Bacillus subtilis DprA Recruits RecA onto Single-stranded DNA and Mediates Annealing of ...

15 Pages·2013·2.6 MB·English

THEJOURNALOFBIOLOGICALCHEMISTRYVOL.288,NO.31,pp.22437–22450,August2,2013
©2013byTheAmericanSocietyforBiochemistryandMolecularBiology,Inc. PublishedintheU.S.A.
Bacillus subtilis DprA Recruits RecA onto Single-stranded DNA
and Mediates Annealing of Complementary Strands Coated
by SsbB and SsbA*
Receivedforpublication,April17,2013,andinrevisedform,June11,2013Published,JBCPapersinPress,June18,2013,DOI10.1074/jbc.M113.478347
TribhuwanYadav‡1,BegoñaCarrasco‡,JamesHejna§,YukiSuzuki§2,KunioTakeyasu§,andJuanC.Alonso‡3
Fromthe‡DepartamentodeBiotecnologíaMicrobiana,CentroNacionaldeBiotecnología,ConsejoSuperiordeInvestigaciones
Científicas,28049Madrid,Spainandthe§GraduateSchoolofBiostudies,KyotoUniversity,Yoshida-Konoe-cho,Sakyo-ku,
Kyoto606-8501,Japan
Background:DifferentmediatorsassistRecAtocatalyzegeneticrecombination.
Results:DprAfacilitatesthedisplacementofbothSSBs(SsbBandSsbA),increasesRecAnucleationontoSSB-coatedssDNA,
andmediatesDNAstrandannealing.
Conclusion:DprAfacilitatesRecA-mediatedstrandexchangeandannealscomplementarystrandscoatedbyanSSBprotein.
Significance:RecA-dependentchromosomaltransformationandRecA-independentplasmidtransformationrequirethecom-
petence-inducedDprAmediator.
Naturally transformable bacteria recombine internalized segmentsamongbacteria(1,2).Withfewexceptions,natural
ssDNA with a homologous resident duplex (chromosomal transformation, which is a widely distributed mechanism for
transformation)orcomplementaryinternalizedssDNAs(plas- geneticrecombination(GR)4inmanybacterialgenera,istran-
midorviraltransformation).Bacillussubtiliscompetence-in- siently induced (competence state) (1, 3). The competence
ducedDprA,RecA,SsbB,andSsbAproteinsareinvolvedinthe machineryactivelyprocessesexogenousdsDNAandtakesup
early processing of the internalized ssDNA, with DprA physi- the internalized ssDNA to replace homologous (or partially
callyinteractingwithRecA.SsbBandSsbAbindandmeltsec- homologous) chromosomal sequences in a mechanism cata-
ondarystructuresinssDNAbutlimitRecAloadingontossDNA. lyzedbyRecAwiththehelpofaccessoryfactors(2,3).TheGR
DprAbindstossDNAandfacilitatespartialdislodgingofboth mechanism, which entails unidirectional recombination of
single-stranded binding (SSB) proteins from ssDNA. In the genesbetweendifferentlineages,representsaformofbacterial
absence of homologous duplex DNA, DprA does not signifi- sexthatcontributestodiversity,adaptation,andalsotheemer-
cantlyincreaseRecAnucleationontoprotein-freessDNA.DprA gence of pathogens and antibiotic resistance variants (2). In
facilitatesRecAnucleationandfilamentextensionontoSsbB- Bacillussubtilis,onlyasmallfractionofcellsdifferentiateand
coatedorSsbBplusSsbA-coatedssDNA.DprAfacilitatesRecA- become naturally competent (1). These genetically pro-
mediatedDNAstrandexchangeinthepresenceofbothSSBpro- grammed cells have distinct physiological characteristics that
teins.DprA,whichplaysacrucialroleinplasmidtransformation, includethetransientinabilitytosynthesizeDNAofitssingle
annealscomplementarystrandspreferentiallycoatedbySsbBto genome or undergo cell division (1, 2). A common core of at
form duplex circular plasmid molecules. Our results provide a least 20 pole-localized proteins, which are either associated
mechanistic framework for conceptualizing the coordinated withthemembraneorcytosolic,aretemporarilyexpressedand
eventsmodulatedbySsbBinconcertwithSsbAandDprAthatare collectively dedicated to the internalization, processing, and
crucial for RecA-dependent chromosomal transformation and recombinationoftheincomingssDNAwiththeresidentchro-
RecA-independentplasmidtransformation. mosomeorwithitselftopromoteplasmidestablishment(1,2).
The membrane-associated proteins, which form the DNA
uptakemachinery,bindenvironmentaldsDNA,degradeoneof
Naturaltransformationandvirus-mediatedtransductionare thestrandstoproducessDNA,andinternalizeitintothecyto-
the main routes of horizontal transfer of chromosomal DNA sol(1).ThefunctionalDNAuptakemachineryisrequiredfor
thelocalizationofthecytosolicrecombinationproteinstothe
cellpole(4).
*ThisworkwassupportedinpartbyMinisteriodeEconomíayCompetitivi-
Thecytosoliccompetenceproteinsthatformtherecombi-
dad,DirecciónGeneraldeInvestigación,GrantBFU2012-39879-C02-01;
ComunidaddeMadridGrantS2009MAT-1507(toJ.C.A);Grant-in-aidfor nationmachinerycanbedividedintothosethatlocalizeatthe
Scientific Research on Innovative Areas “Virus-Host Cell Competency” entrypoleevenintheabsenceofexogenousdsDNA(e.g.SsbB,
(24115003)oftheMinistryofEducation,Culture,Sports,Science,andTech- DprA(alsotermedSmforCilB),RecA,CoiA,RecU,andRecX)
nology,Japan(toK.T.);andJapan-SpainBilateralJointProjectAwardJSPC-
andthosethatformadiscretefocusatthepole-localizedDNA
CSIC2010JP0017(toK.T.andJ.C.A.).
1Ph.D.FellowoftheInternationalFellowshipProgramofLaCaixaFoundation
(LaCaixa/CNB).
2Presentaddress:Dept.ofChemistry,GraduateSchoolofScience,KyotoUni- 4Theabbreviationsusedare:GR,geneticrecombination;AFM,atomicforce
versity,Sakyo-ku,Kyoto606-8502,Japan. microscopy;jm;jointmolecules;lds,lineardsDNA;lss,linearssDNA;nc,
3Towhomcorrespondenceshouldbeaddressed.Tel.:[phone];Fax: nickedcircular;css,circularssDNA;nt,nucleotide(s);SSA,singlestrand
34-91585-4506;E-mail:[email protected]. annealing;SSB,single-strandedbinding.
AUGUST2,2013•VOLUME288•NUMBER31 JOURNALOFBIOLOGICALCHEMISTRY 22437
This is an Open Access article under the CC BY license.
DprAHasTwoActivities
uptakemachinerywhendsDNAisadded(e.g.RecNandRecO) inhibitionexertedbytheSSBprotein(s).Duringdouble-strand
(4–7).Athirdcategorycomprisesproteins(e.g.SsbAandSms) break repair, RecBCD (counterpart of AddAB) resects the
Eco
whose polar location has not been studied but whose role is brokenendsandloadsRecA directlyontothessDNAasitis
Eco
assumed because their expression is induced during compe- generated, whereas with the RecOR or RecFOR media-
Eco Eco
tenceinsomebacterialspecies(2).InB.subtilis,RecA,SsbA, tors,endprocessingandrecombinaseloadingareuncoupled.In
RecU,RecO,RecN,RecX,andSmsareexpressedduringexpo- thatcase,RecOR loadsRecA ontoSSB -coatedssDNA
Eco Eco Eco
nentialgrowth,withRecAandSsbAalsotransientlyup-regu- ends, and RecFOR recruits RecA to the ssDNA/dsDNA
Eco Eco
lated during competence. In contrast, SsbB, DprA, and CoiA boundariesofgappedDNAcoatedbySSB (reviewedinRefs.
Eco
are specifically induced during competence development 14,18,19,21,and22).InB.subtilis,RecOaloneorinconcert
(reviewedinRef.2).Notethatunlessstatedotherwise,theindi- withRecRloadsRecAontoSsbA-coatedssDNA(11,23).Itwas
catedgenesandproductsareofB.subtilisorigin.Thenomen- hypothesized that AddAB and RecFOR might facilitate RecA
clatureusedtodenotetheoriginofproteinsfromotherbacteria loadingontogappedorprotein-freessDNA,respectively,but
isbasedonthebacterialgenusandspecies(e.g.Escherichiacoli theyarepoorlycharacterizedinB.subtilis(24–26).
RecAisreferredtoasRecA ). Unlike recombinational repair (see above), the DNA sub-
Eco
With few exceptions, competent bacteria express two SSB strateduringGRislinearssDNAthatcanbecoatedbySsbA,
proteins(SsbAandSsbB)(2).SsbAisanessential172-residue SsbB,oranyotherssDNA-bindingproteinuponexitfromthe
polypeptide that shares a significant degree of identity with entry channel (2, 3). Genetic data suggest that RecA, DprA,
SSB (8). SsbB is a 113-residue polypeptide that shares 63% SsbB, and RecO are necessary for natural transformation (2),
Eco
identity with the N-terminal DNA-binding domain of SsbA whereastheroleoftheessentialSsbAwasnottested.Togain
(aminoacids1–106),butithasadifferentC-terminaldomain. insight into the mechanism of RecA loading during chromo-
LikeSSB (reviewedin8,9),SsbAandSsbBaretetramersin somaltransformationandinplasmidestablishment,theroleof
Eco
solution and bind ssDNA as tetramers (10, 11). The crystal theubiquitousDprAproteinwasinvestigated.Weprovidebio-
structureoftwopoly(dT)35-ntoligomers(dT )boundtothe chemicalevidencethatDprAhastwodistinctactivities:(i)to
35
surface of tetrameric SsbB was determined (11). The overall facilitateRecAnucleationandRecA-ssDNAfilamentextension
arrangement of monomers within the SsbB-ssDNA complex onto ssDNA coated with SsbB and SsbA and (ii) to mediate
stronglyresemblesthatofSSB andHelicobacterpyloriSsbA ssDNAannealingofcomplementarystrandscoatedpreferen-
Eco
(SsbA )boundtossDNA(11–13),suggestingthatthestruc- tiallybySsbB.ThefirstactivityisessentialforRecA-dependent
Hpy
ture of SsbA will probably resemble SsbB (11). The primary chromosomaltransformation,andthesecondactivityofDprA
ssDNAbindingmodesofabacterialSSBproteinaredenotedas isrequiredtofacilitatetheannealingofcomplementarystrands
the SSB and SSB modes, where the subscript reflects the with subsequent dislodging of the SSB protein (RecA-inde-
65 35
average number of nt occluded by each tetramer in the SSB- pendentplasmidtransformation).
ssDNAcomplex(reviewedinRefs.8,9,and14).SsbAandSsbB,
EXPERIMENTALPROCEDURES
whicharestablehomotetramers,formedmixedcomplexeson
ssDNA (11), but the formation of heterotetramers was not Bacterial Strains and Plasmids—All B.subtilis strains used
detectedwhenSsbAandSsbBwereco-assembledonthesame are listed in Table 1. E.coli BL21(DE3)[pLysS] cells bearing
ssDNA molecule (11). SsbA binds homopolymeric dT in a pCB722ssbA,pCB777ssbB,orpCB596ssb underthecon-
80 SPP1
concentration-dependentmanner,withaK of(cid:1)0.2nMin trol of a phage T7 promoter were used to overexpress SsbA,
D(app)
thepresenceorabsenceofMg2(cid:2),whereasa6–8-foldexcessof SsbB, and Ssb proteins, respectively, as described (11, 27,
SPP1
SsbBwasneededtoreachitsrespectiveK undersimilar 28).pBT61,containingrecAunderthecontrolofitsownpro-
D(app)
experimentalconditions(11). moter,wasusedtooverexpressRecAinBG214cells(29).The
Recombination reactions catalyzed by the RecA family of PCR-amplified dprA gene was joined to NcoI-XhoI-cleaved
proteinsformanintegralpartofDNAmetabolisminallfree- pET21d to yield pCB888. The dprA construct includes an
living bacteria, archaea, and eukaryotes (15, 16). During the added hexahistidine affinity tag. The presence of a hexahisti-
natural transformation process, the major role of RecA is to dine tag at the C terminus of DprA rendered a protein that
form a helical nucleoprotein filament on the internalized complementedthenulldprAmutant((cid:3)dprA)strainforchromo-
ssDNAandtocatalyzeintegrationofthehomologousssDNA somaltransformation(datanotshown).PlasmidpCB888,carrying
intothegenomeofthecompetentcell(2,17).Theassemblyof thedprAgeneunderthecontrolofaphageT7promoter,wasused
the first subunits of RecA (nucleation) onto a ssDNA tract to overexpress DprA in E.coli BL21(DE3)[pLysS] cells. CsCl-
coatedbyaSSBproteinisrelativelyinfrequentandthusrate- purified pGEM3 Zf((cid:2)) plasmid dsDNA and circular pGEM3
limiting(15,18–20).AsetofRecAaccessoryfactorsisrequired Zf((cid:2))andpGEM3Zf((cid:4))ssDNAwereusedasrecombination
to stimulate RecA nucleation and filament extension. The substrates. If not stated otherwise, DNA quantities are
accessoryfactorsthatactbeforeRecAnucleationcanbesubdi- expressedasmolofnt.
videdintotwoclasses:theSSBproteins(SsbAandSsbB)that Enzymes,Reagents,andProteinPurification—Allchemicals
limit RecA nucleation, thereby suppressing unwanted RecA usedinthisstudywereofanalyticalgrade.Isopropyl1-thio-(cid:1)-
nucleoproteinfilaments,andtheRecAmediatorsthatfacilitate D-galactopyranosidewasfromCalbiochem,andPEI,DTT,and
RecAassembly(11,15,19,20). dATPwerefromSigma.DNArestrictionenzymes,ligase,etc.
DependingontheparticularsubstrateontowhichRecAisto were supplied by Roche Applied Science, New England Bio-
be loaded, different mediators are required to overcome the Labs, or Fermentas. DEAE, Q- and SP-Sepharose, Sephadex
22438 JOURNALOFBIOLOGICALCHEMISTRY VOLUME288•NUMBER31•AUGUST2,2013
DprAHasTwoActivities
G-100,andSuperose12werefromGEHealthcare,andphos- constant of 33–62 newtons/m. The scanning frequency was
phocellulose was from Whatman. [(cid:2)-32P]ATP was from 2–3Hz,andimageswerecapturedwiththeheightmodeina
PerkinElmerLifeSciences. 512(cid:5)512-pixelformat.Theobtainedimageswereplane-fitted
SsbA (18.7 kDa), SsbB (12.4 kDa), Ssb (17.0 kDa), and and flattened by the computer program accompanying the
SPP1
RecA(38.0kDa)proteinswerepurifiedasdescribed(11,28,29). imagingmodule.The“tipeffect”wasremovedusingtheappar-
DprA (32.7 kDa) protein was purified as follows. E.coli entsizeofDNAasareference.Volumeanalysiswascarriedout
BL21(DE3)[pLysS]/pCB888cellsweregrownat25°Ctomid- using Image SXM software (S.D. Barrett; available on the
exponentialphase,andexpressionwasinducedbyadding0.4 World Wide Web), and the histograms and Gaussian curves
mM isopropyl 1-thio-(cid:1)-D-galactopyranoside. Rifampicin (200 were drawn using Origin version 6 software (32). Image pro-
(cid:3)g/ml)wasaddedafter30minofisopropyl1-thio-(cid:1)-D-galac- cessingofthetopographsandheightmeasurementswereper-
topyranoside induction. Cells grew for an additional 180 min formedasdescribed(33).TherateofdissociationoftheDprA-
andwereharvestedbycentrifugation.Thecellpelletwasresus- ssDNAcomplexeswasmeasuredbyusingalkali-treatedfilters
pendedinBufferA(50mMpotassiumphosphatebuffer,pH7.5, asdescribedpreviously(11).
1mMDTT,10%glycerol)containing1mMPMSFand1MNaCl RecA dATP Hydrolysis Assays—The ssDNA-dependent
and lysed by sonication. After centrifugation, the 32.7-kDa dATP hydrolysis activity of RecA protein was performed as
DprAproteinwasfoundinthesolublefractionandloadedonto described previously (11). Absorbance measurements were
anNi2(cid:2)-chelatingcolumnequilibratedwithBufferAcontain- takenwithaShimadzuCPS-240Adualbeamspectrophotome-
ing1MNaCland5mMimidazole.DprAwaselutedwithalinear terequippedwithatemperaturecontrollerandsix-positioncell
gradient from 10 to 200 mM imidazole in Buffer A with 1 M chamber,andthecellpathlengthandbandpasswere1cmand
NaCl. Fractions containing DprA were pooled and dialyzed 2nm,respectively(11).TheregenerationofdATPfromdADP
againstBufferAcontaining400mMNaCl.DialyzedDprAwas and phosphoenolpyruvate driven by the oxidation of NADH
diluted 1:3 and loaded onto an SP-Sepharose column equili- canbefollowedbyadecreaseinabsorbanceat340nm.Ratesof
bratedwithBufferAcontaining150mMNaCl.DprAwaseluted ssDNA-dependent RecA-mediated dATP hydrolysis and the
with a linear gradient from 200 mM to 1 M NaCl in Buffer A. lagtimesweremeasuredinBufferD(50mMTris-HCl(pH7.5),
Fractions containing DprA were pooled and dialyzed against 1mMDTT,90mMNaCl,10mMMgOAc,50(cid:3)g/mlBSA,5%
Buffer A containing 300 mM NaCl and stored at (cid:4)20°C in glycerol)containing5mMdATPfor25minat37°Cina50-(cid:3)l
BufferAcontaining300mMNaCland50%glycerol. reaction volume. The orders of addition of 3,199-nt pGEM3
Allproteinswerepurifiedtogreaterthan98%homogeneity. Zf((cid:2))ssDNA(10(cid:3)M),thepurifiedproteins,andtheirconcen-
The NH termini of the purified proteins were sequenced by trationsareindicatedthroughout.AdATPregenerationsystem
2
automatic Edman degradation. The corresponding molar (0.5 mM phosphoenolpyruvate, 10 units/ml pyruvate kinase)
extinctioncoefficientsforSsbA,SsbB,Ssb ,RecA,andDprA and a coupling system (0.25 mM NADH, 10 units/ml lactate
SPP1
were calculated as 11,400, 13,000, 15,300, 15,200, and 45,000 dehydrogenase)werealsoincluded(11).TheamountofdADP
M(cid:4)1cm(cid:4)1,respectively,at280nm,asdescribedpreviously(29). wascalculatedasdescribedpreviously(11,34).
Theproteinconcentrationsweredeterminedusingtheafore- RecA-mediated dATP-dependent DNA Strand Exchange—
mentioned molar extinction coefficients. Quantities of RecA Under physiological Mg2(cid:2) ion concentrations ((cid:6)2 mM),
andDprAareexpressedasmolofproteinasmonomers,and RecA (reviewedinRef.18)orRecA(28)proteiniscompletely
Eco
quantitiesofSsbA,SsbB,andSsb areexpressedasmolof inactive for recombination activities, and the SSB proteins
SPP1
proteinastetramers.Throughoutthetext,theproteinconcen- occlude35nt(SSB bindingmode).However,underoptimal
35
trations are expressed in their stoichiometric ratios with RecAconditions(10mMMg2(cid:2)),theSSBproteinsocclude65nt,
ssDNA,whichisexpressedasmolofnt,whereasinthecorre- with the ssDNA wrapping around all four subunits of the
spondingfiguresandlegends,themolarconcentrationsofpro- tetramer(SSB )(8,9,14).Inthisstudy,theexperimentswere
65
teinsandssDNAarepresented. performedunderoptimalRecAconditions;hence,theSSBpro-
For DprA and RecA association, the proteins were loaded teinswereexpectedtobeintheSSB bindingmode.
65
ontoanNi2(cid:2)columnequilibratedinBufferB(50mMTris-HCl The3,199-bpKpnI-cleavedpGEM3Zf((cid:2))dsDNA(20(cid:3)Min
(pH7.5),1mMDTT,50mMNaCl,10mMMgOAc,5%glycerol) nt) and homologous circular 3,199-nt pGEM3 Zf((cid:2)) ssDNA
containing 2 mM dATP and washed and eluted as described (10(cid:3)Minnt)wereincubatedwiththeindicatedconcentrations
under“Results.” ofproteinorproteincombinationsinBufferDcontaining2mM
Protein and DNA Interactions—The formation of SsbA-, dATPfor60minat37°Cinafinalvolumeof20(cid:3)l.AdATP
SsbB-, or DprA-ssDNA complexes was measured by AFM in regenerationsystem(8units/mlofcreatinephosphokinaseand
BufferC(5mMHEPES(pH7.5),5mMEDTA,65mMNaCl,5% 8mMphosphocreatine)wasincludedasindicated.Thesamples
glycerol) containing 50 (cid:3)M spermidine. The circular 3,199-nt were deproteinized as described (27, 35) and separated on a
pGEM3Zf((cid:2))ssDNAwasincubatedwiththeindicatedprotein 0.8%agarosegelwithethidiumbromide.Thesignalwasquan-
for10minat37°Cina20-(cid:3)lreactionmixture.Afractionofthe tifiedusingaGelDoc(Bio-Rad)systemasdescribed(23).
samplewasdepositedonafreshlycleavedmicasurface,andthe DprA-mediated DNA Strand Annealing—A linear 440-bp
samplewasprocessedasdescribedpreviously(30).AFMobser- [(cid:2)-32P]dsDNA(8(cid:3)Minnt),derivedfrompGEM3Zf((cid:2)),was
vations were performed on a Nanoscope IIIa (Digital Instru- heat-denaturedfor10minat100°Candimmediatelyshiftedto
ments)inairusingthetappingmode.Thecantilever(OMCL- icewaterfor2min.Heat-denaturedssDNAwasincubatedwith
AC160TS-W2,Olympus)was160(cid:3)minlengthwithaspring increasing DprA concentrations or preincubated with SsbB,
AUGUST2,2013•VOLUME288•NUMBER31 JOURNALOFBIOLOGICALCHEMISTRY 22439
DprAHasTwoActivities
SsbA,orSsb atafixedconcentration(0.15(cid:3)M)for10minat unit.Totestthishypothesis,thevolumeoftheSsbAorSsbB
SPP1
30°CinBufferE(50mMTris-HCl,pH7.5,1mMDTT,2mM beadswasestimated.TheobservedvolumesofSsbA((cid:1)135(cid:7)
EDTA,100mMNaCl,50(cid:3)g/mlBSA,5%glycerol)asdescribed 30nm3)andSsbB((cid:1)109(cid:7)22nm3)wereingoodagreement
(10). Variable amounts of DprA (0.05–0.8 (cid:3)M) were then with(i)thetheoreticalvolumeoftetramericSsbA((cid:1)120nm3)
added,andreactionswereincubatedfor60min.Thecomplexes and SsbB ((cid:1)82 nm3) and (ii) the volume determinations for
formedweredeproteinizedasdescribed(27)andfractionated SsbAaswellasvolumesthatwerededucedfromtheco-crystal
through 6% PAGE. The signal was quantified as described structuresofSsbB-ssDNA,respectively(10,11).Atlowratios(1
above. SSB/320 to 64 nt), the SsbA or SsbB beads had a height of
ForAFManalysis,the3,199-ntpGEM3Zf((cid:2))anditscom- (cid:1)1.6(cid:7)0.20nm,andthisheightslightlyincreasedto(cid:1)1.8(cid:7)
plementarypGEM3Zf((cid:4))ssDNAwereincubatedinBufferC 0.18nmatahigherprotein-ssDNAratio(1SsbA/32nt)(Fig.
containing50(cid:3)Mspermidinewiththeindicatedprotein(s)for 1C).Theresults,showingbeadedmorphologyalongthecircu-
30minat30°Cina20-(cid:3)lreactionmixture.Afractionofthe larDNA,suggestthatssDNAwoundaroundanSsbAorSsbB
samplewasdepositedonafreshlycleavedmicasurface,andthe tetramer.Atpresent,wecannotruleoutthepossibilitythatat
samplewasprocessedasdescribedpreviously(10). lowSSB/ssDNAratios,bothproteinsmaybindssDNAinone
binding mode and that at high SSB/ssDNA ratios, another
RESULTS
bindingmodemaybefavored.
Analysis of SsbA and SsbB Bound to ssDNA—Previously, it To establish whether there is a coordinate interaction
wasshownthatSsbA,SsbB,SSB ,andSsbA interactwith betweentheSSBproteins,SsbAorSsbBwasboundtossDNA,
Eco Spn
ssDNAintheSSB bindingmodeintheabsenceofMg2(cid:2)and andthenSsbBorSsbAwasaddedtomaintainthestoichiometry
35
with the SSB mode in the presence of Mg2(cid:2), but SsbB (1SSB/32nt).Regardlessofwhichproteinwasaddedfirst,there
65 Spn
bindshomopolymericssDNAinanSSB bindingmodeinthe were(cid:1)24(cid:7)3(SsbAaddedfirst)and(cid:1)26(cid:7)3(SsbBaddedfirst)
65
presenceortheabsenceofMg2(cid:2)(seeRefs.8,14,and36).We SSB beads/ssDNA molecule (data not shown). Because the
usedAFMtogaininsightintothemechanismbywhichssDNA numbersofSsbAorSsbBbeads,aloneorinconcert,observed
interactswithSsbAand/orSsbB.SSB-ssDNAbindingexperi- perssDNAmoleculearesimilarbut(cid:1)3-foldlowerthanifthe
mentswerecarriedoutintheabsenceofMg2(cid:2)(SSB mode) ssDNA lacked secondary structures (i.e. 3,199 nt/SSB (cid:1)90
35 35
withtheaimofdetectinganypossibledifferencebetweenSsbA beads),itislikelythatSsbAandSsbBbindtopreexistingssDNA
and SsbB proteins when bound to ssDNA (see Ref. 2). The tracts.
3,199-ntcircularssDNA(1nMinmolecules)behavedasadis- DprAPreferentiallyBindstossDNA—Toanalyzethemech-
ordered coil that made length measurements difficult. The anism by which DprA interacts with ssDNA, increasing con-
ssDNA usually appears smaller than normal in AFM images, centrationsoftheproteinwereincubatedwithssDNA,andthe
andthemeanheightofthecollapsedpGEM3Zf((cid:2))ssDNAwas complexeswerevisualizedbyAFM.DprA-ssDNAcomplexfor-
(cid:1)0.4nm,whichdeviatedfromthetheoreticalheightforssDNA mationwasdetectedatDprAconcentrationsaslowas0.1nM(1
(1nm). DprAdimer/ssDNAmolecule),butformationofDprA-dsDNA
SsbAorSsbBspecificallyboundtossDNA(Fig.1,AandB) complexes was not observed even at DprA concentrations as
butfailedtoformastablecomplexwithduplexDNA(10).At highas20nM(datanotshown).
low ratios (1 SsbA tetramer/320 nt), SsbA extended the col- DprA-boundssDNAformeddiscreteglobularstructureson
lapsedstateofthessDNAandfacilitatedtheformationofdis- ssDNA.Atlowratios(1DprA/320nt),DprAformedprotein-
crete beads of ssDNA-protein complexes, with an average as ssDNA complexes with an average of (cid:1)4 (cid:7) 1 DprA/ssDNA
lowas(cid:1)6(cid:7)2SsbAbeads/ssDNAmolecule(Fig.1A,n(cid:8)200). molecule (Fig. 1C, n (cid:8) 100), but at higher protein/ssDNA
At a ratio of 1 SsbA/64 nt, circular beaded complexes were ratios,DprAformedstructuresthatwerelargerthanexpected
moredenselypacked,withanaverageof(cid:1)24(cid:7)4SsbAbeads/ foradimer(Fig.1C,n(cid:8)200).BecausetheDprA-ssDNAcom-
ssDNA molecule (Fig. 1A). The number of SsbA beads plexincreasedinsizewithincreasedproteinconcentrations,we
per ssDNA molecule did not significantly increase at higher termed them “blobs” to differentiate them from the SSB-
SsbA/ssDNAratios(1SsbA/32nt)(Fig.1A). ssDNAbeadsthatweresimilarinshapeandsize.Atratiosof1
SsbBshowsa6–8-foldloweraffinityforssDNAwhencom- DprA/64to16nt,DprAformedlargeblobsonssDNAwithan
paredwithSsbA-ssDNA(11).InthepresenceoflimitingSsbB average of 1.2 DprA complexes/ssDNA molecule (Fig. 1C, c),
concentrations(1SsbB/320nt)onlyssDNAwasobserved(data implyingthatDprAboundtossDNAinteractedwithDprAfree
notshown).Ataratioof1SsbBtetramer/64nt,therewerean insolutiontoformadiscretehigherorderDprA-ssDNAcom-
averageof(cid:1)10(cid:7)2SsbBbeads/ssDNAmolecule(Fig.1B,n(cid:8) plex rather than nonspecific aggregates of proteins on DNA
150).Ataratioof1SsbB/32nt,thisincreasedto(cid:1)24(cid:7)4SsbB (see“Discussion”).
beads/ssDNA molecule (Fig. 1B), with no further increase at In our AFM images, the DprA structures were globular in
higherSsbA/ssDNAratios(1SsbA/16nt)(datanotshown). shape, and their height and width increased with increasing
The morphologies of the SsbA-ssDNA and SsbB-ssDNA proteinconcentrations(Fig.1C,candd).Thetheoreticalvol-
complexesweresimilarintheseexperiments(Fig.1,AandB) umeofDprAwas(cid:1)54nm3foramonomerand(cid:1)108nm3fora
andwereconsistentwithobservedAFMimagesoftetrameric dimer.ThevolumeoffreeDprA,whichdidnotvarywithpro-
SSB bound to different circular ssDNA molecules in the teinconcentrationundertheseexperimentalconditions,corre-
Eco
presence or absence of Mg2(cid:2), respectively (37). It is likely, latedwithDprAmonomers(Fig.1C,d,denotedbyanarrow).
therefore,thatanSsbAorSsbBtetrameristhessDNAbinding ThevolumeofDprA-ssDNAcomplexes,however,variedwith
22440 JOURNALOFBIOLOGICALCHEMISTRY VOLUME288•NUMBER31•AUGUST2,2013
DprAHasTwoActivities
FIGURE1.BindingofSsbA,SsbB,orDprAtossDNA.InA–C,3,199-ntpGEM3Zf((cid:2))ssDNA(0.1nMinssDNAmolecules)wasincubatedwithincreasing
concentrationsofSsbA(A),SsbB(B),orDprA(C)inBufferCcontaining50(cid:3)Mspermidinefor10minat37°C.InDandE,3,199-ntssDNAwaspreincubatedwith
5nMSsbA(D)orSsbB(E),for5mininBufferC,andthenincreasingamountsofDprAwereadded,andthereactionwasfurtherincubatedfor5minat37°Cin
a20-(cid:3)lvolumeinBufferCcontaining50(cid:3)Mspermidine.Afractionofthesamplewasdepositedontofreshlycleavedmicaandprocessedasdescribedunder
“ExperimentalProcedures.”Eachexperimentwascarriedoutmorethanthreetimeswithsimilarresults.InBandC,awhitearrow denotestherespectivefree
proteininsolution,andinC,thewhitearrowheadindicatestheintermolecularbridgingofatleasttwossDNAmoleculesbyaDprAblob.Scalebars(cid:8)100nm.
protein concentration. At low DprA concentrations, two dis- number of SsbA and SsbB beads per ssDNA molecule was
creteDprAsubpopulationswithvolumesof(cid:1)80and140nm3 reducedto15(cid:7)3and13(cid:7)2,respectively(Fig.1,D(c)andE
that might correlate with DprA monomers and dimers were (c)). The DprA blob increased in size with increasing protein
observed (Fig. 1C, a). At high protein concentrations, large concentrations.At2:1DprA/SSBratios,theclearlydistinguish-
DprAaggregatesboundtooneormoressDNAmoleculeswere ableDprAblobsfurtherdislodgedSSBbeadsfromthessDNA.
observedwithavolumeincreaseof10–60-fold(Fig.1C,d). InthepresenceofDprA,thenumberofSsbAorSsbBbeadsper
DprA Promotes Limited SSB Dislodging from ssDNA—To ssDNAmoleculewasreducedfrom(cid:1)24(cid:7)4to(cid:1)13 (cid:7)1and
gaininsightintotheDprAinteractionwithssDNAprecoated (cid:1)10(cid:7)2beads/ssDNAmolecule,respectively(Fig.1,D(d)and
bySsbAorSsbB,ssDNAwaspreincubatedwithanSSBprotein E (d)). Similar results were observed when stoichiometric
at ratios of 1 SSB/64 nt, and then variable amounts of DprA amounts of SsbA and SsbB (1 SSB/32 nt) were co-assembled
were added (Fig. 1, D and E). At low (1:10) DprA/SSB molar ontossDNAandthenDprA(1DprA/(cid:1)64nt)wasadded(data
ratios, the SsbA or SsbB beaded complexes were similar to notshown).
thoseformedintheabsenceofDprA(Fig.1,AandB).Whenthe DprADoesNotFacilitateRecANucleationontossDNA—Rel-
DprAconcentrationwasincreasedupto1:1ratios,theaverage ative to RecA assembly and RecA-mediated DNA strand
AUGUST2,2013•VOLUME288•NUMBER31 JOURNALOFBIOLOGICALCHEMISTRY 22441
DprAHasTwoActivities
exchange with dATP as a cofactor, rATP-bound RecA
decreases filament formation onto SsbA-coated ssDNA by
about 8-fold and impairs the DNA strand exchange between
SsbA-coatedssDNAandhomologousduplexDNAbyroughly
15-fold; thus, dATP may be the preferred cofactor for RecA
activitiesatleastinvitro(28).Therefore,RecAnucleationand
filamentgrowthwereanalyzedinthepresenceofdATP.
IntheabsenceofhomologousduplexDNA,dATPbindingby
RecAisessentialforRecAnucleationontossDNA,anddATP
hydrolysismakesRecA-ssDNAfilamentsdynamic;bothsteps
arecrucialforasuccessfulGRreaction(reviewedin2).Mea-
suringtheinitialrateofdATPhydrolysisprovidesanindirect
measure of RecA nucleation, and the dynamic self-assembly
ontossDNAwithsubsequentdisassemblyfromthisssDNAlat-
tice is inferred by steady state of dATP hydrolysis (RecA-
ssDNA filament formation) (11, 28, 38–40). The lag time of
nucleation is derived from the time intercept of the linear
regression based on the steady state rate of dATP hydrolysis
(34).
Ataratioof1RecAmonomer/12nt,therateofRecAnucle-
ation onto ssDNA and subsequent filament formation was
biphasic,withaslownucleationstep(4(cid:7)0.5-minlagphase).
Afternucleation,theRecAfilamentsformedquickly,hydrolyz-
ingdATPnearthepreviouslyobservedK of17.1(cid:7)0.4min(cid:4)1
cat
(Fig.2A)(28).
TotestwhetherDprAfacilitatesnucleationand/orRecAfil-
amentextensionontossDNA,therateofdATPhydrolysiswas
determined. In control experiments in the absence of RecA,
DprA(1DprA/11nt)didnotexhibitdATPhydrolysisactivity
whencomparedwiththemockreactionintheabsenceofboth
DprAandRecA(datanotshown),indicatingthatthehydrolysis
ofdATPobservedintheassayscanbesolelyattributedtothe
RecAprotein.
TheadditionoflowDprAconcentrations(1DprA/125to66
nt)didnotsignificantlyfacilitateRecAnucleationontossDNA
whencomparedwithRecAalone(Fig.2A,inset).DprAdidnot
FIGURE2.DprAinteractionwithRecAisnotsufficienttofacilitateRecA
significantly stimulate the rate of dATP hydrolysis when nucleationontossDNA.A,the3,199-ntpGEM3Zf((cid:2))ssDNA(10(cid:3)Minnt)
compared with RecA alone (Fig. 2A). It is likely that low waspreincubatedwithDprAinBufferDcontaining5mMdATP.ThenRecA
(0.8 (cid:3)M) was added, and the absorption was monitored for 25 min. The
DprAconcentrationsmightnotcontributetotheremovalof amountofdADPwascalculatedasdescribed(45).Allreactionswererepeated
regions of secondary structure within ssDNA to facilitate threeormoretimeswithsimilarresults.ThessDNA-dependentdATPhydro-
lysisbyRecAaloneisdenotedasabrokendottedline.Themeasuredlagtimes
RecAnucleation.
areplottedagainsttheconcentrationofDprA(inset).B,His-taggedDprA
WhentheDprAconcentrationapproachedparitywithRecA alone(1.5(cid:3)g,lanes2–4),RecAalone(1.5(cid:3)g,lanes5–7),orHis-taggedDprA
(1DprA/16ntand1RecA/12nt),thelagtimewassignificantly mixedwithRecA(1.5(cid:3)geach,lanes8–10)wereloadedontoa50-(cid:3)lNi2(cid:2)
increased(8(cid:7)0.6-minlagphase)(Fig.2A,inset),butthefinal mreticarionceodlupmronteaintsrowoemreteelmutpeedrawtuitrhe5in0(cid:3)BulofffeBruBf.fAerftBercoexntteaninsiinvegw1aMsNhianCgl,atnhde
rate of RecA dATP hydrolysis did not significantly change 0.5Mimidazole,separatedbySDS-PAGE,andstainedwithCoomassieBlue.In
(16.7(cid:7)0.5min(cid:4)1)(Fig.2A).Furthermore,atequimolarcon- lane1,RecAandHis-taggedDprAproteinswereloadedasacontrol(1.5(cid:3)gof
eachprotein).FL,flow-through;W,secondwash;E,elutionwithimidazole.C,
centrationswithRecA(1DprA/11ntand1RecA/12nt),DprA different ssDNA concentrations were preincubated with increasing DprA
reduced nucleation (10.5 (cid:7) 0.3-min lag phase), and the final concentrations(5min),andthenRecA(0.8(cid:3)M)wasadded,andthessDNA-
rateofRecAdATPhydrolysisalsodecreased(13.8(cid:7)0.2min(cid:4)1) dependentdATPaseactivityofRecAwasmeasuredfor25minat37°C.The
activityofRecAaloneisdenotedasabrokendottedline.
(Fig.2A).ItislikelythatDprA,ataboutequimolarconcentra-
tionswithRecA,antagonizeditspolymerizationontossDNA. withtheNi2(cid:2)columnwasobserved(Fig.2B,lane10),indicat-
Previously,itwasshownbyFörsterresonanceenergytrans- ingthattheinteractionofRecAwiththeresinwasdependent
ferthatDprAinteractswithRecAinvivo(4).Totestwhether onthepresenceofDprA.However,theadditionofRecApar-
DprAphysicallyinteractedwithRecAinvitro,weusedaNi2(cid:2) tiallydestabilizedtheHis-taggedDprA-matrixinteraction(Fig.
column and purified His-tagged DprA and native RecA (Fig. 2B,lane8),suggestingthatDprAmakesspecificprotein-pro-
2B).Asexpected,DprAwasretainedontheNi2(cid:2)column(Fig. teininteractionsthatsequesterRecA.Indeed,whenthestoichi-
2B,lane4),butRecAwaspresentintheflow-through(Fig.2B, ometryofthereactionwasaltered,byincreasingRecA(datanot
lane5,FT).InthepresenceofDprA,theassociationofRecA shown)orthessDNAconcentration(Fig.2C),theantagonism
22442 JOURNALOFBIOLOGICALCHEMISTRY VOLUME288•NUMBER31•AUGUST2,2013
DprAHasTwoActivities
exerted by DprA on RecA nucleation and filament extension
wasoverridden.ThesedatasuggestthattheinhibitionofRecA
loadingontossDNAathighDprAconcentrationsmightbedue
toaninteractionbetweenDprAandRecAthatformsaninac-
tivebinarycomplex.Thisseemsmoreplausiblethanthepossi-
bilitythattracesofaputative“inhibitoryfactor”presentinthe
DprApreparationinhibiteddATPhydrolysis.
DprAandSsbBInteractionFacilitatesRecANucleationonto
ssDNA—Förster resonance energy transfer suggested that
DprA physically interacts with SsbB in vivo during GR (4),
althoughthisinteractionwasnotdetectedinourinvitrostud-
ies. To test whether DprA stimulated RecA nucleation onto
ssDNAcoatedbyaSSBprotein,thekineticsofDprA-mediated
nucleationofRecAontoSsbB-orSsbA-coatedssDNAwasana-
lyzedbymeasuringRecA-mediatedhydrolysisofdATP(Fig.3).
At a ratio of 1 RecA monomer/12 nt, RecA nucleation onto
ssDNA was slow (4 (cid:7) 0.5-min lag phase) (Fig. 3, inset), and
subsequentfilamentextensionapproachedthemaximalrateof
dATPhydrolysis(17(cid:7)0.5min(cid:4)1).Incontrolexperimentsin
theabsenceofRecA,noneoftheSSBproteinexhibiteddATP
hydrolysisactivity(datanotshown).
PreincubationofssDNAwithSsbBorSsbA,sufficienttosat-
uratethebindingsites(1SSB/33nt)delayedthenucleationof
RecAandreducedtheRecAfilamentextensionphase,albeitto
differentextents(Fig.3,AandC,inset)(11).Independentofthe
orderofaddition,SsbB(1SsbB/33nt)andDprA(1DprA/33to
125nt)addedpriortoRecAsignificantlyreducedthelagtimeto
(cid:1)1 min when compared with RecA nucleation onto SsbB-
coatedssDNA((cid:1)8-minlagtime)(Fig.3,A(inset)andB).Under
thisconditionanddependingonthestoichiometryofthereac-
tion, the final rate of RecA dATP hydrolysis approached the
maximumrateof25–30min(cid:4)1(Fig.3,AandB).Theseresults
suggestthatDprA,bypromotingpartialSsbBdislodging(Fig.
1E),facilitatesnewRecAnucleationeventsandtherebyaccel-
erates the dynamic assembly and disassembly of RecA from
FIGURE3.SsbBorSsbAandDprAplayaroleintherate-limitingnucle-
SsbB-coatedssDNAwithsubsequenthydrolysisofdATP(see ationofRecA.A,the3,199-ntpGEM3Zf((cid:2))ssDNA(10(cid:3)Minnt)waspreincu-
Fig.3,AandB). batedwith0.3(cid:3)MSsbB(5min)andincubatedwithincreasingDprAconcen-
trations.ThenRecA(0.8(cid:3)M)wasadded,andthessDNA-dependentdATPase
In vivo, the absence of SsbB resulted in only a 5–10-fold
activitywasmeasuredfor25min.Themeasuredlagtimesareplottedagainst
reduction in chromosomal and plasmid transformation (2), theconcentrationofDprA(inset).Thefilledsquaredenotesthelagtimeof
suggesting that the essential SsbA, perhaps in concert with RecAalone.B,ssDNAwaspreincubated(5min)withincreasingDprAconcen-
trationsandthenincubatedwithSsbB(0.3(cid:3)M),andthessDNA-dependent
SsbB, might play a role in RecA nucleation. Adding SsbA (1 dATPaseactivityofRecA(0.8(cid:3)M)wasmeasuredfor25minat37°C.C,ssDNA
SsbA/33 nt) and DprA (1 DprA/66–125 nt) prior to RecA waspreincubatedwith0.3(cid:3)MSsbA(5min)andincubatedwithincreasing
reversed the SsbA-mediated antagonism of RecA nucleation DprAconcentrations.ThenRecA(0.8(cid:3)M)wasadded,andthessDNA-depen-
dentdATPaseactivitywasmeasuredfor25min.Allreactionswererepeated
(Fig.3C).ThepresenceofDprA(1DprA/125to33nt)didnot threeormoretimeswithsimilarresults.TheamountofdADPandthelagtime
facilitateRecAnucleationontoSsbA-coatedssDNA(lagtime werecalculatedasdescribedinthelegendtoFig.2.ThessDNA-dependent
dATPhydrolysisbyRecAaloneisdenotedasabrokendottedline,andthe
(cid:1)5(cid:7)1min)whencomparedwithRecAalone(Fig.3C,inset).
hydrolysisinthepresenceofanSSBproteinisrepresentedbyabrokenline.
InthepresenceoflowDprAconcentrations(1DprA/125to66
nt),thefinalratesofdATPhydrolysis(17.2(cid:7)0.3min(cid:4)1)were ysis((cid:9)9min)andthefinalrate(13(cid:7)0.5min(cid:4)1)decreased
comparablewiththefinalratewithRecAalone(Fig.3C).The to levels comparable with those of reactions lacking DprA
addition of DprA (1 DprA/33 nt) to the preformed SsbA- andcontainingSsbA(Fig.3C).
ssDNA complex increased the final rate of dATP hydrolysis DprA Facilitates RecA Nucleation onto SsbB- and SsbA-
(25 (cid:7) 0.5 min(cid:4)1) to a level comparable with the rate in the coatedssDNA—TotestwhetherbothSSBproteins(1SSB/33or
presence of SsbB (Fig. 3A). These results reveal a difference 16nt)affectedtheabilityofDprAtofacilitateRecAnucleation,
betweenDprAandDprA .UnlikeDprA(Figs.2and3),itwas SsbAwaspreincubatedwithssDNA,andSsbBwasadded(or
Spn
suggested that DprA binds to the protein-free ssDNA and vice versa), followed by the addition of DprA. RecA was then
Spn
nucleatesRecA ontossDNA(seeRef.41).WhentheDprA added, and RecA-mediated dATP hydrolysis was monitored
Eco
concentrations approached the RecA concentrations (1 (Fig.4A).AddingSsbAandSsbBpriortoRecAdelayednucle-
DprA/16to11nt),thelaginRecA-mediateddATPhydrol- ationby2.5–3-fold(Fig.4A)andreducedittolevelscompara-
AUGUST2,2013•VOLUME288•NUMBER31 JOURNALOFBIOLOGICALCHEMISTRY 22443
DprAHasTwoActivities
DprAOvercomestheInterferenceofaNon-cognateSSBPro-
tein with RecA Nucleation—To test whether DprA bound to
ssDNAconstrainedthediffusionofSSBalongthessDNAlat-
ticeandfacilitateditsspontaneousdislodging,thesteadystate
rateofRecA-mediateddATPhydrolysiswasmeasuredinthe
presenceofanon-cognateSSBprotein(Ssb )(Fig.3B).Of
SPP1
interestisthefactthatSsbAandSsbBshareasimilardegreeof
identitywithSSB (177residueslong)andSsb (B.subtilis
Eco SPP1
SPP1virus-encodedprotein,159residueslong)attheirN-ter-
minaldomains.SsbAshares39and40%identity,respectively,
inthefirst130residues,andSsbBshares40and39%identity
with SSB and Ssb in their first 105 or 111 residues,
Eco SPP1
respectively. Little homology was revealed at the C-terminal
domain except for the acidic C-terminal tail. It is absent in
SsbB.
PreincubationofssDNAwithsufficientSsb tosaturate
SPP1
the binding sites (1 Ssb /33 nt) retarded the nucleation of
SPP1
RecA onto ssDNA, with a lag phase of 7–8 min, and also
reducedtheRecAfilamentextension(12.4(cid:7)0.4min(cid:4)1)(Fig.
4B)(23).ThepresenceofDprAreversedtheinhibitoryeffect
exerted by Ssb on RecA nucleation onto Ssb -coated
SPP1 SPP1
ssDNA(Fig.4B),suggestingthatDprAmightfacilitatepartial
displacementofanon-cognateSSB(Ssb protein).
SPP1
DprAFacilitatesRecA-mediatedDNAStrandExchange—To
FIGURE4.DprAfacilitatesRecAloadingonSsbA-ssDNA-SsbBcomplexes. understand the role of DprA in RecA-mediated DNA strand
A,the3,199-ntpGEM3Zf((cid:2))ssDNA(10(cid:3)Minnt)waspreincubatedwithSsbA exchange, we performed three-strand exchange assays in the
andthenwithSsbB(0.3(cid:3)M)inBufferDcontaining5mMdATP.Increasing
DprAconcentrationswereaddedandincubatedfor5min.RecA(0.8(cid:3)M)was presenceofdATPasanucleotidecofactor(Fig.5).RecAiniti-
thenadded,andtheabsorptionwasmeasuredfor25min.B,the3,199-nt ates DNA strand exchange by pairing the free end of the lds
swsiDthNAD(p1r0A(cid:3).MThinennt)RweacsApr(e0i.n8cu(cid:3)bMa)tewdawsitahd0d.e3d(cid:3),MaSnsdbStPhPe1asnsdDtNhAe-ndienpceunbdateendt withthehomologouscircularcss,leadingtojmintermediates.
dATPaseactivitywasmeasuredfor25min.Allreactionswererepeatedthree Continued strand exchange in these hybrid complexes ulti-
ormoretimeswithsimilarresults.ThessDNA-dependentdATPhydrolysisby
matelygeneratesncdsDNAandlssproducts(Fig.5A).Inthe
RecAaloneisdenotedasabrokendottedline,andthehydrolysisinthepres-
enceofanSSBproteinisshownbyabrokenline. strand exchange reaction, the displaced lss product could be
furtherengagedinareversereactionwithsubsequentreversion
ofthencproducttotheinitialsubstratesoranetworkofinter-
blewiththoseofSsbA(seeRef.11).TheadditionofDprA(1
linkedintermediatesifanSSBproteinisomittedinthereaction
DprA/33–66nt)tothepreformedSsbB-ssDNA-SsbAcomplex
mixture(reviewedinRef.21);hence,thestrandexchangereac-
markedlyshortenedtherate-limitingRecAnucleationtimeto
tionrarelygoestocompletion,andthespecificroleoftheRecA
(cid:1)1minandfacilitateddATPhydrolysis(RecApolymerization)
accessoryproteinisnotalwayseasytoevaluate.
(Fig. 4A), and the final rate increased (28(cid:7) 0.4 min(cid:4)1) com-
Invitro,RecApromotesasetofDNAstrandexchangereac-
paredwithSsbB-ssDNA-DprAcomplexes(Fig.3A).Thiseffect
tionsthatmimicsitspresumedroleinGR.Totesttheroleof
was independent of the order of addition of SsbA or SsbB.
DprAonRecA-mediatedDNAstrandexchange,wefirstper-
BecausethesecondSSBproteinwasaddedafterthefirstwas formedthethree-strandexchangeassaysinthepresenceoflow
already in a complex with ssDNA, formation of heterotetra- RecAconcentrations.LimitingRecA(1RecA/28nt)catalyzed
mericproteinswasunlikely. DNAstrandexchangebetweenthecssandtheldsDNA,con-
Collectively, these results suggest that (i) SSB-mediated verting(cid:6)10%ofthesubstrateintojmintermediatesduringa
removalofsecondarystructures,inthepresenceofDprA,was 60-minreaction(Fig.5B,lane2).Theadditionofhalf-saturat-
sufficient to accelerate RecA nucleation onto SsbB-coated or ingtosaturatingamountsofSsbA(1tetramer/66,33,or22nt)
SsbBplusSsbA-coatedssDNAand(ii)thespecificinteraction priortoRecAstimulatedRecAstrandexchange6–9-fold,with
of DprA with RecA and perhaps SsbB facilitates RecA nucle- ncproductsamountingto32.7(cid:7)0.2to42.9(cid:7)0.5%ofthetotal
ation onto a DprA-SsbB-ssDNA-SsbA complex more effi- (Fig. 5B, lanes 3–5). When SsbB replaced SsbA, a significant
ciently than a DprA-ssDNA-SsbA complex (Figs. 3, A and C, stimulation of RecA-mediated DNA strand exchange (4–5-
and4A).Alternatively,RecAnucleatesontotheDprA-ssDNA- fold)wasalsoobserved(ncproducts,21.8(cid:7)0.7to23.3(cid:7)0.4%)
SsbA complex with similar efficiency as the DprA-ssDNA- (Fig.5B,lanes6–8).ItislikelythattheSSBproteinaidsRecA-
SsbB,butintheformercase,thenegativeeffectexertedbySsbA mediated DNA strand exchange by facilitating spontaneous
was larger. The answer to this question is complex, because meltingofsecondarystructureonssDNAand/orbysequester-
varyingtherelativeconcentrationoftheSSBproteinsfacilitates ing the displaced lss DNA strand, albeit with different
RecAnucleationsimilarly. efficiency.
22444 JOURNALOFBIOLOGICALCHEMISTRY VOLUME288•NUMBER31•AUGUST2,2013
DprAHasTwoActivities
FIGURE5.DprAfacilitatesRecA-mediatedDNAstrandexchangeinthepresenceofbothSSBproteins.A,schemeofthethree-strandexchangereaction
betweencircularssDNA(css,inred)andthelinearduplex(lds,inblackandred)substrateandtheexpectedintermediates(jm)andfinalproducts(nc andlss)by
RecA-mediatedDNAstrandexchange.B,circular3,199-ntpGEM3Zf((cid:2))ssDNA(10(cid:3)Minnt)andhomologousKpnI-linearizeddsDNA(20(cid:3)Minnt)were
preincubatedwithincreasingconcentrationsofSsbA(0.15,0.3,and0.45(cid:3)M;lanes3–5),SsbB(0.15,0.3,and0.45(cid:3)M;lanes6–8),andDprA(0.15,0.3,and0.45
(cid:3)M;lanes9–11)for10minat37°CinBufferDcontaining2mMdATP.ThecircularssDNAandhomologousdsDNAwerepreincubatedwithaconstantamount
ofSsbA(0.15(cid:3)Minlanes12–14)orSsbB(0.15(cid:3)Minlanes15–17).ThenincreasingconcentrationsofDprA(0.15,0.3,and0.45(cid:3)M)wereaddedandincubatedfor
10minat37°C.ThenaconstantamountofRecA(0.35(cid:3)M;lanes2–17)wasadded,andthereactionwasincubatedfor60minat37°C.C,circularssDNAand
homologouslineardsDNAwerepreincubatedwithdecreasingconcentrationsofSsbBandthenincreasingconcentrationsofSsbA(lanes3–6)orviceversa
(lanes8–11)for5minat37°CinBufferDcontaining2mMdATP.ThecomplexwasincubatedwithaconstantamountofDprA(0.1(cid:3)M;lanes3–12)for5minat
37°C,followedbytheadditionofaconstantamountofRecA(0.7(cid:3)M;lanes2–12)andincubatedfor60minat37°C.Theproductsofthereactionswere
deproteinized,separated,andquantifiedasdescribedunder“ExperimentalProcedures.”Thepositionsofthebandscorrespondingtocss,cds,lds,nc,andjm
areindicated.(cid:2)or(cid:4),presenceorabsenceoftheindicatedprotein.Dataofrecombinationproducts(jm,nc,orthesumofboth)areindicatedaspercentages
andaretheaveragevaluesobtainedfrommorethanthreeindependentexperiments(theresultsgivenarewithina5%S.E.).
TheadditionofDprA(1DprA/66,33,or22nt)priortolim- tion (with average nc products, 21.6 (cid:7) 0.8%) (Fig. 5B, lanes
iting RecA stimulated RecA-mediated DNA strand exchange 9–11). It is likely that DprA facilitates RecA-mediated DNA
(4–5-fold),butthiseffectwasinsensitivetoDprAconcentra- strandexchange,butthemechanismispoorlyunderstood.To
AUGUST2,2013•VOLUME288•NUMBER31 JOURNALOFBIOLOGICALCHEMISTRY 22445
DprAHasTwoActivities
test whether DprA could recruit RecA onto SsbA- or SsbB- TABLE1
coated ssDNA, RecA-mediated strand exchange in the pres- TheabsenceofRecAandDprAimpairsgeneticrecombination
ence of a constant amount of SSB protein (1 SSB/66 nt) and Normalized Normalized
Genotype chromosomal plasmid
increasingDprAconcentrationswasanalyzed.Inthepresence
(strain) transformationa transformationb
ofSsbAandlowDprAconcentrations(1DprA/66nt),theaccu-
rec(cid:2)(BG214)c 100 100
mulation of nc product by RecA-mediated DNA strand (cid:3)recA(BG190) (cid:6)0.01d 97d
exchange was increased and reached levels comparable with (cid:3)recO(BG439) 48d 3.0d
(cid:3)dprA(BG1163) 1.7d 2.5d
RecAplusSsbA(Fig.5B,lanes3–5and12).Inthepresenceof (cid:3)recO(cid:3)dprA(BG649) (cid:6)0.01d (cid:6)0.1d
SsbA and higher DprA concentrations (1 DprA/33 or 22 nt) (cid:3)recO(cid:3)recA(BG1165) (cid:6)0.01d 44d
(cid:3)dprA(cid:3)recA(BG1291) (cid:6)0.01 0.5
equimolar with RecA (1 RecA/28 nt), the accumulation of nc
aTheyieldofmet(cid:2)transformants(chromosomaltransformation)wascorrected
productsdecreased(35.0(cid:7)0.1and33.1(cid:7)0.4%)(Fig.5B,lanes forDNAuptakeandcellviability,andthevaluesobtainedwerenormalizedrela-
13and14).ItislikelythatinthepresenceofSsbA,anexcessof tivetotherec(cid:2)strain,takenas100.Theresultsaretheaverageofatleastfive
independentexperimentsandarewithina10%S.E.
DprA contributed to RecA dislodging, as described for RecA bTheyieldofpUB110kanamycin-resistanttransformants(plasmidtransforma-
nucleation(seeabove).WhenSsbBreplacedSsbA,DprAmod- tion)wascorrectedforDNAuptakeandcellviability,andthevaluesobtained
werenormalizedrelativetotherec(cid:2)strain,takenas100.Theresultsaretheav-
erately increased RecA-mediated DNA strand exchange (Fig. erageofatleastfiveindependentexperimentsandarewithina10%S.E.
5B,lanes15–17). cThegenotypeoftherec(cid:2)strainanditsisogenicrec-deficientderivativesistrpCE
metA5amyE1ytsJ1rsbV37xre1xkdA1attSP(cid:1)attICEBs1.
ThepresenceofsuboptimalRecA(1RecA/14nt)catalyzed dThetransformationfrequenciesof(cid:3)recA,(cid:3)recO,(cid:3)dprA,(cid:3)recO(cid:3)dprA,and
strandexchangebetweenthecssandtheldsDNA,converting (cid:3)recO(cid:3)recAwerereportedelsewhere(11,31,46)anddeterminedhereagain
27.4(cid:7)0.6%ofthehomologousldsDNAintoncandlssprod- fordirectcomparison.
uctsduringa60-minreaction(Fig.5C,lane2).Totestwhether
limiting DprA concentrations could facilitate RecA-mediated pressed the need for RecO during plasmid transformation
DNAstrandexchangewithanssDNAsubstratecoatedbySsbA (Table 1) (11). However, plasmid transformation was also
andSsbB,thessDNAwaspreincubatedwithvariousconcen- impairedin(cid:3)dprA(cid:3)recAcells(Table1),suggestingthatDprA
trationsofbothSSBproteins(1tetramer/66,33,or22nt).SSB- isrequiredforplasmidtransformation.
boundcssDNAwasincubatedwithldsDNAandlimitingDprA DprA Facilitates Annealing of Protein-free or SSB-coated
concentrations (1 DprA/100 nt), followed by the addition of DNAStrands—Aboveitwasshownthat(i)SsbAorSsbBbound
suboptimalRecAconcentrations(1RecA/14nt).Intheabsence tossDNAfailedtobridgetwonon-complementaryDNAmol-
of SSB proteins, the effect of DprA on RecA-mediated DNA eculesbyadirectprotein-proteininteraction(Fig.1,AandB),
strandexchangewasnotmarkedlyincreasedwhencompared (ii) DprA on one ssDNA molecule interacted with DprA-
withRecAalone(Fig.5C,lanes2and12).TheadditionofDprA ssDNA on a second ssDNA molecule and bridged them (Fig.
tothepreformedSsbA-ssDNA,SsbB-ssDNA,orSsbB-ssDNA- 1C,denotedbyanarrowhead),and(iii)DprAisnecessaryfor
SsbA complexes significantly increased RecA-mediated DNA effective plasmid transformation (Table 1). However, it was
strandexchange(Fig.5C,lanes3–11).Theorderofadditionof reported that DprA was unable to catalyze ssDNA annealing
theSSBproteinsmightplayaminorroleifany(Fig.5C).Itis underconditionswhereDprA does(seeRef.3).Wetested
Spn
likely that the substrate for RecA-mediated DNA strand whether DprA is able to catalyze SSA, using heat-denatured
exchange is SSB-coated ssDNA rather than protein-free 440-nt-longcomplementaryssDNAstrandscoatedornotbyan
ssDNA. SSBproteinassubstrate.
DprA Is Important for Plasmid Transformation—DprA is InthepresenceorabsenceofMg2(cid:2),spontaneousreanneal-
requiredforchromosomaltransformationindifferentbacterial ingoftheprotein-free440-ntDNAstrandswasmeasuredtobe
species.IntheabsenceofDprA,chromosomaltransformation (cid:1)16and(cid:1)22%,respectively(Fig.6)(10,11).DprA(1DprA/160
isreduced10–100-fold(2,3),buttherequirementofDprAfor nt) facilitated the annealing of both complementary DNA
plasmidtransformationislessclear.Plasmidtransformationis strandsby(cid:1)2-foldwhencomparedwiththeabsenceofDprA
marginallyreducedifatallin(cid:3)dprA or(cid:3)dprA compe- (Fig.6,A(lane4)andE),butincreasingDprAconcentrations(1
Hpy Hin
tentcells(3),butitis40-foldreducedin(cid:3)dprAcells(11).Sim- DprA/80 to 10 nt) did not further enhance the SSA reaction
ilarly, the absence of the strand-annealing protein RecO (Fig.6,A(lanes5–8)andE).
resulted in a 30-fold reduction in plasmid transformation. As reported previously (10, 11), the presence of any of the
Chromosomal and plasmid transformation, however, were SSBproteins(1SSB/53nt)preventedspontaneousreannealing
blockedina(cid:3)recO(cid:3)dprAdoublemutantstrain(Table1)(11), ofthecomplementarystrands(Fig.6,B–D,lane2).Ataratioof
suggestingthatbothDprAandRecOarecrucialforefficientGR 1DprA/80nt,DprAfacilitatedtheannealingofthetwocom-
inotherwiserec(cid:2)cells. plementary ssDNAs coated by SsbB (1 SsbB/53 nt), and at a
ToconfirmtheroleofDprAinplasmidtransformation,we ratio of 1 DprA/40 nt, the SSA reaction reached its maximal
constructed a (cid:3)dprA (cid:3)recA double mutant strain (Table 1). level(Fig.6,B(lane5)andE).WhenSsbBwasreplacedbySsbA,
ChromosomaltransformationisRecA-dependent,andplasmid (cid:1)30%ofthecomplementarystrandswerereannealedatratios
transformation is a RecA-independent reaction (see Table 1) of1DprA/80to20nt(Fig.6,C(lanes4–7)andE).Bycontrast,
(2).Asexpected,chromosomaltransformationwasblockedin DprAproteinannealedSsb -coatedssDNAcomplexesvery
SPP1
the(cid:3)dprA(cid:3)recAor(cid:3)recO(cid:3)recAcontext(Table1).In(cid:3)recO poorly, and a high DprA/ssDNA ratio (1 DprA/20 nt) was
cells, defects in plasmid transformation were not due to the needed(Fig.6,D(lane6)andE).BecausethessDNAwasthe
unavailability of ssDNA, because the absence of RecA sup- samefortheSsb andtheSsbBexperiments,itislikelythat
SPP1
22446 JOURNALOFBIOLOGICALCHEMISTRY VOLUME288•NUMBER31•AUGUST2,2013