DNA Replication in the Archaea - Microbiology and Molecular

12 Pages·2006·0.68 MB·English

MICROBIOLOGYANDMOLECULARBIOLOGYREVIEWS,Dec.2006,p.876–887 Vol.70,No.4
1092-2172/06/$08.00(cid:1)0 doi:10.1128/MMBR.00029-06
Copyright©2006,AmericanSocietyforMicrobiology.AllRightsReserved.
DNA Replication in the Archaea
Elizabeth R. Barry and Stephen D. Bell*
MRCCancerCellUnit,HutchisonMRCResearchCentre,HillsRoad,CambridgeCB22XZ,UnitedKingdom
INTRODUCTION.......................................................................................................................................................876
REPLICATIONORIGINS.........................................................................................................................................876
ORIGINBINDINGBYOrc1/Cdc6...........................................................................................................................878
REPLICATIVEHELICASE.......................................................................................................................................879
LoadingoftheReplicativeHelicase.....................................................................................................................879
StructureandFunctionofMCMProteins..........................................................................................................880
Hel308a.....................................................................................................................................................................880 D
SSBs..............................................................................................................................................................................881 o
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PRIMASE.....................................................................................................................................................................881
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GINS.............................................................................................................................................................................881 lo
REPLICATIVEDNAPOLYMERASES...................................................................................................................882 a
SLIDINGCLAMPS....................................................................................................................................................882 d
e
RFC—theClampLoader.......................................................................................................................................883 d
PCNA-InteractingProteins....................................................................................................................................883 f
r
ROLEOFARCHAEALCHROMATIN...................................................................................................................884 o
m
REFERENCES............................................................................................................................................................885

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INTRODUCTION (47).Thismodelhasprovedextremelyaccurateforbacteria. :
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Bacterial chromosomes contain a single replication origin, m
SincethepioneeringworkofCarlWoeseinthelate1970s,
oriC,whichconsistsofanA-T-richregionofDNAcontain- m
it has been well established that the archaea constitute a de-
ingmultiplecopiesoftheDnaAbox,whichisboundbythe b
fined domain of life (118). With the availability of archaeal initiatorproteinDnaA.Inmanyspecies,thegeneforDnaA r.a
genomesequencesinthemid-tolate1990s,itbecameappar- s
is carried adjacent to the origin, so the two may be coregu- m
entthatthearchaealDNAreplicationmachineryhasstriking
lated (75). .
similarity to that in eukaryotes and is evolutionarily distinct o
In contrast to those of bacteria, eukaryotic chromosomes r
from that in bacteria. How this curious dichotomy arose in a g
contain multiple replication origins. So far, only Saccharo- /
process central to the very propagation of life has been the o
myces cerevisiae (budding yeast) has been shown to have n
subject of much debate. A wide range of theories have been
clearly defined replication origins, known as autonomously A
put forward to account for this observation, ranging from the
replicating sequences. These contain conserved sequence p
proposal that DNA replication arose twice in cellular organ- r
isms, suggesting that the last common ancestor of all living elements,similartothesituationinbacteria,andarebound il 1
by the origin recognition complex (ORC). The ORC con- 4
organismsmaynothavehadaDNAgenome,tothepossibility
tains six separate polypeptides, Orc1-6, several of which ,
thatthelastcommonancestorhadadefinedreplicationsystem 2
contain AAA(cid:1) (ATPases associated with various cellular ac- 0
butthatitwasdisplacedbynonorthologousgenetransferfrom, 1
tivities) ATPase domains. Interestingly, Orc1 is also closely
for example, a viral source (26, 65, 82). Regardless of the 9
related to another replication factor, Cdc6 (Cdc18 in Schizo- b
derivationofthearchaeon-eukaryoteDNAreplicationsystem, saccharomyces pombe), which presumably is indicative of the y
itisapparentthatthearchaealmachineryisasimplified,and g
presumablyancestral,formofthatineukaryotes.Theorgani- derivation of Orc1 and Cdc6 from a common ancestor. Al- u
thoughORCactsasasequence-specificDNAbindingcomplex e
zationalsimplicityofthearchaealmachinery(Fig.1),coupled s
inbuddingyeast,inS.pombeandhighereukaryotesthereisno t
with the biochemical advantages conferred by the study of
clear consensus sequence for origins, although in many cases
hyperthermophilicarchaea,hasledtoconsiderableinterestin
theydotendtobeA-T-richregions.Indeed,inXenopuslaevis
thearchaealmachineryasamodelofthatineukaryotes.
eggs, any sequence seems capable of initiating DNA replica-
tion(16).Insteadofrelyingonsequence-specificDNArecog-
REPLICATIONORIGINS nition by ORC, a growing body of evidence suggests that in
higher eukaryotes, origins are defined by facilitated recruit-
Therepliconhypothesis,proposedbyJacobetal.,predicted
mentofORCbyavarietyofotherDNAbindingproteins.The
that a trans-acting initiator protein binds to a cis-acting repli-
extenttowhichthisisadirecteffectormediatedbysecondary
cator DNA sequence to initiate DNA replication in bacteria
chromatinalterationsisnotfullyunderstood(98).
Itwasinitiallythoughtthatbecausethechromosomestruc-
tureinarchaeaissimilartothatinbacteria,archaealchromo-
*Correspondingauthor.Mailingaddress:MRCCancerCellUnit,
someswerealsolikelytocontainoneoriginofreplication.The
HutchisonMRCResearchCentre,HillsRoad,CambridgeCB22XZ,
UnitedKingdom.Phone:[phone].Fax:[phone]. firstorigintobeidentifiedwasthesingleoriginofPyrococcus
E-mail:[email protected]. abyssi (80, 89). The origin binding proteins in archaea are
876
VOL.70,2006 DNA REPLICATION IN THE ARCHAEA 877
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FIG. 1. Components of the archaeal DNA replication machinery and chromatin proteins. Structure figures were prepared using Pymol
(www.pymol.org),usingthefollowingPDBcoordinates:PyrobaculumCdc6,1FNN;SulfolobusSSB,1O71;PyrococcusRFCsmallsubunit,1IQP;
SulfolobusPolB1,1S5J;PyrococcusPCNA,1ISQ;ArchaeoglobusFen1,1RXW;SulfolobusAlba,1H0Y;SulfolobusSul7d,1WTP;andMethano-
thermushistoneHmfB,1BFM.TheimageoftheligasestructurewassuppliedbyY.Ishino(Fukuoka,Japan),andweobtainedtheimageofthe
primasecomplexincollaborationwithL.Pellegrini(Cambridge,UnitedKingdom).
homologuesoftherelatedeukaryoticOrc1andCdc6proteins inHalobacterium(121),ageneticscreenfoundthatonlyoneof
(discussedbelow).TheorigininP.abyssiislocatedadjacentto them had autonomously replicating sequence activity (3).
thegeneforOrc1/Cdc6,inasituationsimilartothatforDnaA However, two origins of replication were subsequently found
inmanybacteria.AlthoughbioinformaticanalysisusingtheZ andmappedforSulfolobussolfataricusbytwo-dimensionalgel
curvemethodshowedthattherewerelikelytobetwoorigins analysis(99).S.solfataricushasthreeOrc1/Cdc6genes,encod-
878 BARRY AND BELL MICROBIOL.MOL.BIOL.REV.
ing Cdc6-1, Cdc6-2, and Cdc6-3. The two identified origins, TABLE 1. GenesforOrc1/Cdc5,MCM,andPCNAinsequenced
oriC1andoriC2,arelocatedupstreamofthegenesforCdc6-1 archaealgenomesa
andCdc6-3,respectively(99).Nooriginwasfoundadjacentto
No.ofgenes
the gene for Cdc6-2. A third origin, oriC3, was subsequently Organism
Orc1/Cdc6 MCM PCNA
identified in both S. solfataricus and Sulfolobus acidocaldarius
by marker frequency analysis but was located at least 50 kb Aeropyrumpernix 2 1 3
away from Cdc6-2 (69). Thus, at least some archaea contain Archaeoglobusfulgidus 2 1 1
Cenarchaeumsymbiosum 1 1 1
multipleoriginsofreplication.
Haloarculamarismortui 17 3 1
FinemappingofthethreereplicationoriginsinS.solfatari- Halobacteriumsp.strainNRC-1 9 1 1
cus led to the identification of origin recognition boxes Ignicoccussp.strainKin4-1 2 1 3
(ORBs), which are inverted repeat sequence elements bound Methanobacteriumthermautotrophicum 2 1 1
Methanococcoidesburtonii 2 1 1
by Cdc6-1, at oriC1. These sequence elements are well con-
Methanococcusjannaschii 0 4 1
served across many archaeal species, although most archaeal
Methanococcusmaripaludis 0 4 1
origins have not been proven experimentally. oriC2 in S. sol- Methanopyruskandleri 0 2 1
D
fataricus contains sequences homologous to the central ele- Methanosarcinaacetivorans 2 2 1 o
mentsofORBs.Thesesequences,termedmini-ORBs,arealso Methanosarcinabarkeri 3 1 1 w
Methanosarcinamazei 2 1 1 n
found in the predicted origin of Methanobacterium therm- Methanospirillumhungatei 2 1 1 lo
autotrophicum. It therefore seems that, like the case for bac- Methanosphaerastadtmanae 2 1 1 a
d
teriaandS.cerevisiae,archaealoriginsaredefinedbyspecific Nanoarchaeumequitans 1 1 1 e
sequenceelements(99). Natronomonaspharaonis 5 2 1 d
Ineukaryotes,notalloriginsareusedineachSphase,and Picrophilustorridus 1 1 1 fr
Pyrobaculumaerophilum 1 1 2 o
those that are used are fired asynchronously. Whether an or- Pyrococcusabyssi 1 1 1 m
igin is used and whether it fires early or late in S phase vary Pyrococcusfuriosus 1 1 1 h
dependingonchromatinstructure,thetranscriptionalstatusof Pyrococcushorikoshii 1 1 1 tt
thesurroundingregions,andthedevelopmentalstageandcell Sulfolobussolfataricus 3 1 3 p:
Sulfolobusacidocaldarius 3 1 3 //
type for higher eukaryotes (98). Marker frequency analysis m
Sulfolobustokodaii 3 1 3
combined with computational modeling suggested that all Thermococcuskodakarensis 1 3 2 m
three origins in S. acidocaldarius and S. solfataricus fire syn- Thermoplasmaacidophilum 2 1 1 b
r
chronously in all cells and that all three are used in each cell Thermoplasmavolcanium 2 1 1 .a
s
cycle (69). However, some differential origin usage cannot be aCrenarchaeal species and their values are indicated in bold. Genes were m
ruledout,especiallyasthethreeCdc6proteinsinS.solfatari- identified by Blast searching using the server at http://www-archbac.u-psud.fr .
o
cusbindwithdifferentaffinitiestothedifferentoriginsinvitro /projects/sulfolobus/Blast_Search.html. r
g
(99;discussedbelow). /

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ORIGINBINDINGBYOrc1/Cdc6 was shown by chromatin immunoprecipitation to bind the re- A
gioncontainingthesingleknownP.abyssiorigin(80).There- p
r
Intherepliconhypothesis,Jacobetal.proposethatatrans- fore, the Orc1/Cdc6 proteins are thought to act as the origin il
1
actingfactorrecognizesandbindsthereplicatorsequenceand recognition and binding proteins in archaea. In addition, 4
recruitsotherreplicationfactors(47).Asmentionedabove,in Cdc6-1fromS.solfataricuscanbindtoORBelementspresent ,
2
bacteria this protein is DnaA, multiple monomers of which intheHalobacteriumNRC1andP.abyssioriginsinvitro(99), 0
1
bind the DnaA boxes at the origin and melt the DNA. In supporting the idea that archaeal Orc1/Cdc6 proteins recog- 9
eukaryotes, the ORC, consisting of proteins Orc1-6, binds at nize specific sequence motifs and that these motifs are con- b
replication origins. In many eukaryotes, ORC remains bound servedacrossarchaea.ItisnotknownwhethermultipleOrc1/ y

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throughoutthecellcycle,whereasinbacteriaDnaAisreleased Cdc6 proteins bind to each origin or whether binding is u
as replication starts and then rebinds before the next round cooperative,butthishasbeensuggestedbasedonthestructure e
s
(70,75).ORCrecruitsmanyproteinstothereplicationorigins, ofOrc1/Cdc6andthesymmetryofORBelements(110). t
including Cdc6 (Cdc18 in S. pombe). With the exception of Mostarchaealgenomescarryfromone(Pyrococcusspecies)
three methanogenic archaeal species, all archaeal genomes to nine (Halobacterium) Orc/Cdc6 genes. Sequence analysis
sequencedtodatecontainatleastonegenewithhomologyto hasshownthatthesecanbeclassifiedintothreemajorgroups
both Orc1 and Cdc6 (Table 1). The identities of the initiator (3),andallspeciesthathavemorethanoneOrc1/Cdc6protein
proteinsinthethreemethanogenexceptionsremainunknown. have at least one from the SsoCdc6-1 and SsoCdc6-2 sub-
Although all archaeal Orc/Cdc6 genes contain regions of ho- groups (99). The three Orc1/Cdc6 proteins in S. solfataricus
mology to both ORC and Cdc6 genes, in different archaeal show different DNA binding footprints for the origins, which
genomesequencestheyaregenerallyannotatedaseitherOrc-x suggests that they could function differently or play different
or Cdc6-x. Like components of the eukaryotic ORC and the roles in replication. Work with S. acidocaldarius, which also
Cdc6protein,archaealOrc1/Cdc6proteinsaremembersofthe contains three Orc1/Cdc6 proteins, showed different patterns
AAA(cid:1)proteinfamily.ThethreeOrc1/Cdc6proteinsfromS. of protein levels following perturbation of the cell cycle by
solfataricus,i.e.,Cdc6-1,Cdc6-2,andCdc6-3,havebeenshown treatment with acetic acid. Treatment of Sulfolobus cells with
to bind to origins, with Cdc6-1 binding specifically to ORB low concentrations of acetic acid leads to an accumulation of
elements(99).ThesingleOrc1/Cdc6proteinfromPyrococcus cellsintheG periodofthecellcycle.Followingwashingofthe
2
VOL.70,2006 DNA REPLICATION IN THE ARCHAEA 879
cellsandtransfertofreshmedium,cellsreenterthecellcycle. to be required for replication elongation and meiotic recom-
However, this entry appears to be very asynchronous. Never- bination(5,73).ThefunctionofMCM9,whichispresentonly
theless, it was demonstrated that Cdc6-1 and Cdc6-3 levels inhighereukaryotes,iscurrentlyunknown(74).
were elevated in G - and S-phase cells, whereas the Cdc6-2 There is considerable debate concerning whether MCM is
1
level was highest in G -arrested cells. This observation, along thereplicativehelicaseineukaryotes.Mcm2-7areessentialfor
2
withthefactthatCdc6-1andCdc6-3bindingsitesoverlapwith the initiation and elongation phases of DNA replication in
Cdc6-2 binding sites in S. solfataricus origins, suggests that yeast and Xenopus (58, 91, 106). They are recruited to the
Cdc6-2mayactasarepressorofreplication.Theexpressionof replicationoriginbyORC,Cdc6,andCdt1(discussedbelow),
all three proteins was also reduced in stationary-phase cells and blocking this recruitment completely inhibits replication.
comparedtothatinexponentiallygrowingcells(99). However, the complex has no ATPase or helicase activity in
The structures of an Orc1/Cdc6 protein from Pyrobaculum vitro.AsubcomplexofMCM4,-6,and-7hasweak3(cid:2)-5(cid:2)heli-
aerophiliumandofAeropyrumpernixORC2(itshouldbeem- caseactivityinvitro,andthushasbeensuggestedtoformthe
phasized that this protein is related to S. solfataricus Cdc6-2, activecomplex,whereasMCM2,-3,and-5areregulatory(45,
noteukaryoticOrc2)havebeensolved.Thesestructuresshow 63,64).Allsixproteinsarerequiredforreplicationelongation,
D
that the C-terminal region of Orc1/Cdc6 contains a winged- however. In this light, a weak helicase activity was recently o
helix(WH)domain,andsequencealignmentsshowthatthisis found to be associated with an endogenous purified complex w
n
conserved throughout archaeal and eukaryotic Cdc6 proteins from Drosophila melanogaster containing MCM, Cdc45, and lo
(67, 110). This domain is found in several DNA binding pro- the GINS proteins (see below) (87). Many MCM molecules a
d
teins and thus has been postulated to be the region of Cdc6 (10to100insomeorganisms)arealsoloadedateachorigin(7, e
responsibleforcontactingDNA.Insupportofthishypothesis, 29),incontrasttothecaseinEscherichiacoli,whereonlytwo d

mutation of WH domain residues in S. solfataricus Cdc6-1 molecules of DnaB are loaded, with one for each replication fr
o
reduced its ability to bind origin DNA (99), and the WH fork.Inaddition,manyimmunofluorescencestudiesofhigher m
domain from A. pernix ORC2 was shown to be necessary and eukaryotes have found that the majority of MCM does not h
sufficientforDNAbinding(110). colocalize with replication forks but, instead, associates with tt
p
The crystal structure of A. pernix ORC2 was determined in unreplicated DNA (24, 56, 71). However, a model has been :
/
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the presence of both ADP and the nonhydrolyzable ATP an- proposed for MCM function at a distance from forks (dis- m
alogueADPNP.Thestructuresshowedsubstantialconforma- cussedbelow),sothisdoesnotnecessarilyprecludeafunction m
tional flexibility in the ADP-bound form, but all ADPNP- forMCMasthereplicativehelicase. b
r
boundproteinsadoptedthesameconformation.Thissuggests All archaeal genomes sequenced to date have at least one .a
s
that ATP binding may stabilize a single conformation of MCM homologue (Table 1). In contrast to eukaryotes, how- m
ORC2.Theinvivorelevanceofthisisunclearasyet,butATP ever, many archaea contain only one MCM gene, and the .
o
binding and hydrolysis are likely to play an important part in protein forms homohexamers in vitro. Like the eukaryotic r
g
Cdc6functioninthecell(110). MCM proteins and bacterial DnaB, archaeal MCM is an /

The two Orc1/Cdc6 homologues from M. thermautotrophi- AAA(cid:1)protein.Invivo,MCMinteractsfunctionallywithCdc6 on
cum and P. aerophilium Cdc6 have been shown to autophos- (22, 23, 51, 108) and, via GINS, with the primase (76). It A
phorylateatserineresidues.Thisautophosphorylationactivity localizestoreplicationoriginsinP.abyssi(80);however,since p
r
isinhibitedbyDNA,butthisinhibitionisseverelyreducedin genetic systems for archaea are still in their infancy, it is not il
1
theabsenceoftheWHdomain.Thisfurthersupportstheidea knownwhetherMCMisessentialforreplication.Despitethis, 4
thattheWHdomaininteractswithDNA.Itisinterestingthat the very conservation of MCM from archaea to eukaryotes ,
2
theS.pombeCdc6homolog(calledCdc18)canalsoautophos- argues for an essential role, such as that of a replicative heli- 0
1
phorylate, but it is not clear what functional significance this case. 9
autophosphorylationactivitymighthave(40). b
TheORC2structurealsorevealsremarkablesimilaritiesto y
DnaA.BothDnaAandORC2areAAA(cid:1)proteins,andboth LoadingoftheReplicativeHelicase gu
containaC-terminalDNAbindingdomain,althoughinDnaA Ineukaryotes,MCMisloadedontoDNAinaprocessthat e
s
thisisahelix-turn-helix,notWH,domain.Itisthereforelikely requiresORC,Cdc6,andCdt1(70).Cdc6bindingtoDNAis t
that despite the lack of homology between the two proteins anATP-independentprocess;however,MCMloadingrequires
beyondtheirAAA(cid:1)domains,theyfunctioninsimilarways. ATPhydrolysisbyCdc6butnotbyMCM(67,116).
In bacteria, both DnaA and DnaC are required to load
DnaB onto DNA at origins. Like Cdc6, DnaC is an AAA(cid:1)
REPLICATIVEHELICASE
protein.DnaCbindshexamericDnaBand,presumably,alters
In bacteria, the replicative helicase is DnaB, an AAA(cid:1) the conformation of the ring, thereby facilitating its loading
proteinwhichfunctionsasahomohexamer.Ineukaryotes,the ontoDNA.ItisthoughtthatDnaCbindsDnaBwhileboundto
bestcandidateforthereplicativehelicaseisMCM(minichro- ATP. This increases its affinity for single-stranded DNA
mosome maintenance complex). The MCM proteins were (ssDNA),butDnaC-ATPinhibitsDnaBhelicaseactivity.Once
originally identified in yeast, in a screen for genes whose mu- DnaBisloadedattheorigin,thepresenceofbothDnaBand
tationabrogatedtheabilityofthecellstomaintainaplasmid ssDNA stimulates the ATPase activity of DnaC, causing it to
containingacentromereandareplicationorigin(72).MCMin stimulateinsteadofinhibitDnaB(20,21).
eukaryotes is a heterohexamer of MCM2-7. MCM8, another For archaea, little is known about MCM loading. Open
memberoftheMCMfamily,wasrecentlyidentifiedandseems forms of the hexameric MCM ring from M. thermautotrophi-
880 BARRY AND BELL MICROBIOL.MOL.BIOL.REV.
helicase, and thus it probably tracks along the leading strand
duringreplication.
ThecrystalstructureofthedodecamericN-terminalregion
of M. thermautotrophicum MCM has been solved, as has the
EM structure of the full-length protein (30, 92). These struc-
FIG. 2. DomainorganizationofMCMproteins.TheN-terminalre- turesrevealedalarge,positivelychargedchannelinthecenter
gion,consistingofthreedomains,A,B,andC,ispoorlyconservedbe- ofthering,withadiameterofbetween23Åand47Å,which
tweendifferentMCMproteinsandisthoughttobeinvolvedinregulation. is easily wide enough to accommodate single- or double-
EukaryoticMCMproteinsoftenhaveanadditionalN-terminalextension.
stranded DNA. The mechanisms by which ATP hydrolysis is
ThecatalyticAAA(cid:1)domainisshowninblue-green.Thehelix-turn-helix
coupled to helicase activity are unclear. However, the crystal
(HTH)domainattheCterminusisnotinvolvedinDNAbindingbutmay
playaroleinregulationofthecomplex. structure of the N terminus revealed a conserved (cid:3)-hairpin
motif.AnalysisoftheC-terminalsequenceshowedthatthere
was another conserved insertion likely to form a (cid:3)-hairpin in
the AAA(cid:1) domain. Mutation of conserved basic residues in
cumhavebeendetectedbyelectronmicroscopy(EM).These D
either of these had only a modest effect on the ability of the o
may represent a loading intermediate, as the MCM ring may proteintobindDNA.Mutationofboth,however,causedaloss w
bebrokenandreformedaroundDNAinawaysimilartothat n
forDnaB(21,39).ThereisapparentlynohomologueofDnaC o(cid:3)f-hDaiNrpAincbainudseindga. sIlnighatdrdeidtiuocnt,iomniuntahteiolincasoefatchteivitNy,-twehrmerienaasl loa
orCdt1inarchaea,suggestingthattheOrc/Cdc6proteinsmay mutation of the C-terminal (cid:3)-hairpin completely abrogated de
performthefunctionscarriedoutbyORC,Cdc6,andCdt1in helicase activity (83). For the superfamily 3 helicase simian d
eukaryotes. However, the sequence similarity between yeast virus 40 (SV40) large T antigen, a similar (cid:3)-hairpin has been fr
andhumanCdt1proteinsisonlyaround10%,soitisconceiv- o
shown to move upon ATP hydrolysis and thus has been pro- m
able that there is a protein with low or even no homology in
posedtoeffectapowerstroke,drivingtheproteinalongDNA h
archaeawhichperformsthesamefunctionsasCdt1.Likethe (37, 66). It is therefore highly possible that MCM may trans- tt
caseineukaryotes,bindingofarchaealOrc1/Cdc6proteinsto p
locatealongDNAbyasimilarmechanism. :
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origins is apparently ATP independent (99), despite the fact /
The mechanism by which MCM effects unwinding is still m
that they have a functional AAA(cid:1) domain (40, 110). ATP unknown.MCMfromM.thermautotrophicumisabletotrans- m
hydrolysisbyCdc6maybeimportantforMCMloading,similar locatealongdouble-andsingle-strandedDNA,aswellasbeing b
tothesituationineukaryotes.TheCdc6proteinsinM.therm- able to unwind a forked substrate (109). It may act as a mo- r.a
autotrophicum have been shown to inhibit MCM helicase ac- s
lecular bulldozer, separating strands as it translocates along m
tivityinanATP-dependentmanner,buttheinvivosignificance
DNA. Consistent with this possibility, the EM structure of .
of this is not clear. Interestingly, MCM from M. therm- o
MCMrevealedthepresenceofholesinthesideofthecomplex r
autotrophicum also modulates autophosphorylation of Cdc6-1 g
which may act as exit pores for DNA (92). Alternatively, a /

andCdc6-2(51,108). o
rotary pumping model for eukaryal MCM function at a dis- n
tancefromforkshasbeenputforward.Thisproposesthatafter A
MCMproteinsareloaded,theytranslocatealongdsDNAaway p
StructureandFunctionofMCMProteins r
fromtheorigininbothdirectionsandarethenimmobilizedby il
1
AllMCMproteinshaveaconserveddomainstructure(Fig. attachment to the nuclear matrix. After immobilization, fur- 4
2).TheC-terminalAAA(cid:1)catalyticdomainiswellconserved thertranslocationcausestheDNAtorotateandthusleadsto ,
2
betweenMCMproteins.TheNterminioftheproteinsareless unwinding at the fork (62). This would explain why many 0
1
well conserved and are thought to be responsible for multi- MCM proteins are loaded per origin and why they do not 9
merizationandregulation(30,33).IneukaryoticMCMproteins, colocalize with replication forks. It remains to be seen which b
the phosphorylation sites for cyclin-dependent kinases and model is correct and, indeed, whether MCM functions in the y

g
otherregulatorykinasesaremostlylocatedinthisregion(54, samewayinarchaeaandeukaryotes. u
86). There is also a helix-turn-helix domain at the extreme C e
s
terminusoftheprotein.Thisdoesnotseemtoberesponsible t
Hel308a
forDNAbindinginS.solfataricusMCM,anditsinvivofunc-
tionisunknown. ItislikelythatotherhelicasesalsofunctioninarchaealDNA
In solution, archaeal MCM proteins usually form double replication.ItwasrecentlyshownthatHel308a,ahelicasefrom
hexamers, although single hexameric, heptameric, and fila- M.thermautotrophicum,interactswithstalledreplicationforks
mentousformshavealsobeenreported(12,15,30,31,39,92, in vivo in E. coli and in assays performed in vitro. Strikingly
120). Unlike the heterohexameric eukaryotic complex, the similar results were observed with the Pyrococcus homolog,
double hexamer has DNA-stimulated ATPase and helicase termedHjm.WhenexpressedinanE.colistrainlackingRecQ
activities in vitro (13, 52, 83, 107). The double- and single- (aDNAhelicaseassociatedwithrecoveryofstalledreplication
hexamerformsofM.thermautotrophicumMCMhaveequiva- forks), Hel308a/Hjm complemented the recQ phenotype,
lentATPaseandDNAbindingactivitiesinvitro,butthedou- stronglysuggestingthatHel308a/Hjmmayplayasimilarrolein
blehexamerisamoreactivehelicasethanthesinglehexamer, archaea(6,35,42).Interestingly,insomearchaeatheHel308a
implying that this may be the form responsible for active un- homologue is encoded within an operon-like structure along
windinginthecell(31).IncontrasttoDnaB,a5(cid:2)-3(cid:2)helicase, withMCMandGINS,suggestiveofalinkedfunctionofthese
butlikeeukaryoticMCM-4,-6,and-7,archaealMCMisa3(cid:2)-5(cid:2) proteins.
VOL.70,2006 DNA REPLICATION IN THE ARCHAEA 881
SSBs ArchaeahavehomologuesofeukaryoticPriSandPriL,but
they lack Pol(cid:4) and the B subunit. The small subunits of pri-
Single-stranded binding proteins (SSBs) are present in all
masefrombothS.solfataricusandPyrococcusspeciescansyn-
threedomainsoflife.Theyprotectsingle-strandedDNAfrom
thesize both RNA and DNA primers in vitro (59, 68, 79).
nucleasedegradationandchemicalmodificationduringDNA
Pyrococcus furiosis PriS preferentially synthesis long (up to 6
replication,recombination,andotherprocesseswhichrequire
kb) DNA oligonucleotides. However, the addition of PriL in-
DNA to be unwound. All SSBs bind DNA via a common
creasestheRNApolymeraseactivity,decreasestheDNApoly-
oligonucleotide/oligosaccharide binding (OB) fold (88). Bac-
merase activity, and decreases the average product length,
terialSSBisahomotetramer.EachmonomercontainsanOB
suggesting that PriL plays a regulatory role (68). Despite the
fold for contacting DNA and an acidic C-terminal domain
fact that S. solfataricus PriS can synthesize both DNA and
(CTD) responsible for protein-protein interactions (94, 95).
RNA in vitro, it has a higher affinity for nucleoside triphos-
EukaryotescontainaheterotrimercalledreplicationproteinA
phates than for deoxynucleoside triphosphates, so it probably
(RPA)thatcontainsfourDNA-bindingOBfolds.Thelargest
makes RNA primers in vivo (59, 81). In addition, Okazaki
subunit, RPA70, contains two of these in addition to a zinc
fragments have been isolated from archaea and found to be
D
binding motif, whereas the smaller two subunits, RPA30 and RNA at the 5(cid:2) end (80). Any in vivo relevance of the DNA o
RPA14,bothcontainsingleOBfolds. polymerase activity of PriS is unclear, but given the lack of a w
oveArraclhlatheaeycsohnotawinmaorvearsiiemtyilaorfitySStoBeaurkraanrygoetmicenRtPs,Aaltthhaonugtho Pploaly(cid:4)sahormoloeloinguperiminerareclohnaegaa,tioitniasnpaolosgsiobulesttohathtattheofpProiml(cid:4)asine nloa
bacterial SSBs. The best-studied archaeal SSB is the 16-kDa d
eukaryotes.However,thereiscurrentlynoevidenceforthis. e
singleSSBofS.solfataricus.ItcontainsasingleOBfoldand The structures of the P. furiosis and Pyrococcus horikoshii d
constitutes 2 to 5% of total soluble protein in the cell. The primase small subunits and the S. solfataricus heterodimer fr
sequenceshowsthatthedomainstructureismostsimilarto o
havebeensolved(1,46,60)Inallcases,PriSconsistsofalarge m
thatofabacterialSSB,anditcontainsanacidicCTDsimilar (cid:4)/(cid:3)domaincontainingthecatalyticprimdomainandasmaller h
tothatofE.coliSSB(114).LikethecaseinE.coli,thisCTD (cid:4)-helicaldomain.Italsocontainsazincbindingmotifwhichis tt
is not required for DNA binding but mediates protein-pro- p
conservedineukaryotesandhasbeensuggestedtobeinvolved :
/
teininteractions(96,114).However,thecrystalstructureof /
ininteractionoftheenzymewithDNAinS.solfataricus.PriL m
S. solfataricus SSB revealed that the OB fold is actually is largely made up of an (cid:4)-helical domain with a small (cid:4)/(cid:3) m
more similar to that of human RPA than to those of bacte- domainthatmediatesinteractionwithPriS.Theinterfacebe- b
rial SSBs (53). tween the two subunits is conserved between archaea and r.a
PyrococcusfuriosishasthreeSSBs,namely,RPA41,RPA32, s
eukaryotes.ThestructureoftheheterodimershowsthatPriL m
and RPA14, which form a heterotrimer with high affinity for
does not directly contact the active site and can probably in- .
ssDNA. RPA41 shows homology to eukaryotic RPA70, and o
teractwiththeprimeronlyonceitreachesalengthof7to14 r
likeRPA70itcontainsazincbindingmotif(55).Methanosar- g
bp. This may trigger handoff to the polymerase and may ex- /

cina acetivorans also has three SSBs, namely, RPA1, RPA2, o
plainwhyPriLinhibitstheproductionoflongerprimers(60). n
and RPA3. However, unlike the Pyrococcus proteins, they do Archaeal PriS also contains a conserved catalytic triple-as- A
notinteract,andallformhomodimers(97). partatemotifwhichisstructurallysimilartothatfoundinthe p
In addition to its role in stabilizing ssDNA, S. solfataricus PolXfamilyofDNApolymerases.However,secondarystruc- ril
SSB has also been implicated in DNA damage recognition 1
ture elements surrounding this motif are very different, sug- 4
(17). The M. acetivorans SSBs have also been shown to stim- gestingconvergentevolution(60).TherearefiveknownPolX ,
ulatetheprimerextensionactivityofpolymeraseB1(PolB1) 2
familymembersinmammaliancells,andtheyfunctioninpro- 0
(97). There is some debate over the effect of SSB on MCM. 1
cesses involving DNA replication, repair, and recombination. 9
AlthoughonegroupreportedthatSSBstimulatesMCMheli- WiththeexceptionofM.thermautotrophicum,archaeadonot b
caseactivity(10),mostdatasuggestthatthepresenceofSSB containPolXfamilyproteins,sothesimilaritybetweenthese y

atlowconcentrationshasnoeffectonMCM,whereasathigher g
proteinsandprimasehasledtosuggestionsthatarchaealpri- u
concentrationsitisslightlyinhibitory(52,77). masemayplayaroleinDNArepair(61).Thismayexplainwhy e
s
itpossessesfunctions,suchasDNApolymeraseand3(cid:2)-nucleo- t
tidyl terminal transferase activities, not normally associated
PRIMASE
withprimases.
DNA polymerases are unable to initiate synthesis de novo
andthereforerequireaDNAorRNAprimerwhichtheycan
GINS
elongate.Thisissynthesizedbyaprimase,whichineukaryotes
andbacteriaisaDNA-dependentRNApolymerase.Bacterial The heterotetrameric eukaryotic GINS (go, ichi, nii, san
primaseisDnaG,amonomer,whereaseukaryoticprimaseisa [five,one,two,threeinJapanese])complexwasfirstidentified
dimer consisting of a small catalytic (PriS) and a large non- in yeast and Xenopus and consists of SLD5, PSF1, PSF2, and
catalytic (PriL) subunit. This associates with Pol(cid:4) and the B PSF3 (38, 57, 112). The complex is essential in yeast and
subunit to form the Pol(cid:4)/primase complex (34). The primase interactswithMCMandCDC45(38,57).Morerecently,itwas
synthesizesRNAprimersof8to12nucleotides(nt)inlength. shown that GINS is necessary for the inclusion of MCM in
Thesearethenelongatedtoaround30ntbyPol(cid:4)toproduce replisomeprogressioncomplexes,whichincludeseveralrepli-
aDNA-RNAhybridbeforehandofftothereplicativepolymer- cationandcheckpointproteins,duringreplication(38).
ase(34). AnarchaealhomologueofGINSwasoriginallyidentifiedin
882 BARRY AND BELL MICROBIOL.MOL.BIOL.REV.
Organisms in the euryarchaeal phylum contain polymerases
belongingtotwodistinctfamilies,i.e.,theubiquitousfamilyB
polymerases and a family that thus far appears unique to the
euryarchaea, called family D. The family D polymerases, first
discoveredbyIshinoandcoworkers,aretwo-subunitenzymes
comprised of subunits DP1 and DP2 (9). It appears that the
polymerization activity is contained within the larger subunit,
DP2. The sequence of DP2, however, is very distinct from
those of other DNA polymerases. Interestingly, the smaller
subunit, DP1, possesses recognizable sequence homology to
the noncatalytic B subunits of several eukaryotic family B
DNApolymerases.Recentworkhasindicatedthatthissubunit
possesses intrinsic 3(cid:2)-to-5(cid:2) exonuclease activity and that this
FIG. 3. ModelofthearchitectureofthearchaealDNAreplication activity is highest on substrates with mispaired nucleotides or
fork.ParentalDNAisindicatedbyblacklines,andnewlysynthesized D
DNA is shown in red. RNA primers, synthesized by primase, are single-stranded DNA, leading to speculation that this may be o
shown in blue. MCM is shown as a yellow hexameric assembly sur- important for proofreading by the archaeal family D poly- w
n
rounding the leading-strand template. We propose that the MCM merases(50). lo
helicasetranslocatesalongthisstrand,unwindingtheparentalduplex In addition to the family D polymerases, euryarchaea pos- a
aheadofthereplicationfork.Single-strandedDNAisboundbySSB d
(Sulfolobusnomenclature),shownaspinkcircles.MCMinteractswith sess DNA polymerases belonging to family B, suggesting that e
the two classes of polymerases may play distinct roles in the d
thearchaealGINScomplex(brown),andGINS,inturn,isadditionally
capableofbindingprimase(lightblue).WeproposethatGINSactsto cell.Indeed,ananalogymaybefoundintheapparentdiscrim- fr
o
coupleMCMtranslocationontheleading-strandtemplatewithdepo- ination between leading- and lagging-strand polymerases in m
sitionofprimaseonthelagging-strandtemplate.DNApolymerase(in Bacillussubtilisandeukaryotes.Arecentstudyofthebiochem- h
salmonpink)actstoextendtheRNAprimers,andweindicatethattwo
polymerasesarecoupled,althoughthereiscurrentlynoevidencefor icalbehaviorofPyrococcusDandBpolymerasesbyRaffinand ttp
this in archaeal systems. Each DNA Pol interacts with a trimer of colleaguesprovidedevidenceforamodelinwhichPolBsyn- :/
/
PCNA(brown).PCNAcanactasaplatformforadditionalassemblyof thesizes the leading strand and Pol D replicates the lagging m
theflapendonucleaseFEN1(green)andDNAligase1(Lig1[blue]), strand(43). m
ascartoonedonthelaggingstrand-associatedPCNAonly. The crenarchaea do not carry a family D polymerase but b
r
generallyhavemultiplefamilyBpolymerases.Itisagainpos- .a
s
sible,thoughasyetuntested,thatdifferentcrenarchaealfamily m
ayeasttwo-hybridscreenforinteractionpartnersofMCMin B polymerases have distinct roles on the leading and lagging .
o
S.solfataricus.Theproteinsinteractbothinvivoandinvitro. strands. r
g
Furtherinvestigationshowedthatthisprotein,whichishomol- Oneintriguingfeaturethathasbeendemonstratedforboth /

ogous to all the eukaryotic subunits but which has stronger euryarchaeal and crenarchaeal family B polymerases is the on
homology to the proteins encoded by psf2 and psf3, interacts ability to sense uracil ahead of the polymerase in the DNA A
withanotherGINShomologue,withstrongerhomologytothe templateandstall4ntbeforethatresidue,preventingitscopy- p
r
sld5- and psf1-encoded proteins. The two genes are therefore ing by the polymerase. Work by Connolly and colleagues re- il
known as gins23 and gins15. The proteins interact stably with vealed that this property is conferred upon the enzyme by a 14
RecJdbh, a protein homologous to the bacterial RecJ DNA small,conservedpocketthatliesintheN-terminaldomainof ,
2
binding domain, to form the archaeal GINS complex. The the polymerase (14, 32). This pocket has the ability to bind 0
presence of a RecJ homologue in the complex has led to uracilwithhighaffinity,resultinginstallingofthepolymerase 19
suggestions that the complex may be involved in stalled fork as it moves along the template. Presumably, the polymerase b
processing(76). thensignalstotherepairmachinerytofacilitateremovalofthe y

In addition to interacting with MCM, Gins23 also interacts uracil base and lead to correction of the lesion. How this is gu
with the primase. This solves a puzzle because in many repli- achieved is currently unknown, although it is probable that e
s
cationsystems,primaseinteractswiththereplicativehelicase. some form of replication fork regression may be involved to t
Indeed, a bacteriophage protein has been identified which facilitatetemplaterepair.
containshomologytobothMCMandprimase(82).However,
no interaction could be detected between primase and MCM
SLIDINGCLAMPS
inS.solfataricus.ItthereforeseemslikelythattheGINScom-
plex forms a molecular bridge between MCM on the leading Although the leading-strand DNA polymerase in archaea
strandandprimaseonthelaggingstrand(Fig.3)(76). may,inprinciple,havetosynthesizeoveramillionbaseswith-
outdisengagingfromthetemplate,theintrinsicprocessivityof
purifiedDNApolymerasesisactuallyquitelow.Therequired
REPLICATIVEDNAPOLYMERASES
processivityofpolymerasesisinsteadconferreduponthemby
Asdescribedabove,theroleoftheprimaseistosynthesize associationwithanaccessoryfactor,theslidingclamp.Inbac-
a primer that is extended by a DNA polymerase. In common teria, the sliding clamp is a homodimer, the (cid:3)-clamp. In con-
with bacteria and eukaryotes, archaea possess multiple DNA trast,inarchaeaandeukaryotes,theslidingclamp,proliferat-
polymerases.Interestingly,thereisaclearevolutionarydivide ingcellnuclearantigen(PCNA),isatrimer.However,despite
in the distribution of polymerase families within the archaea. the difference between the subunit compositions of bacterial
VOL.70,2006 DNA REPLICATION IN THE ARCHAEA 883
FIG. 4. Cartoonoftheclamploadingprocess.ApentamericRFC(gray)containingonelargesubunitandfouridenticalsmallsubunitsbinds
ATPandinteractswitharingofPCNA(blue)(step1).RFCopensPCNA34Åintheplaneofthering,andDNAentersthering(steps2and
3).ThePCNAringthenclosesaroundtheDNAbutleavesanout-of-planegapofapproximately5Å(step4)beforesealingshut(step5).ATP
isthenhydrolyzedbyRFC,andRFCleavesPCNAboundattheprimer-templatejunction.
D
and eukaryal/archaeal clamps, both classes of protein possess RFC—theClampLoader o
quasi-sixfold symmetry. As with the phylogenetic distribution w
ThePCNAtrimerisatoroidalmoleculethatencirclesDNA, n
ofDNApolymerases,thereappearstobeadivisionwithinthe therebytetheringitsclientproteinstotheDNAsubstrate.How- lo
archaeawithregardtotheidentitiesofgenesencodingPCNA a
ever, PCNA does not normally spontaneously assemble around d
(Table1).Inalmostalleuryarchaea,asineukaryotes,thereis
e
asinglePCNAhomolog,andtheproteinformsahomotrimer. DNA, but rather requires a specific loading factor, replication d
Asingleeuryarchaealspecies,Thermococcuskodakarensis,has factor C (RFC), to facilitate appropriate placing of the sliding fr
clampontotheDNAmolecule.RFCcatalyzesopeningofPCNA o
twoPCNAhomologues,butithasbeenproposedthatoneof m
and deposition of PCNA around double-stranded DNA at the
these (TK0582) may have been deposited in the genome rel-
h
atively recently via a lateral gene transfer event (36). In con- site of a double- to single-strand transition, such as a primer- tt
templatejunction(49).ThisprocessisdependentonATPbinding p
trast,themajorityofcrenarchaeaforwhichgenomesequences :
byRFC.ArchaealRFCisapentamercontainingfouridentical //
areavailablehavemultiplePCNAhomologues.InAeropyrum m
pernix,therearethreePCNAhomologs,andthesehavebeen copies of a small subunit (RFCS) and a single copy of a large m
showntobecapableofbothhomo-andheteromultimerization subunit(RFCL).BiochemicalandstructuralanalysesoftheRFC b
(19).Strikingly,S.solfataricusalsopossessesthreePCNAho- of Archeoglobus fulgidus by Wigley and colleagues, along with r.a
structural studies of the Pyrococcus homolog by Ishino and col- s
mologs, but in this case they are only capable of heterotri- m
leagues,inconjunctionwithstructuralandbiophysicalstudiesof
merization. In general, archaeal PCNAs have the capacity to .
yeastRFCandPCNA,haveshedconsiderablelightonthemo- o
interact with and stimulate the processivity of the replicative r
lecular mechanisms of RFC action (84, 85, 90, 103–105). The g
polymerases.Inaddition,asreviewedelsewhere,PCNAinter- /
RFCsubunitsaremembersoftheAAA(cid:1)familyofATPases,and o
actswithanumberofotherfactorsinvolvedinreplicationand n
repairofDNA(113,115). incommonwithothermembersofthisfamily,ATPisboundat A
TheinteractionbetweenPCNAandaclientproteinisusu- thejunctionoftwosubunits(8,103–105).TheintactRFCcom- p
allymediatedviaashortrecognitionmotif,termedthePCNA- plexformsarisingright-handedspiralandthereforehasfoursites ril
betweensubunitsthatcanbeoccupiedbyATP.ATPbindingby 1
interacting protein (PIP) motif, most commonly found at ei- 4
ther the N or C terminus of the protein (115). The structural all four sites is required for clamp loading. Fluorescence reso- ,
nanceenergytransferstudiesofyeastRFCandPCNAindicated 2
basisoftheinteractionwasrevealedwiththeelucidationofthe 0
thatfollowingbindingofPCNAbyRFC-ATP,PCNAisopened 1
structure of human PCNA bound to the PIP peptide of p21.
9
Importantly,thisstructureshowedthatonepeptidecouldbind about34Åintheplaneofthering(122).Thisstructureislikely b
perPCNAmonomer(41).ItisthereforepossiblethatPCNA theconformationthatcanbindDNAandmediateloading.The y

loading reaction then progresses via an intermediate where the g
is able to interact simultaneously with multiple partner pro- u
teins,formingamolecular“toolbelt.”Interestingly,analysisof PCNAringisheldopenwithagapofabout5Å,asseeninanEM e
structureofPyrococcusPCNA(85).Thisappearstobeastructure s
the heterotrimeric Sulfolobus PCNA led to the discovery that t
akintoalockwasher,withPCNAbeingopeninandoutofplane
distinct PCNA subunits within this complex each have pre-
configuration(Fig.4).PreciselyhowbindingofATPcausesthese
ferred client proteins. More specifically, Flap endonuclease 1
modulations in the structure is not fully understood, but
(FEN1),DNApolymeraseB1,andDNAligaseIinteractpref-
arginine fingers in the RFCS subunits that act to communi-
erentiallywithdistinctsubunits.Furthermore,affinitychroma-
cate with the ATP binding site of the neighboring subunit
tography indicated that the heterotrimeric Sulfolobus PCNA
appear to play a pivotal role in the process (105). As stated
couldbridgebetweenFEN1andligaseorpolymerase(25).It
above, ATP hydrolysis is not required for PCNA loading per
appears,therefore,thatanindividualPCNAringcanorganize
se.Rather,hydrolysisdrivesthefinalstageoftheprocess,i.e.,
and coordinate the activities of multiple factors simulta-
releaseoftheclamploaderfromtheloadedPCNA.
neously. The crystal structure of the Sulfolobus PCNA1-
PCNA2heterodimerincomplexwithFen1wasrecentlyeluci-
dated (27). This has revealed that the basis of discrimination
PCNA-InteractingProteins
between PCNA subunits and client proteins lies in distinct
geometries being adopted by the PIP motif-binding interdo- Once loaded, PCNA can then bind DNA polymerase, and
mainconnectorloopontheindividualPCNAsubunits. theprimercanbeextended.Asalludedtoabove,PCNAalso
884 BARRY AND BELL MICROBIOL.MOL.BIOL.REV.
acts as an adaptor for a range of additional proteins. These spitetheirdiscoveryover16yearsago,littleisyetknownabout
include the flap endonuclease FEN1 and DNA ligase I (113, theroleofarchaealhistonesinvivo.Differencesintheexpres-
115).Duringlagging-strandsynthesis,downstreamRNAprim- sion of histone variants have been observed during culture
ers must be removed in order for Okazaki fragments to be growth, suggesting that alterations in levels of compaction
joined.ThesetasksaremostlikelyperformedbyRNaseH2,an and/or distribution of histone subtypes may modulate gene
RNase that cleaves RNA in RNA-DNA hybrids, and FEN1. expressionorevenreplicationrates.Biochemicalworkusinga
FEN1 interacts with and is stimulated by PCNA. The crystal highly defined in vitro transcription system derived from M.
structures of a number of archaeal and eukaryal FEN1 en- thermautotrophicumhasrevealedthatanarchaealnucleosome
zymes have been solved, both alone and in complex with the positioned at an artificially selected high-affinity site has the
DNAsubstrateandwithPCNA(11,44,100). capacitytoslowtranscriptionthroughthenucleosome(119).It
ThecomplexofhumanFEN1withPCNArevealedthateach iscurrentlyunclearwhethertranscriptionthroughthenucleo-
subunitofthehomotrimericPCNAboundtoadifferentmono- somedisplacesitfromthetemplateorifitremainsbound.
mer of FEN1. Interestingly, each individual PCNA subunit- Intriguingly, with the exception of some mesophilic marine
FEN1 interaction showed a distinct geometry courtesy of a organisms,histonesareabsentfromthecrenarchaea(18).The
D
highlyflexiblehingeregionadjacenttothePCNAinteraction most highly studied crenarchaeal chromatin proteins come o
site (100). Two of the FEN1-PCNA monomer complexes did from the Sulfolobus genus. Species within this genus have a w
n
notinterferewiththepotentialinteractionbetweenPCNAand Sulfolobus-specificchromatinprotein,Sul7d.Inaddition,there lo
DNAbutheldFEN1insuchapositionthatitwasunlikelyto isasecond,abundant,nonspecificnucleicacidbindingprotein, a
d
becapableofcatalyzingDNAstrandcleavage.Thishasledto Alba. Alba is additionally found in a broad range of both e
speculation that these geometries may correspond to translo- crenarchaea and euryarchaea (117). Alba has both DNA and d

cating forms of the PCNA-FEN1 complex, with FEN1 in a RNA binding activities but has been found to be associated fr
o
“locked-down”andinactiveconformationthatwouldthenun- with a range of genomic loci in Sulfolobus, suggesting that it m
dergoastructuraltransitioninthepresenceoftheappropriate hasasignificantroleasachromatinprotein(78).Recentwork h
DNAsubstrate,therebyactivatingFEN1.Intherecentstruc- hascharacterizedasecondhomologofAlbafoundinSulfolo- tt
p
tureofSulfolobusFEN1incomplexwithPCNA1andPCNA2, buscells(48).Interestingly,Alba2formsobligateheterodimers :
/
/
FEN1wasboundonlytoPCNA1,inagreementwithprevious withAlba1andappearstoalterhigher-orderDNApackingby m
biochemical studies, and was positioned in the complex in an Alba1. It is possible that differential levels of expression of m
orientationthatwascompatiblewithitsaccessingDNA. Alba1 and Alba2 could modulate nucleoid structures in Sul- b
r
Theconceptofcarrierandactiveconformationsofproteins folobus. .a
s
on PCNA rings probably has relevance beyond the case of In contrast to the situation with archaeal histones, both m
FEN1.Forexample,SulfolobusPCNAwasfoundtobeableto Sul7dandAlbashowposttranslationalmodificationinSulfolo- .
o
simultaneously bind DNA ligase 1 (Lig1) and FEN1 in solu- buscells.Sul7dshowsmonomethylationoflysineresidues,but r
g
tion. However, the structure of human Lig1 in complex with the consequence (if any) of this modification and the identity /

o
DNAwasrecentlysolved,anditwasseenthatLig1effectively of the methyltransferase are currently unknown (28). Alba1 n
encircled the entire DNA molecule, such that even if it was hasbeenshowntobeacetylatedataninternallysineresidue, A
bound to only a single subunit of PCNA, it would effectively lysine16(2).TheeffectofacetylationofK16istolowerAlba’s p
r
stericallyoccludeaccesstoPCNAbyotherfactors(93).How- affinityforDNA.EnzymesthatacetylateanddeacetylateAlba il
1
ever,ifLig1canadoptacarrierconformationonPCNA,like havebeenidentified(78).Interestingly,theacetylase,Pat,and 4
the case for FEN1, it may dock down on the substrate only the deacetylase, Sir2, are conserved in many bacteria, where ,
2
transiently to catalyze the ligation step at the final stage in theyacttoregulateacetyl-coenzymeAsynthetasebyreversible 0
1
lagging-strand maturation. Indeed, it is tempting to speculate acetylation (111). It therefore appears that Sulfolobus may 9
that the steric clash that would be induced by Lig1 engaging have coopted this bacterial regulatory system to generate a b
with the DNA template may displace other proteins from primitive and simplified form of chromatin regulation. It is y

g
PCNA. This may permit access to PCNA by RFC after Lig1 of particular interest that Sir2 is well conserved in eu- u
hasjoinedOkazakifragments,whereuponRFCcouldunload karyotes,whereithasexpandedintoaproteinfamilywhose e
s
thePCNAring. membersplayrolesinavarietyofcellularprocesses,includ- t
ing regulation of chromatin structure, microtubule dynam-
ics, and life span (4).
ROLEOFARCHAEALCHROMATIN
Recent work has revealed that both Alba and Sul7d can
The vast majority of studies that have been performed on inhibittranslocationbypurifiedSulfolobusMCMhelicase(77).
the archaeal DNA replication machinery have used naked AcetylationofAlbabyPatalleviatedthisrepression,leadingto
DNA templates. Yet it is clear that within the cell, DNA is speculation that mechanisms may exist within Sulfolobus cells
compactedbyassociationwitharangeofsmallbasicproteins tocoupleAlba-modifyingor-displacingactivitytoprogression
andthattheseproteinshavethepotentialtomodulateaccess ofthereplicationforkinvivo.
to the DNA template. As recently reviewed elsewhere, ar- Considerableprogresshasbeenmadeinunderstandingthe
chaealcellsutilizeanintriguingvarietyofdifferentproteinsto formandfunctionofarchaealDNAreplicationandchromatin
mediategenomecompaction(102,117).Themajorityofeury- proteins.Itisclear,however,thatmuchremainstobediscov-
archaeahavehomologuesofeukaryotichistones,andextensive ered about how these proteins interact in the context of the
biochemical studies have revealed that these form structures macromolecular assemblies found at replication origins and
analogous to the eukaryotic H3/H4 tetrasome. However, de- duringprogressionoftheDNAreplicationfork.Furthermore,
VOL.70,2006 DNA REPLICATION IN THE ARCHAEA 885
the manner in which these proteins are regulated during the 25. Dionne,I.,R.K.Nookala,S.P.Jackson,A.J.Doherty,andS.D.Bell.2003.
course of the cell cycle in archaeal species remains largely A heterotrimeric PCNA in the hyperthermophilic archaeon Sulfolobus
solfataricus.Mol.Cell11:275–282.
unexplored.ForSulfolobus,inparticular,wheremultipleDNA 26. Dionne,I.,N.P.Robinson,A.T.McGeoch,V.L.Marsh,A.Reddish,and
replication origins are used, it will be of great interest to S.D.Bell.2003.DNAreplicationinthehyperthermophilicarchaeonSul-
folobussolfataricus.Biochem.Soc.Trans.31:674–676.
determine whether a mechanism exists to allow coordinated
27. Dore,A.S.,M.L.Kilkenny,S.A.Jones,A.W.Oliver,S.M.Roe,S.D.Bell,
regulationoforiginactivity,andifso,howthisismediated. and L. H. Pearl. 2006. Structure of an archaeal PCNA1-PCNA2-FEN1
complex:elucidatingPCNAsubunitandclientenzymespecificity.Nucleic
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