Mammalian Rad51 protein: A RecA homologue with pleitropic functions

Iliochimie ~ 1997) 79, 5,~7-592 © Soci6td fi'aq~'aise de I'fiochimie ct bioMgie moldcukfire / l'~lne~ icr, Paris

Revie~

M a m m a l i a n RadS1 prote~n: A RecA homo[ogue w~th ple~otropk functions S Visp6, M Defais :+: lustitut de P h a r m t . ' o l o g i c el +h, Biologic Strttct~n'ah,. CNRS, U P R 9062, 205. route de N a r h o m w . 31077 l m d . . ~ , , ~ c,A'~. I +,m,

tReceivcd 4 June 1997: accepted 13 November 1997) Sl.|nlll|llr) ...... Dinhie tile last years, hont~.~Jogucs of E colt RecA have t3ccil cloned in nuiB,2rotls species includin,g tllilll +[hc.,u RaC5 [ pn~lcin.~ share SCtlUence as well as functional homologies witll the bacterial prolein, litmlan RadSI (llsRadSI I is able Io ealal~,.,'t: .,tland t \~ h;til+!c U; vitro between holuologous DNAs. but with a lower efficiency compared to lh.[ll of RccA. This nug.ge.4s the requircnlenl ,}i adthli,~nal lllt;hlI'. A ~cry intel'eStillg feature of RadS[ is its essential role ill nlotlse which could Illetln thai il ha,., gilil;C,.I ;tl'l c,-,sClltial ltll+utl<+n ill C¢[i +'l't,'*',th The interaction of HsRad51 with several tumor suppressor genes namely p53. BRCA I and B R C A 2 implies pt~s,,il',lc rolc+,,i ~l fin', piotcm ill ttlnlorigellesis. Thus. tile COlllillued study of RadSI nll,a lid bl'illg inlpol'talll insi~ahts i1o1 tqlly illlO holl)Olo~2tRl', ~¢t'tHIIbHlillioB Hlk't.'hill~i%lll~ but also into ceil pl'ulil~ratitm regulatiun.

Rad51 / homologous recombination/ DNA repair Introduction Homologous recombination is a mechanism shared by every studied ot'ganisna from bacteria to man. Because DNA structure remained constant, it is not surprising that this important process was conserved throughout evolution. Recombination allows horizontal genetic transfer in bacteria as well as repair of some DNA damage like DNA crossliitks, strand breaks and pustreplication gaps. In eul~:|ryotes it is well established that ltomohi, gotlS recombination is es+ scntial during meiosis lilt" correct segregation of hontoloo gobs chromosonles il+s v+cll as l'tn' g¢llClic diversity. However, the role of homoh.,gous recontbination in I)NA repair is rather unclear in higher etlkaryotes. In prokttryoles. homohlgoas recombination is mainly e:ttalyzed by RecA protein. The recent identification of bacterial RecA homo+ logues in several organisms including man, gives the opportunity to gel a better insight into this process I1, 21.

RecA is the key component of homologous reeoml~htation in bacteria Among all the proteius il|volved in homologous recombination in E colt, ReeA protein phtys an essential role. This small protein {37 842 Da) has the property of polymel-izing on single stranded DNA as ,'t nucleoprotein filament which is able to catalyze in vitro reactious consistent with its

+iic-orrespondenceand reprint~

various i , viva functions. It promotes ATP-dependent formatiou of homologously paired joint molecules and ex+ chauge ol'complementary DNA strands. It alno catalyses, the ATP aud single stranded DNA dependem cleavage of tile LexA repressor, larnbda repressor and UmuD protein+ LexA cleavage triggers the induction ol+the SOS response leading to the derepressiou of :tbout 20 geues iuvolved in DNA re. pair aud i l l u l a g e u e s i s alllOtlg 9.rhich tile rvcA ~.'cuc it,,elt RecA proteins ha~e bccn fou|td ill all bacteria 13-5 l. RecA protein houtologues have been chm~'d in ~ca',t 3 ('('revi.~iae (ScRad51 ) Ib.8t anti S / , , , / w I SpRad51 I ]9 I I l. alld ill inany Ili~ll¢l"¢ttkar)'t)t¢,, hlcludinL2 n)alt I lIk +2. I:;! lit this review we will maillly focus oil recent restiIls oil mammalian Rad51 proteius.

SeRadSl is the yeast homologue of bacterial RceA ScRad51 proteiu is involved, along with other proteius frout the Rad52 epistasis group, in homologous t'ecombinatiot~ repair of DNA double strand breaks 16. 12[+ ScRAD51 m u taltts .~tre ~t.'uhili'Y~.' to tlil'l+el'e!tt gellttt+,~,ki¢ a~tYllts ilrZlt:+¢ly ionizing radiations, nlethyl methane sulfonate {MMSt+

e,'oss-linkiug agents and are deficient iu mitotic as well a', meiotic I+oulologous recombination leadiug to inviable spores 16, 14-16 I. S+'RAD51 mRNA accumulates after weal + ment with MMS. [IV and imfizing radiation, and during meiosis 16-8 I. ScRAD51 transcript.,, weir also found to vary throughout the cell cycle, peakiug in late G I to early S 18 I. This ineKvase could be related to a ful|ction of Rad51 ill I)NA replication. In this case. the i+wolved lunction does not seem to he essential siuce ScRAD51 null rrlutants ;+ire

~8 viable as long as they stay in the vegetative replicative cycle. SeRadSI is also, like RecA, proficient in in vio~, DNA ~rand exchange 117-19]. If SeRad51 has both structural a ~ functional homologies with RecA, it has also specific ~ u r e s . The DNA ~ a n d exch~ge reaction mediated by ScR~l$:l,~ngie strar~JedDNA n u e l e o ~ e i n filament is ~Owec and procet~lsin an ~ 0 s i t e polarity to that of RecA [18}; F~l~rmore, under eo~iti0nS: that ~e Optimal for exchange, ScRadhl hydrolyses ATP at only 7.,%,of t ~ rate ~ u ~ for RecA protein [! 71. In vivo S,zRad51 is as~iated with other proteins of the Rad52 epistasis group nantdy Rad52, Rad55, Rad57 and Rad54 constitating a basic complex named the reeombinosome 120-221. This complex would partly catalyze recombinational repair of double strand breaks. Furthermore, no SOS respon~ switch function has been identified for ScRad5 I.

higher eukmLvotes.This is supported by the interaction of HsRad51 with HsRad52 which could stimulate HsRad51 reaction 1331, It is also interesting to note that Rad55-Rad57 heterodimer stimulates the ScRad51 strand exchange reaction. One can then wonder if it is the case for higher eukaryotes. Furthermore, a stimulation of end-to-end ligation duplexes by HsRad51 has been observed, suggesting a direct role of Rad51 in illegitimate recombination repair of double strand breaks 1301. Even though this hypothesis still needs to be demonstrated, these biochemical features are supported by interesting biological observations described in the following sections.

Hi!~her euka~ote homologues of RecA and ScRad51

Li et a11341 have shown that Rad51 expression is dramatically induced in primary marine B cells cultured with lipopolysacchafides (LPS) to stimulate heavy chain class switch recombination. These results have been confirmed by immunohistochemical observations, Furthermore, a Rad51 mRNA accumulation has been observed in reproductive organs of mouse, chicken and Xenopus but also in the thymus and the spleen of mouse attd chicken 110, 23-251. In addition, in human and mouse spermatocytes Rad51 protein has been shown to be associated with synaptonemai complexes {SCs) 135, 361. SCs formation is necessary to bring homo!ogous DNA segments into close contact for crossing-over and recombimttion to t~cur. These events are crucial for proper segregation of homologous chromosomes and yield of normal haploid gamcles. Thus mammalian Radhl may have functions, yet to be defined, in meiotic homologous recombination and specific recombination:like heavy chain class switch recombination, t!sRad51 has been shown In interact with a human ubiquitin-conjugating enzyme Ubc9, and u ubiquitin like protein Ub!I 137, 391. Ubiquitination is a protein degradation pathway which serves as regulatory machinery for numerous proteins, including DNA repair proteins 1401. Ubc9 has also been shown like Rad51 to be associated with SCs, It can be propo~'d that Rad51 function(s) in meiotic cells ;nay be regulated by ubiquitin mediated Radhi degradation.

U,~ing in most cases degenerated oligonucleotides derived from the yeast Radhl mRNA sequence, several groups succeeded in cloning Radhl homologue cDNAs by PCR amplification from many organisms namely man I IO. 131, mouse [ 10, 231. chicken [241, Xenopus laevis [251, Arabid~#~.ffsthaliima [871, Drosophila melanogaster 126, 271 and hamster (unpublished results), The Radhl homologue of A ¢halia~a has also been cloned using a Sy~techoeoceus recA pn~" 1281, Each of these proteins shows strong amino acid eon~rvation with yeast Radhl protein, More relevant are the conservation of biochemical and biological functions.

Bh~hemleal properties of human Rad$ I pr~Dtein (IhRadS l) H'sRnd~i was fi~t purified fi'om/~"coli, The protein was able {~ bind sidle and double stranded DNA and to form filaments in which DNA is extended and underwound in a manner ~milar to that observed with RecA, Nevertheless, the HsRad5 Itsingle stranded DNA filaments in the presence of ATP/AT~ resemble the inactive form of RecA filament in the ab~t~e of a nuclcotide cofactor [291, it is interesting to note that extended RecA nueleopr~tein filament in the p~sen~'¢ of ATP is th~ ~ict~v~form of the p~tein proficient in DNA strand exchange and LexA and UmuD cleas,age. More recently HsRad51 has been shown to possess a DNA strand exchange activity 130-321, However, the reaction is significantly less efficient than that promoted by RecA in the ~me conditions and HsRad51 is not able to exchange long DNA strands, This could be consistent with the 'inactt e apl~aranc~ o f ~ HsRadhl filament on single stranded DNA, Taken together, th~,~¢results suggest a rt~luiremeut for ~ i t i o n a l proteins to catalyze efficient strand exchange. Actually it is tam~eivable that this reaction carried out by RecA alone in prokaryotes is shared by ~veral proteins in

Role of mammalian RadS! protein in meiotic recombination and heavy chain class switch recombination

RADS! is an e,~nfial gene in mouse. Possible role in

somatic tell growth and DNA repair Two groups have tried to generate RADhl t- knock out mice [41,421. By intercrossing heterozygous mice containing one disrupted RADhl copy they failed to obtain RAD514pups. It appeared that null embryos arrested etwly in development, namely at stage E7.5. This lethal phenotype was associated with decreased cell proliferation and increased cell death, Trophoblast-like cells derived from E3.5 embryos

5~t~ showed ganmta radiation sensitivity compared to the heterozygous or the wild type cells. The authors have also observed multiple chromosome loss in RAD5.t null embryo cells. Taki el at 143] have shown the same proliferation defect and gamma radiation sensitivity using RAD51 antisense oligonucleotides in mouse cells. These results indicate thai Rad51 has an essential role in cell life. Other results indicate that Rad51 protein is upregulated in S phase when cells are stimulated to enter into the cell cycle [44-46]. Chen et al have also shown a cell cycle-dependent expression of Rad51 but also Rad52 proteins in haman and hamster cells [471. They found that their level is maximum in G2/M phases. In S phase nuclei Rad51 localizes to foci I441. Such a localization has been also observed in human cells treated with genotoxic agents 1351. Howevei; tiffs event is not associated with an increase in Rad51 level. We also observed a Rad51 steady level in similarly treated hamster cells (unpublished results). However, the high endogenous Rad51 amount that we measured in these cells could explain that no induction of the protein is required even afte': DNA damaging treatments. One can propose that the knock-out embryo lethality is due to the loss of homologous recombination repair of double strand breaks occurring during DNA replication. The same defect could be responsible for gamma radiation sensitivity, However, it could also indicate that Rad51 has gained a novel function, yet to be defined, essential for mammalian somatic cell growth and different from its repair function.

Rad5! interacts with the tumor suppressor proteins p53, BRCAI and BRCA2. Possible implication in a signal transduction pathway p53 is a transcription fiicto." involved in cell cycle arrest and apoptosis induction in response to DNA ;!amage. It ha,~ been called rite ~Gttardian of the gcnome' because of its role in genomic stability malntenauce [48-501. While looking for a mote direct role of p53 in DNA repair StNrzbccher et a/ found a physical interaction both in vitro and in t,h,o between p53 and HsRad51 and in vitro between purified p53 and E coli RecA [511. They showed that in vitro, purified human p53 protein abolished RecA mediated homologous strand exchange reaction by direct interacthm. Very recently, Buchhop et al mapped the interaction domains of p53 and HsRad51 1521. They propose that p53 binding would disrupt HsRad51 homo-oligomerisatimt, thus inhibiting its biochemical functions. Furthermore, two recent articles indicate that cells expressing mutant forms of p53 have a significant increase in their spontaneous Immologous recombination rate 153.54J. 1;ake,i togetheh these results point to a role of wild type p53 in negatively regulating homologous recombination. This regulation could take place either directly via an interaction with Rad51 or indirectly via p53 el'lectors.

Recent]3 two papers reported physical in~eracti~ms z~,, well as striking homologous beha\i~rs between Rad5t artd BRCAI proteins on one side [551 and Rad51 and BRCA2 proteins on the ~ther [561. BRCA t and BRCA2 are human tumor suppressor genes which ha~'e been invol~ed. ~hen mutated, in breast and ovarian cancer predisposition 157. 581. The disruption of these genes in mouse has the same embryonic lethal phenotype as RAD51 knock out embryos. [56, 59, 60]. BRCA2 deficient embryos were found to be gamma radiation hypersensitive as RAD51 embryonic cells are. BRCA2 has been shown to interact with Rad51 in yeast and mammalian two hybrid screens [561. BRCA1 interacts and colocalizes with Rad51 in S phase nucki and m human spermatocytes SCs 1551. Furthermore, these three genes have been shown to be upregulated in actively divriding cells 144-46, 61,621. These common features along with physical interactions indicate a possible role of a complex includinn at least Rad51, BRCAI and BRCA2 in DNA repair and/or somatic cell orowth. This hypothesis is further supported by the induction of BRCAI hyperphosphorylation during the cell cycle namely near the G I-S phase boundar) and after DNA damage 163]. Dynamic changes of BRCAI subnuclear location following genotoxic treatments have also been reported [64]. To further investigate these roles it would be interesting to measure the repair of DNA lesions such as double strand breaks in cells where one or several of these proteins could be transitorily downregulated. Tumor cells mutated in either BRCA 1 or BRCA2 gene could be useful for such an analysis. A striking observation is the partial rescue of RAD51, BRCAI and BRCA2 knock out leth,'d phcnotype by deletion of both copies of p53. A partial rescue was also observed by deletion of the cell cycle inhibitor p21, a p53 regulated gene, in a BRCA I / kn~ck oUl 14 I. 65, {~t~].To explain these results. Brugart~las and .hicks h'ave proposed that ,'l inatation iu either of these three ~,~,ea¢', in the early embryo would icsult in a DNA repair defect oJ endogenous lesions arising in rapidly proliferating embryonic cells 167]. The unrepaired DNA damage would then activate the p53 dependent growth arrest and apoptotic response. Mutation of p53 or its efl~etorp21 would then alh~w RADS I, BRCA I and BRCA2 null embryos to overcome this response and to continue cellular proliferation until genetic damage becomes incompatible with cell growth. There is some evidence that BRCA I and BRCA2 could be transcripo tion activators [68, 691. Furthermore, haman RadSl has been assnciated with a purified RNA polymerase 11colnpLc~ [701. It could then be conceivable that Rad51 play,~ a role in a transduction signal pathway involving transcription aCo tivation of DNA repair and/or cell growth genes mediated by BRCAI and/or BRCA2 for instance. Considering the role of Rad51 in recombination and potentially in cell pro~ lil'eration, and its association with both BRCAI and BRCA2. it is possible that mutations in either gene c~mld increase genomic instability and/or disturb the cell cycle. leading to tumorigenesis. To support this hypothesis it would be interesting to look for RAD51 mutations in lt|nlor

5~ cells, Among the functions of Rad51 possibly involved in tumorigenesis, a candidate could be recombination since it has been proposed to play a role in this phenomenon I711. Pad$!

~la~

F!ygare et at 1721 have observ~ proteolytic cleavage of HsRadSl protein during apoptosis in human peripheral . ~ u J lymphocytes and in two T-lymphocyte cell lines. y1 ~ th~ltcleavage of HsRad51 in response to substanfial DNA damage would inhibit repair, stop further progression of the cell cycle and favor the characteristic DNA fragmentation associated with apoptosis. Although this cleavage could be s~cific to the cell tyl~.~. it has also been observed for another DNA repair enzyme, the PARP q~ly(ADP-ribese) polymerase) protein 173 t.

Role of (~lher Red homologues of the )'east Rad52 epistasis group cDNAs 5~r humau and mouse Rod52 and Rod54 homoIogues as well as a chicken Red52 homologue have been cloned 174-761, In yeast the corresponding proteins are involved along with Red51 in double strand breaks repair and homologous recombination [ 16, 771. It has been shown that overexprossion of the human RAD52 homologue eDNA in monkey cells could enhance gamma ray resistance aud could illduge homologous intrachromosomal recombination [781, RAD54 ¢ knock out mice are viable but exhibit !oniz~ ing radiation, m0omy¢in C and methyl methane snllbnate sensitivity as well as reduced rate of homologous rccombio nation 1791, The ~ame phenotype has I~en observed iu hoo nlczygous R A D 5 4 ' chicken D140 cells 180[, These resnlts indicate a stla)n~,~conservation of uunlerous proteins and t'~l~fit)n~ involved in recombination repair &ore yeast to human, They also imply that, besides DNA end~joining m ~ a t e d by ONA~depcndent protein kinase homologous ~,combination contributes to the repair of double smmd breaks in mammalian tells,

Conclusions Th~.,~ now exists evidence involving mammalian Rad51

protein in homologous recombination and DNA repair. However, its essential role in embryonic developorent and its association with tumor suppres~w proteins and a RNA polymerase II transcription complex tend to indicate that RadS I is not only involved in homologous recombination bul could have a pleiotropic role in rite cell. in particular its association with p53 is reminiscent of the role of RecA nucl¢opcotein filament in the induction of the SOS response in E coli mediated by the transcription repressor LexA cleavage, in this view it would be of great interest to (dent-

ify the proteins specifically associated with the Rad51 nueleoprotein filament in mammalian cells, Because RAD51 is essential in mouse it wonld be interesting to construct and express dominant negative RAD51 mutants in order to dissect the different functions and domains of the protein. If Rad5t plays a role in recombinmion, it may not be the only recombinase. Other RecA homologues have been identitied in yeast. DMCI,/'or instance has sequence homologies with ~ecA and has been specifically involved in meiotic homolngous recombination [81, 82]. Mouse and human DMCI homologues have been cloned suggesting a conservation of at least part of its functions 1831. Interestingly, it has been shown recently that the human Dmc I protein (HsDmc I ) catalyses the formation of Dloops as well as strand exchange between singleostranded and double-stranded oligonucleotkles 1841. Human XRCC2 and XRCC3 genes have been cloned o, |he basis of their ability to complement ionizing radiation and cross-linking agent sensitivities of hamster mutant cell lines 12, 85. 861, The corresponding proteins share some sequence homology with Rad51 protein. However, no evidence fi~r homologous recombination activity Ires been associated with these proteins. Even though it is possible that Rad51 could have some specific features following the studied organisms, the high conservation of its sequence allows to reasonably extend to human results obtained in nlammalian systems, The continued study of the role of this protein should bring important insights not only into homologous recombination but also into cell proliferation regulation.

References i l~d¢Intaun ~, K|i;2heiL~tfqai K I lqJ{'}~) Ride ol [ectnubinalioll cnl.viues l'h.-aaps~,t L|! t i'49(~} O".id..',ce fi~at |nallil!!tili[ui L'etl'~po~se~..~honlo-

Ingoas t'~2cotilhitl,~ltiol|idrepair pathways. Mttlal I~c.~3(13, 77..-88 3 Roca Al. Cox MM (1~}97)ReeA protein: slrilcnIFe, I'|mctiotl. alld rt)le in recolnhioatioflal DNA repair. P ~ g r Nm'h'ic A<'id Res Mal I~in156. 129~223 4 West SC t 1~)2) Enzymes and m(flecular mechanisms of genetic recombination. Atom Rev Bbwhem 61,603-640 5 Kowalczykowski SC. Dixon DA, Eggleston Ale,, l,auder SD, Rehtoner WM (1994) Biochemistry of htnllologoas feconlbhlation ill I~s'chef(chin coil, Mh'nddol Rev 58. 4(11--465 6 Shinohara A, Ogawa H, Ogawa T ( 19921 Rad51 protein iuvoived in repair and recombination in S cerevishw is a RecA-like protein. Cell 69, 457-470 7 Ab(~ussekhra A, Chanet R, Adjiri A. [:abre F (1992) Sere(dominant suppressors of Srs2 helicase mutations of Saccha~m~vces cererisiae map in tile RAD51 gene, x~.hoxe sequence predicts a protein with slmilaril(e~ l(~ pr~vcatyoticReeA proteins. Mol Cell Biol 12. 3224-+ 3234 S Basile G, Aker M, Mortimer RK (1992} Nuuleotide Sctlnenee anti Ilxmst.'rii)lioi~alrcgaialion ot tim yeast i'co,)nlhinaliOlla[ repair gelle RADSI, M,I Cell Ilhd 12, 3235-3246 9 Jang YK, Jin YH, Kim EM, Fabre E Hong SH, Park SD (1994) Cloniltg and sequence analysis of rhp5 I+, a Schi:osacchanm~vces pmnbe homolog of the Sm'charomyces cel~'vlshw RAD51 genel Gem, 142, 207-211

591 Ill Shinohara A. Ogal~ a I I. Mai~uda Y. LMm~ N. Ikeo K. ()~;ma ] t !9~;'3~ ('h;niug tff hilnlilll, nliiH%eand linshnl yeaxl [e~'lllllbinlniol'l gellex all) m,,h,gtmn m RAD51 and ~v,,.4, N.: d,,,,,: ,L 239 24 1 I I Muris DF. Vreeken K. C a n AM. B~'~m~hton B('. I_chnlaml AR. I.~hman PII. Pa,d,ink A 11q931 (.'h~nlllg lhe R.\D5~ homotogue t~l ,St'h/~'i~Xilt'('t~I'l~#gP, l'~ "~. p l ' t ~ b c Nm'h'i~ Ac&A Rcs 2 I. 4586-4591 I 2 0 g a ~ x a "|'. Shh'~ohara A. Nabelani A. Ike}:l T. Yu X. |'.gehlmn hH. Ogawa H t 1993) RecAqike i~,¢tllnllillalitnl protein,, m ctlkai~,acn: lilnCliOllSand stru¢l~.~res ~tff RAD51 geaes. Cohl Spri,,e Harb S3m P Onanl Bh# 58. 567-576 13 Yoshimtlrn Y, Morila T, Yamamolo A, Malsushiro A i 19931Cloniag anti ~,cquence tff lhc hnman RecA-like gene cl)NA, Nnch'ic Acids Re~ 21, 1665 14 Jachymczyk WJ, vtm Borstel RC, Mo,aat MR, ltastin~s PJ 119811 Repair of inlerSll'and cross-links in DNA of S~wchat~:mty~'c,~ cerevisiac requires tv, o syslems for DNA repair: the RAD3 syslenl anti tl~e RAD51 syslem. Meal Gelt Ch,~wl 182, 196-205 15 Resnick MAt 19871 Invesligating the geueli¢ conlml of biochenfical e%elllS ill Illeh'~lJ¢ rt.,c~,nllbiualion. IrE "]']lf h.l~'i~,~i~ {MoellS Pit. ed) Academic Press. New-¥in'k. 157-2111 16 i't.le~. TD. Malone Rh. Symmgtt,1 I.S t 1991 ). I.: TIic moh.ctth, hi. elegy and celhdar biolog3 o! the yeas! N~('~ Ila~m13ce*: grmmu' dy~1,111ic~. prol¢#: ~3"ntht'.~'L~a~ld e,cr~eti~'.'; {Bleach J R. Pri n gle J. Jones E. eds) Cold Spring Itarbor Lab. NY. 407-521 17 Sullg P (1994) Cahd) six t~l .,\TP-del)endcni honloh~[!lms DN/~ pairing aad nlri|nd exchange by 3¢a'-.I RADS[ protein, Scicm e 265. 12411243 18 Snng P. Robberson DL t 19951 DNA slrand exchange mediated by a RAD51-ssDNA nacleopmlein filament ~ilh polarily opposilc Io lhal of Re,2A. Cell 82,453 461 19 Sung P, SIl'it[Ion SA(1996) Y~asl Rad51 re¢onll'~ifulse nletliales polar DNA stnmd exchange m Ihe absence ~ff ATP hydrob sis..I Biol Che,I 27 I. 27983-27986 2{} Clever B, Inlerthal H. Sclunuckli-Maurer .I, King J, Sigrisl M, Ileyer WD 119971 Re¢ombiuational r,2pair in yeast: functional intera¢tionr, between Rad51 nntt Rad54 piotcins. EMBO .I {x 2535-2544 21 I lays SI., Firnlenieh AA. Berg P (19951 ('omplex t'ta'alali(al in yeaM d~nhle-slnmd break repair: parlicipalion of RadSI. Rad52. Rad55. and Rad57 r,'oleins. Pro(' Natl Acad Sci I/S.,~t 92, ('¢)25.6929 22 ,liaug FI, Xie Y. Ilouslon P, Stcmke-lhd`2 K. Mortcnscn UIt. R~thnl¢in R. Kt~dadek T { 199('0 Dire¢l asst~¢iation Ilelween the yeasl I~.:ad51 and Rad54 i'e¢tnnhinalitnl proteins. ,I Biol Chem 271, 33181 33186 23 Morila T, Yoshinn.a'a Y, ~l'alnalntllo A, MLllilia K. Muri M, Yillllanlolo tl. MalsLishirll A (It)93) A inotl~,e honn~h~g of lhe I'~ II,'~i~hi~t colt li*l'A ilUd .~th'l'Jhlt'o#tl~t'l's c,',vu/~/,a' RADSI ]k'll¢'~, t'll~l N"II ..h ,;d ,%'c/USA 91). 0577-0581) 24 liezztlbuva O, Shintflrm~ A. Mu¢ller RG. (.)gawa II. Bncrslcthle JM (1993) A chicken RAD51 htnntflt~u¢ i~ expressed at high levels in lynlphokl and reprodn¢tiv¢ orguns, Nm'h'ic Acids Res 21, 1577-1580 25 M~leshilna K. Mormlatsu K, Shinohara A. Horii T (1995) RAD51 htmlnlogues Jn Xenopu,~ hwv/s: 1we distin¢l [!enes al'e highly expressed in ovary and lestis. Ge/w 160. 195-2110 26 McKee BD, Ren X, Hong C (1996) A ~vcA-like gene in Drosoph/h~ mekmogasler lhal is expressed al high levels in female but nol male meiotic tissnes. CIiroaRisolno j(M, 479-488 27 Akaboshi E. Inoue Y. Rye H (1994) Cloning of the eDNA and generate DNA that correspoud to the wcA-like gene of Drosop/tih~ ,telanogaster. .Ipn J Genet 69, 663..-670 28 Ce~'LltliH, Osnlan M, Gnulth~ni P, Jilg¢lldl)rJ' AT (1992) A Iitnnnh~g of Esrherichia coli RecA protein in plastids of higher planls. Proe Natl Acad ,~'i USA 89. 8068-8072 29 Benson FE. Slaslak A, Wesl SC (1994) Purifiealion and characterization of Ihe hunlan R:~d51 protein, an analogue of E colt ItecA. EMBO ,I 13, 5764-577 I 31) B:nnnann P, Benson FE. Wesl SC (1996} Ihunan Rad51 prolein pwmotes ATP-dependent honlol,agoas pairing mid stnmd transfer reactions ill vitro. Cell 87, 757-766 31 Baumann P. West SC (1997)The human Rad51 protein: polarily of slrand tnnlsfcr aud stinmlalion by hRPA. EMBO J 16. 5198-5206

~2 (;uPL~ R('. galem,~l¢ I R . (i~qud~ I I b~addlag i x ~ t bJ971 \~I~ ilL~ Of hLInNIl~leclll~Ji*lil/Lllie~npllDlelil RddSa Piit~ ~,¢it/ ~l lad %¢1 ~ k .~ ~14 4h.' 46.'4

33 Shell Z, ('loud K(L I.]hcn D.|. P/ilk MS { 191}(l~h~lk'llth. Itl[~A~li, lll" bel'~.een die hkllllliil I~[AI)51 Lind RAI)52 plOlc'in, J' i¢i,!1 d blHt 271. H8-152 ~-1 I,i ~,1, Ileilknlilll ~|('. (;*~lttb El. Redd ~, (i. \~.did D(" R:iddJl/g (%1 Maizcls N tl9961 Rad5t cxplcsnion dild hie.ill/alton m B ~,:LL~ cart3 ing out class switch i-¢¢Olllhilhlliolt PI~I .Vat/ 4~eul S, i 158.1 93. 10222-10227 35 Haaf T, Golub El. Reddy (3. Radding CM. \~,ard DC i t995} Nuclcal ibci of illanllllaJian Rad51 reconlbinalion ['~l'(llc'inill ,OIIlil/it; cells ariel DNA dalllag¢ and ils k;cMizalion m s MiLiplollenlal ~omplexe, Pr., ~'%¢ltlAcad Sci I'SA 92. 2298 2302

36 Barlow AI.. Benson F. \Ve~i SC. Hulton MA 119971 Disiribumm oi lhe Rad51 recombinane in IltnllLlll and nloust_' Spelnlidoc}l¢~. I M B O .I 16. 5207-52t5 37 Kovalenko OV. Phlg AW. HaM "1\ Gonda DK. Aqfl¢5 T. \Vaid I)('. Raddmo CM. (;olal~, El (i9~16) Mammalian ubiquitu~-c~mlugalin~ ell/ynle Uhc9 inlel'tlClS with RadSI i'Ccolnbillalilnl pli)lcin and Iocilii/¢s ill s~llal'nonCnlal c'olnplexes. Proc NaIl .lclal 511 I %A Ok

2958 2963 l,g Shell ~', Ilaildin~hlll-JJtlrl~lntln PI]. (Tomeaux .1('. Xhl',/i~ RK, ('hvl/ DJ 1199¢'.) Anso..:ialionn of UBE21 with RAD52. U B I I . p53. mid RAD51 pn,einsinayeasllwo-h3brkls}slenI. G c m . m ~ 3 7 . 183 lSh 30 Sties Z. Pardinghm-Ptn'lynmn Pk. ('OllleinlX J([. ).lo)/in RK. (]hell DJ (199()1 U BLI, it hulniln uhiquilin-likc protein klx',ocldlln~2 I,~llh hanlan RADSI/RAD52 pl'oteillS.Gellolnics 36. 271 279 4(} flershkn A. Cieehanover A 11992) The uhiquilin ,,3stem t~r pmlcm dcgradalioa. Alnttt Rc.v Biocllem 61. 761 11117 41 LimDS, HastyPl1996)An~ulalioninmouserad51 re'.uh~,iuaneart5 etnbrvouic lethal that is suppres~,ed by il mutation in p5%..1¢ol Ucll Biol i6, 7133--7143 42 T,.uzuki '1".Fujii Y, Sakumi K. Tomiuaga Y. Nakal~ K. Sekiguchi M. Matr..ushirn A. ¥oshinlura 'f. Miwilal" 119961 lal~cted dlsiuplit,I el 111,2Ilad51 gene leads to lethality in en]brytmie mice. Pr¢~c .'Vail Ac ad Sci USA 93. 6236-6240 43 Taki I. Ohni,dn I. Y;nnanum~ A, Hiraga S. Arita N. I/ninuto bl. llayakav, a T. Morita T (19961 Anlinem, c inhibili~m el the RADSI enhallCCs ra,,liosenniti'.,ily. Bio~ l,,m tli~q#t~ ~ Rc~ Cerumen 223. ,134 4aS -14 rl'a,~hil'OS. Kt~hlnnH'i!N. Nhnlohlnll m. 'lhllakll K. t .2da K. Kanilad;i N I it)96) .% Ilhip.¢H3`2cifi¢ lill'lllillit)ll el Ih`2htanan l,~iltJ51l'~lolelllInltl%~iIl ' fiwi in lyinI!hn¢)Icn Om .grin' 12. 21652171! -15 'Y;,nan~.I. A, TaM T. Ya~.,iIt. IIabu I. "(t~qml;~ K. Y.~himura Y. ;'~mla molo K..Malnunhiltl A. Niqliunun¢ "', Molna I ~19*m)( 'ellQtlc tic

46 47

48 49 50

51

52

pendenl expt'es~inn el the nn~t,,,2 RadSI gent in pro[ili~rating c¢lb, M~I ~h', (h,m't 251, 1=12 Flygare J. Bensnn F. HellLtren J) 119%} Ibq3r¢~qon nl lhe humml RAD51 g0ne dnring the ¢¢11¢ycle it| I~rimary hinllan p,2riplleral hlnml ly mphn,2yt¢~. Bloc'him Biophy,~ Acre 1312.23 I, 2 ~(~ Chcn I< Naslasi A. Shen Z. Brennt~nlau M. (;Ini%sl]|all It. Chca DJ ( 19971 Cell cycle-dependent protein expressinn tlf nmmmalian homo logs of yeast DNA dottble-slrand break repair geacs Rad51 and Rad52 Ilu Process Cimtionl. Mr#st Res 384.2(!5-211 Kastan MB, Onyekwcre O. Sidransky 13. V~geb, tem B. Craig I1~' (1991) Participaliim of p53 protein in 111`2eelluhu' respnn~e In DNA damage. Cma'er Rex 5 I. 6304-631 [ Lall`2 DP t 19931 Cau¢cr, A death iu 111`2lil~.' t'd p53 I n e ~ ; ¢llllnnelltl, Nntl,v 302. 786-787 Hanseu R. Oren M t 1997) p53: fronl inducti~ e siL-'nalm cellular ¢t'1¢c1. Cm'r Opin Genet Dev 7, 40-51 Slurzb`2,,:htrrHW. rhmzehnann B. Henning W. Knipp~,chihl U. Bud~. Ill)1"~ S (199fl~ p53 is linked dire¢tl.', to [lt)uloh)gntl~, recnnlbinatitm pn~cess`2s rla Rad5111t¢¢A t'n'ol`2iu inlcraclion, hMHIJ ,/ 15. 191)2 2(1112 Buchh~p S. Gibson MK. Wang XW. Wagner P. Slurzb`2¢her IIW. Harris CC ~19971 Inlera¢lion of p53 with the human rad51 Illoleiu l In Pmc`2ss Citationl. Nm'h,ic Acids Res 25. 3808. 3874

592 53 I~rtrm~t P~ Roailla¢d D, Iloulel A. Levalois C, Soussi 1", Lopez BS 11997) Increav~ of slx}ntal~tms intrachromosomal homologous rec * ~ r a t k m }n mammalian cells expressing a mutant p53 protein. Oneugene 14. I I 17~ I 122 54 Meked KL, Tang W, Kachnie LA, Lap CM, DeFrank iS, Powell SN ( 19971 lubrication of p53 results in high rates of homologous recombi.~.~m, O m ~ , ! ~ !847+!857 55 S¢ldl~.'~ Chin .I; Plug A, Xiao Y. Weaver D. Feunteun J. Ashley T. [ivi~.~oa ~ I ~ ) As~iation of BRCAI with Red51 in mitotic and ~ k ' a i c cells; Cell 88~ 265-275 56 ~ SK, ~ m a l s u M. Albrecht U, Lira DS. Regel E. Dinh C. A. Ekt~le G. Hagy P. Bradley A (1997I Embryonic lethality radiatt~ hYl~-r~nsitivity mediated by RadSI in mice lacking l~'~a2. Nat~n" 386. 80-4-8Ill 57 Fetml¢tm J; I . ~ i r GM { 1 ~ } BRCAI. a gone invoh.ed in inherited p~d~%po~it~ to breast cud o~arian cancer. Bi~whim Bh~phys At:at 1242, 177- ~ 58 CJaylhcr SA. Mention J. Rus~U R Seal S. Bnrfi~ot R. Ponder BA. gwarton MR. Eaton D ( 10971 Variati{mof risk..;of breast and ovarian carKer a~ociatcd with different gennline mutations of the BRCA2 gone. h'a~ Goner 15. 103-105 5~ S~.~t.ukiA. de la Pompa JL Hakem R. Ella A. Yo~ida R. Mo R. Nishina H. Ch~ang 1". Wakeb..am A. Itie A. Koo W, Billia P, Ha A. Fukunvolo M, Hut CC, Mak TW (It~71 Been2 is required for embrymaic cnHlularproliferation in the mouse. Gene~ Dec I I, 1242-1252 ¢~1 Hakem R. de la Pompa JL, Sirard C. Mo R. Woo M. H~em A. Wake,ham A, Potter L Rei~mair A, Billia F, Firpo E, Hai CC. Roberts J. Ros~nt J; Mak TW {1996) The tumor suppres~r gone Brcul is I"¢qui~d fi~" ¢mlx3'o~ic cellular proliferation in the mouse. Co//85. 61 Rajah JV. W~ng M. ,Marquis ST, Ch~x:loshLA 119961 Been2 is e~mrdh~te|y regulated with Breal during proliferation and differentiatio, hi r~,m~l~, epithelial cells. Pmc Nail Acad &'i USA 93. 13(178130~3 R ~ , JV. MaRlUi~ST, Gardner HP, Chodo~ LA l 1~)7 ) l~elopment~l ~ s s i o n of BI~2 ¢olo~altz~s with Brcal and is usst~iuted with pmllfera~k~ and difl'~l~nll~lion ill muh!pl,0 tb,sues, l~.v liial 184, 39.~=-401 63 'l't~mlls JE, Sl~ilh M. Tt~ktnson JL. Rubinf¢ld B. Polakix O ( 1997; l~u..:tion of pho~phorylution ~m BRCAI durin~ the cell c~.cle aod al'tcr DNA d a a ~ e . Cell ¢imu'th Differ 8, 801-~i}~ M .%ally R. Cher~ J, ~ h s RL. Keegan K. Ih'.¢kslra M. Feaal~.aa J, l.iviugstntt DM Hq~?} Dynamic changes of BRCAI sabouclear I,., .:alion and phosldlorykRlou ~tate u~ iahialcd by DNA dalol!ge. ('ell ~-.'~ tt~kent R. d~ la P.,nl~ JL. ~lia A. I~tter L Mak TW 11997t Partial ~ of B~al i5-61 early embryonic lelhallty hy p53 or p21 null mul~llon. Ndt P~¢t~¢tI~ 298~3(t2 kud~wi~ T, Chapman DL. i%pai(~nnou VE, I~.l'slratiadisA I tt197~ l ~ t e d mulalio~ of b~ast cancer snscelribility gene hornpipes in m~e: klh~l p l ~ y p e s of Breul, Brca2. Brcal/Brca2, Brealtp53. a~l B~a~l~3 nulli~ygous embryos, Genes I)¢r I I, 122f~ 12.11 h7 Btugur~.~l~sJ, Jacks T 11997) I~uble i~demnily: p53. BRCA and ca~e¢, p33 mutation partially re~ncs developmental ~ s l in Breal at~ Bp~a2 uullmi~e, su~esting a role l\r" familial breast cancer gene~ in DNA damu~ ~B~ir. Nat Med 3. 726722 ~;~ Mil~er J. P ~ r B, Huglves.Davies L, Seltmauu M, Kouzarides T {Iq97l Tran~ril~inual activation I'unctio~s in BRCA2. Nutan. 31.16. ~2~773 69 Mont¢ir~ AN. August A. Hanafusu H t 19961Evidence fiwa transcript i r o l t~tivatkm tu~tion of BRCAI Cqermiaal rectum. Pna" A~etl Ac~ul,~'i USA 93, 13595~135~) 71t Mald~m~.OoE, Shiekhatlar R. Sheldon M. Cho II. Drapk,~ R. Rickert P, I.~x E. A ~ c ~ m CW. Ioinn S. Reinl~rg D t I ~ 0 A human RNA

polymemse II complex associated v;ith SRB and DNAqepair pro. teins. :~,kmov 381, 86-89 71 Sengstag C (1994) The role of mitotic re¢oalbination in carcinogenesis. Crit Rer T.xk'..124. 323-353 72 Flygare J. Orsan S. Hellgrea D ( 19971Exprcsslon of Red51 in human cells. Muu~t Res Fund Mol Meeh Muulgen tsuppl I ) 379. $62 73 Kaufmann SH. Desnayers S. Ouar/ano Y Davidson NE. Poirier GG t 1993) Specific proteolytic cleavage of poly (ADP-ribose) polymerage: an early marker of chemotherapy-induced apoptosis. Cancer Res 53. 3976-3985 74 Bezzubova OY. Schmidt H. Ostermann K. Heyer WD. Bue~tedde JM t 19931 Identification of a chicken RAD52 homologue suggests conservation of the RAD52 recombination pathway, throughout the evolution of higher eukaryotes. Naeleic Achls Res 2 I, 5945-5949 75 Muris DF. Bezzubova O. Buerstedde JM. Vreeken K. Balajee AS. Osgood CJ. Troelstra C. Hoeijmakers JH. Ostermann K. Schmidt H et al (19941 Cloning of human and mouse genes homologons :o RAD52. a yeast gone involved in DNA repair and recomh~:talion. Murat Res 315. 295-305 76 Kanaar R. Troelstra C. Swagemakers SM. Essers J Sum B. Franssen JH. Postink A. Bezznbo~a OY. Buerstedde .IM ~ lever B. Heyer WD. Hoeijmake~ JH ( 19961 Human and mou.:.: homolegs of the Saceharam)cox cereviskte RAD54 DNA rep:~r gone: evidence for functional conservation. Cure Bi.I 6, 828-83g 77 Friedherg EC. Siede W. Cooper AJ ( 1991 t. In: The molecular biology and cellular biology of the yeast S.cdt.mmyces: genuine dynamics. protein synthesis and energetics (Broach JR. Pringle J. Jones E. eds) Cold Spring Harbor Lab N¥. 147-192 78 Park MS (19951 Expression of human RAD52 confers resistance to ionizing radiation in mammalian cells, d Biol Chem 270. 154671J470 79 lessees J, Hendriks RW. Swagemakers SM. Troelstra C. de Wit J. Btmtsma D. Ht~ijmakers JH. Kanaar R ( 19971 Disruptkm of mouse RAD54 reduces ionizing radiation resistance and homologous ta:cmubiuation. CoX 89. 19.5-204 80 Bezzubova O. Silborgleil A. Yamaguchi-lwai Y. Takeda S. Buerstedde JM (1997) Reduced X-ra~_resistance and homologous recmnbJuation frequencies in a RAD54 - mutant of the chicken DT4(I cell line. Cell 89, 185 ~.!t13 81 Bisbap DK. Park D. Xu L. K!eckner N t It~921DMCI: a meiosis-specific yeast honlo[og of l: ~oli t~.c..I reqtlired for re¢onthhllnh)n, synap|oaemal complex, lbrmalion, and cell cycle progression ('¢tl 69. 439-.~51. 8~ Bishop DK t [i}t~) RecA hoaloh~gs I)n|ci tllltl RiRl.'ilit|tercel to h}rin multiple nuclear complexes prior to meiotic chromoson!e synupsis. Cell 79. 1081~!092 83 Habu ~KTaki T West A. Nishimuue Y. Morita 1" 09961 The mouse attd human hornpipes of DMCI. the yeast meiosis-sl~eifie homologous recombination gone. have a COUllrlonunique l'orm of cXOllskipped tran~ript in meiosis. Nach.u" Acids Res 2'4. 470~477 84 Li Z. Golub E. Gupta R. Radding C ( 1997} Recombiuation activities of HsDmel protein, the meiotic human honmlog of RecA protein. Pn~" Nail Acad Sci USA 94. 11221-11226 85 Tebbs RS. Zhao Y. Tucker JD. Seheerer JB. Siciliano MJ. Hwang M. Liu N. Legerski RJ. Thompson LIt ( It.~15}Correction of chromosomal instability and ~nsitivity to diverse mutagens by a cloned eDNA of the XRCC3 DNA repair geue. Pine Nail Acad Sci USA 92. 6354-6358 86 Liu N. Lamerdin JE. Sici[iano MJ. Carrauo AV (It.~SI Molecular cloning of the XRCC2 human DNA relxlir gone. which is similar to RAD51 of S cerrrishte, Am J ttam Gem,t (suppl 571. A 147 87 Doutriaux ME Coulcau E Befgougnioux C. White C 119981 Isolation and characterization.of the RAD51 and DMCI homologues fronl Arcthhkq~sis thtdimm, Mol Gen Goner, in press