swi5-Swi6 complex
plays a role
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in mating-type switching (160), whereas theSfr1-Swi5 complex is
needed for mitotic andmeiotic HR (64, 160). The sfr1 and swi5
nullmutants are partially impaired for the abil-ity to assemble DNA
damageinduced fociof Rph51 (which is the S. pombe Rad51
or-thologue), and the DNA repair defects ofthese mutant cells can
be partially suppressedby the overexpression of Rhp51. The
Sfr1-Swi5 complex appears to provide a functionin HR similar to
that of the Rhp55-Rhp57complex (which is orthologous to the S.
cere-visiae recombination mediator Rad55-Rad57complex), as swi5,
rph57 double mutant cellsare more severely HR impaired and
decientin DNA damageinduced Rph51 focus for-mation than the single
mutants (161). Takentogether, the genetic and cytological
observa-tions suggest that Swi5-Sfr1 regulates Rph51presynaptic
lament assembly and/or main-tenance, and that it acts independently
of theRph55-Rph57 complex in this regard (161).
The Sfr1-Swi5 complex (which harborsone Sfr1 molecule and two
Swi5 molecules)has been expressed in E. coli, puried,
andcharacterized by Haruta et al. (64). The Sfr1-Swi5 complex
physically interacts with bothRph51 and Dmc1 through Sfr1 (64,
160).Sfr1-Swi5 enhances the homologous DNApairing and strand
exchange activity of Rph51and Dmc1 and can function in
conjunctionwith both recombinases in the displacementof RPA from
ssDNA. Thus, the biochem-ical analyses of Haruta et al. (64) reveal
arecombination mediator activity in the Sfr1-Swi5 complex and also
an ability of this com-plex to stimulate the homologous DNA
pair-ing and strand exchange potential of the tworecombinases.
RELATIONSHIP OF THES. CEREVISIAE MEI5-SAE3COMPLEX TO THE S.
POMBESWI5-SFR1 COMPLEX
The S. cerevisiae MEI5- and SAE3-encodedproteins are
structurally related to the S. pombeSfr1 and Swi5 proteins,
respectively (74). Un-
like their S. pombe counterpart, the expres-sion of Mei5 and
Sae3 proteins is restricted tomeiosis (74, 162). Deletion of MEI5
or SAE3impairs meiotic HR and the ability to mountnuclear Dmc1 foci
in response to meiotic DSBformation. Taken together, the mutant and
cy-tological analyses provide evidence that Mei5,Sae3, and Dmc1
proteins operate in the samerecombination pathway and suggest a
criti-cal role of Mei5 and Sae3 in the delivery ofDmc1 to the HR
substrate (74, 162). Mei5and Sae3 proteins form a complex that
phys-ically interacts with Dmc1 (74). Consideringwhat is known
about the functional proper-ties of the S. pombe Sfr1-Swi5 complex
(64),it will be particularly relevant to test whetherthe Mei5-Sae3
complex enhances the recom-binase activity of Dmc1 and Rad51 and
pro-vides a recombination mediator activity forthe two
recombinases.
BIPARTITE ACTION OF THEHOP2-MND1 COMPLEX
INRECOMBINASEENHANCEMENT
That HOP2 and MND1 genes are critical formeiotic recombination
was demonstrated ingenetic studies in S. cerevisiae and mice
(163169). Based on extensive genetic analyses in S.cerevisiae, it
has been deduced that the Hop2and Mnd1 proteins function with Rad51
andDmc1 to ensure the timely formation of DNAintermediates critical
for the completion ofmeiotic recombination (163165, 167,
168).Although the expression of HOP2 and MND1is restricted to
meiosis in S. cerevisiae, thesegenes are also expressed in somatic
tissuesin plants, mice, and humans (165, 169171).This latter
observation hints at the possibil-ity that in higher eukaryotes,
the HOP2- andMND1-encoded products inuence mitoticHR as well.
The Hop2 and Mnd1 proteins can becoimmunoprecipitated from
meiotic S. cere-visiae cell extract, indicating that they exist ina
complex (167). Consistent with this nding,when coexpressed in E.
coli, Hop2 and Mnd1
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proteins assemble into a stable, heterodimericcomplex (163, 172,
173). The Hop2-Mnd1complex binds dsDNA preferentially over ss-DNA
(42, 163, 173) and appears to have aneven higher afnity for
branched DNA (172).Studies using puried components revealedthat the
mouse Hop2-Mnd1 complex directlyinteracts with Rad51 and Dmc1 (174)
but notwith E. coli RecA (42). Although Hop2 andMnd1 proteins can
individually bind DNAand interact with Rad51 and Dmc1 (42,
173),Hop2 has much higher afnity for DNA andMnd1 possesses greater
avidity for Rad51(42). The Hop2-Mnd1 complex from mam-malian and
yeast species strongly stimulatesthe recombinase activity of Dmc1
(163, 172,174), and the mouse and human Hop2-Mnd1complexes are just
as active toward Rad51 inthis regard (172, 174). Because
Hop2-Mnd1does not enhance the recombinase activity ofthe E. coli
RecA protein (42), physical associa-tion of Hop2-Mnd1 with Rad51
and Dmc1 islikely important for functional interaction tooccur.
Recent studies by Chi et al. (42) andPezza et al. (43) have
revealed that recom-binase enhancement afforded by the Hop2-Mnd1
complex occurs at two critical stagesof the homologous DNA pairing
reaction.First, Hop2-Mnd1 stabilizes the presynapticlament of Rad51
and Dmc1, as shown us-ing a variety of approaches (42, 43) when
thepresynaptic lament is rendered stable by theuse of a
nonhydrolyzable ATP analogue, cal-cium ion, or the Rad51 K133R
protein (whichbinds but does not hydrolyze ATP), Hop2-Mnd1 still
exerts a strong stimulatory effect onDNA joint formation, leading
to the deduc-tion that it must also act at a stage subsequentto
presynaptic lament assembly. Importantly,Chi et al. and Pezza et
al. have shown that theHop2-Mnd1 complex works in conjunctionwith
the presynaptic lament to capture du-plex DNA molecule to
facilitate the assemblyof the synaptic complex (42, 43; P. Chi
& P.Sung, unpublished data). Duplex capture byHop2-Mnd1 and
Rad51 or Dmc1 is not de-
pendent on homology in the incoming duplexmolecule (42, 43) but
requires a functionalpresynaptic lament (42). Thus, Hop2-Mnd1acts
in a bipartite fashion in Rad51/Dmc1-mediated homologous DNA
pairing: stabi-lization of the presynaptic lament and du-plex
capture to enhance synaptic complexformation.
Figure 4 presents a model that depicts thebipartite action of
the Hop2-Mnd1complexin its enhancement of Rad51 and Dmc1activity.
Future studies will determine therelative importance of the
presynaptic la-ment stabilization and duplex capture rolesof
Hop2-Mnd1 in HR reactions. Since theHop2-Mnd1 complex appears to
have ahigh afnity for branched DNA structures(172; J. San Filippo
& P. Sung, unpublishedresults), it remains possible that
Hop2-Mnd1recognizes and stabilizes the nascent DNAloop formed by
the two recombinases.
THE MULTIFUNCTIONAL ROLEOF THE DNA MOTOR PROTEINRAD54 IN HR
Rad54 is a member of the Swi2/Snf2 super-family of proteins and,
similar to other mem-bers of that family, has
dsDNA-dependentATPase, DNA translocase, DNA supercoilingand
chromatin remodeling activities. Recentreviews (175, 176) have
summarized someof the roles of this multifunctional factor inHR.
Notably, Rad54 interacts with Rad51and is required at multiple
stages in HR,in the early stages to promote a search forDNA
homology, chromatin remodeling, andD-loop formation, and in the
postsynapticstage to catalyze the removal of Rad51 pro-tein from
dsDNA. The ability of Rad54 toremove Rad51 from dsDNA is believed
toprevent the nonspecic association of Rad51with bulk chromatin and
to provide DNApolymerases access to the 3-OH primer ter-minus in
the nascent D loop to initiate therepair DNA synthesis reaction
(175). Rad54also mediates the ATP hydrolysis-driven
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Hop2-Mnd1
Rad51 alone
D-loop formationdisfavored
Presynaptic filamentstabilization
Synaptic complex formation
D-loop formation
Hop2
Mnd1
Rad51
Key:
Figure 4The bipartiteaction of theHop2-Mnd1complex
inrecombinaseenhancement.Hop2-Mnd1 actsin two critical stepsto
enhance therecombinaseactivity of Rad51.Hop2-Mnd1 rststabilizes
thepresynapticlament and thencooperates with thepresynapticlament
to capturea dsDNA molecule(42). Hop2-Mnd1also functionallyinteracts
withDmc1 in the samefashion (43).
migration of branched DNAs including theHJ and acts in
conjunction with Rad51 to pro-mote a DNA strand exchange reaction
that in-volves two duplex molecules (47). Under cer-tain in vitro
conditions, Rad54 can dissociatethe D-loop intermediate, an
activity thoughtto be relevant for the promotion of SDSA(177). By
ChIP, the synapsis of the MAT ini-tiator and HML donor sequences
can be de-tected in the absence of Rad54 (76). Whetherthis reects a
role of Rad54 in plectonemicDNA joint formation or the initiation
of re-pair DNA synthesis remains to be established(76).
Rad51 and Rad54 enhance each others ac-tivities (47, 175). Given
the multifaceted role
of Rad54 in HR, one might expect RAD54to be essential to HR and
DSB repair. Whileyeast rad54 mutants are extremely sensitiveto
ionizing radiation and other types of DNAdamage that induce DSBs
and are severely re-duced in spontaneous and induced mitotic
re-combination (2, 3), rad54 mutants are amongthe least debilitated
in meiosis of the HR mu-tants. In contrast to rad51 and rad52
mutants,rad54 mutants are able to form viable meioticproducts,
although with reduced efciencycompared to wild type (178, 179). The
lackof a strong meiotic phenotype of S. cerevisiaerad54 mutant
cells can be attributed to theRad54-related protein Rdh54 (179; see
be-low). Nonetheless, overexpression of Rad54
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can suppress the meiotic defects caused by thedmc1 mutation
(180). In mouse ES cells, lossof Rad54 results in a slightly
reduced HR fre-quency, sensitivity to ionizing radiation
andmitomycin C, and aberrant repair of DNAdamage, whereas rad54/
mice appear to benormal (183, 184). This stands in contrast toother
mammalian HR genes such as RAD51,as loss of Rad51 in mouse ES cells
is lethal andthe mouse rad51/ genotype is an embryoniclethal (6,
185).
The stronger mitotic phenotype of rad54mutants in yeast and
mammals may reecta preferential action of Rad54 in promotingDSB
repair between sister chromatids. In-deed, genetic studies in S.
cerevisiae have sug-gested that Rad54 acts in sister
chromatidrecombination (186). The modest knockoutphenotype in
vertebrates may reect the pre-dominant use of nonhomologous end
joining(NHEJ) to repair DSBs. This suggestionis supported by the
nding that chromo-some loss and rearrangements are increased
inrad54 mutants in yeast and mammalian cells,and that loss of the
NHEJ pathway in combi-nation with a rad54 mutation has a
synergis-tic effect on chromosome instability, DNA-damage
sensitivity, and cell growth (187). Thisshows that mammalian Rad54
is critical forDSB repair and for maintenance of genomicstability.
Several hRAD54 mutations that re-duce or eliminate Rad54 activity
in vitro havebeen found in human tumors, suggesting animportant
role of Rad54 in cancer avoidance(188191).
RAD54-RELATED DNA MOTORPROTEINS: S. CEREVISIAERDH54 AND HUMAN
RAD54B
RAD54 paralogues exist in both yeast andmammals. The mammalian
paralogue iscalled RAD54B. The Rad54B protein has bio-chemical
activities that are similar to thoseof Rad54 (191193). Rad54B
interacts withRad51, has a dsDNA-dependent ATPase ac-tivity, and
can translocate on duplex DNA to
result in topological changes in the DNA andthe transient
opening of the DNA strands.Similar to Rad54, Rad51 enhances the
activi-ties of Rad54B, and Rad54B promotes D-loopformation by Rad51
(193). The recombinaseactivity of Dmc1 is also stimulated by
Rad54B(55, 194).
Mouse ES cells decient in Rad54Bhave no overt HR defect as
measured bygene-targeting frequencies, but Rad54/
Rad54B/ cells are decreased in gene-targeting efciency below
that of theRad54/ single mutant, showing thatRad54B functions in HR
and this is revealedonly when Rad54 is absent (193). Studies
ofionizing radiation and mitomycin C sensitiv-ities showed that
mouse Rad54B has a rolein repairing DNA damage caused by
theseagents. Mice that are decient in both Rad54and Rad54B are very
sensitive to mitomycinC treatment, with particular damage to
thebone marrow. While Rad54/ mice showsome abnormalities in meiotic
chromo-some structure, Rad54/, Rad54B/, andRad54/ Rad54B/ mice are
fertile (193).
S. cerevisiae also harbors a RAD54 par-alogue, called RDH54 or
TID1 (179, 195).However, based on mutant phenotypes, par-ticularly
the meiotic mutant phenotype,RDH54 is not equivalent to RAD54B.
Rdh54protein has functional attributes similar tothose of Rad54,
including dsDNA-dependentATPase, DNA translocase, DNA branch
mi-gration, and DNA supercoiling activities,and also the ability to
enhance the Rad51-mediated D-loop reaction (196199). It alsocan
remove Rad51 from duplex DNA in anATP hydrolysis-dependent fashion
(196), anactivity that may become important in thelater stages of
HR (196), and in the adaptationfrom a DSB-induced checkpoint arrest
(200).Rdh54 seems to be able to remove Dmc1 fromnonrecombinogenic
chromatin sites as well(201; P. Chi & P. Sung, unpublished
results).
Rdh54 interacts with Dmc1 (202). Al-though the meiotic role of
Rdh54 is notfully understood, rdh54 mutants are severely
www.annualreviews.org Eukaryotic Homologous Recombination
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reduced in meiotic viability and the doublemutant rad54 rdh54
fails to form viable mei-otic progeny or repair meiotic DSBs
(179,203). The mitotic phenotype of the dou-ble mutant is most
visible in diploid cells,seen as a failure to grow uniformly
owingto spontaneous DNA damage and check-point arrest. rdh54
mutants are also defec-tive in resuming growth after a
DSB-inducedcheckpoint arrest (200). These ndings indi-cate that
aside from some functional over-lap, RAD54 and RDH54 have
independentfunctions in HR, DSB repair, and otherprocesses.
Genetic studies by several groups havesuggested that in meiosis
Rad54 may havea prominent role in promoting DSB re-pair through HR
between sister chromatids,whereas Rdh54 is required for
interhomo-logue HR (180182, 186). How this distinc-tion is made at
the molecular level is notknown.
While Rad54 and Rdh54 can move Rad51bound to duplex DNA and can
remodel chro-matin in vitro, in vivo other DNA or chro-matin
binding proteins may well be targetsof these proteins. A recent
study (204) hasfound that the meiotic-specic sister chro-matid
cohesion factor Rec8 and the mitoticsister chromatid cohesion
factor Mcd1/Scc1shows aberrant distribution on chromosomesin mutant
rdh54 meioses. Chromosomes aremis-segregated in both of the meiotic
divi-sions, which is attributed to a failure of sis-ter chromatid
separation. Whether Rdh54acts directly to remodel cohesin
throughMcd1/Scc1 and Rec8 is not known. How-ever, loading of
cohesin at DSBs is criticalfor repair of mitotic DSBs (205208), so
itis conceivable that cohesin loading and re-modeling at meiotic
DSBs by Rdh54 is criti-cal for proper meiotic interhomologue
HR.Mcd1/Scc1association on chromatin duringmeiosis in the absence
of Rdh54 appears tobe the cause of chromosome mis-segregation.One
intriguing possibility is that Mcd1/Scc1-mediated cohesion is
important in distin-guishing sister from nonsister chromatids
in
HR. If true, then the deciency of rdh54 mu-tant cells in
interhomologue HR could arisefrom an inability of nonsister
chromatids tointeract due to persistent cohesion of the
sisterchromatids.
CONCLUSIONS
Defects in HR cause genomic instability.When the instability
leads to aberrant ex-pression or regulation of tumor suppressorsor
oncogenes, cell transformation and cancermay ensue. Because HR can
give rise to alter-ations in the genomic conguration, it mustbe
nely controlled to avoid deleterious chro-mosome rearrangements and
the generationof pathological intermediates (8, 9).
As detailed here and elsewhere (8, 9,33), the basic HR machinery
and its mech-anism and regulation are remarkably con-served among
eukaryotes. The HR reactionmediated by either Rad51 or Dmc1 has
atleast two rate-limiting steps, assembly of thepresynaptic lament
and capture of duplexDNA by the presynaptic lament. As has
beenreviewed in this article, recent biochemicalstudies have
unveiled recombinase accessoryfactors that function to overcome
these rate-limiting steps.
One of the most exciting developments inunderstanding HR
mechanism and its healthrelevance is the identication of the
tumorsuppressor BRCA2 as a recombination me-diator. Aside from
interactions with Rad51,Dmc1, RPA, and DNA, additional
domainswithin BRCA2 mediate its association withother factors, such
as PALB2, that are im-portant for its biological functions.
Whetherthese factors inuence the recombination me-diator activity
of BRCA2 is not yet known.Moreover, how the BRCA2-PALB2
complexfunctionally links the HR machinery to the FApathway of
DNA-damage repair and responseremains to be delineated.
Studies on the Swi2/Snf2-like DNA mo-tor proteins Rad54, Rdh54
and Rad54B havebegun to elucidate their multifaceted role inHR.
Rad54 and Rdh54 help determine the
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selection of the sister chromatid or nonsis-ter chromatid as HR
substrate. The geneticconsequences of recombination between
thesister or nonsister chromatids have a profound
effect on sequence variation and meiotic chro-mosome
segregation. How this distinction ismade or regulated will be a
particularly inter-esting problem to tackle.
SUMMARY POINTS
1. Homologous recombination is required for DNA double-strand
break repair, repairof damaged replication forks, repair of
incomplete telomeres, and correct segregationof homologous
chromosomes in meiosis.
2. Homologous recombination is critical for suppression of
genome instability and tumorformation.
3. Homologous recombination is mediated by recombinases, a
conserved group of pro-teins from bacteria to humans. The
eukaryotic recombinases, orthologues of E. coliRecA, are Rad51 and
Dmc1.
4. Rad51 and Dmc1 mediate the homologous DNA pairing and strand
exchange reactionthrough the presynaptic lament, single-stranded
DNA coated with Rad51 or Dmc1.
5. Presynaptic lament assembly is slow and prone to interference
by the single-strandedDNA binding protein RPA and so requires the
involvement of recombination medi-ator proteins, such as BRCA2, for
enhancement.
6. Synaptic complex formation by Rad51 and Dmc1 are enhanced by
the Hop2-Mnd1complex, revealing that capture of homologous duplex
DNA is a rate-limiting step inhomologous recombination.
7. Several DNA motor proteins, such as Rad54 and Rdh54, function
at several steps inhomologous recombination. They promote
homologous pairing and strand exchangeon naked DNA and
chromatinized DNA an