Topoisomerase I sites cluster asymmetrically at the ends of the simian virus 40 core origin of replication.

Vol.63, No. 12 JOURNALOFVIROLOGY, Dec. 1989,p. 5175-5183

0022-538X/89/125175-09$02.00/0

CopyrightC 1989, American Society for Microbiology

Topoisomerase

I

Sites

Cluster

Asymmetrically

at

the Ends of the

Simian Virus

40

Core

Origin

of Replication

SHANLI TSUI, MARY E. ANDERSON, AND PETERTEGTMEYER*

DepartmentofMicrobiology, State University of New York, Stony Brook, New York 11794-8621

Received8 May1989/Accepted 11 August1989

Invivo,topoisomerase I cleavage sites are locatedpredominantly on the strands of simian virus 40 DNA that arethe templatesfor discontinuous synthesis (S. E. Porter and J. J. Champoux, Mol. Cell. Biol. 9:541-550, 1989). This arrangement of sites suggests that topoisomerase I may associate with replication complexes in unique functional orientations at replication forks. We have mapped topoisomerase I cleavage sites in the simian virus 40origin of replication in vitro under conditions suitable for DNA replication. Numerous sites cluster in the invertedrepeat and AT-rich domains at the ends of the core origin and are arranged on the same strands that are cut most frequently in vivo. We propose that cleavage atthese sites would allow bidirectional extension of the replication bubble induced by T antigen within the core origin of replication early in the initiation of DNA synthesis. A mutational analysis of the topoisomerase I sites confirms the importance of positions -4 to -1 and +1 in the consensus sequence5'-A/T-A/G-A/T-T-break-G/A-3'. Surprisingly, more distantnucleotide positions also influence topoisomerase I sites in the inverted repeat and AT-rich domains of the core origin. Theeffects of distant sequences could be mediated by direct interactions with topoisomerase I orby theconformation of DNA in the core origin.

Simian virus40 (SV40)large Tantigen interacts with the

SV40origintoinitiateDNAreplication in cooperation with

hostcellularproteins (17, 33). The origin of replication has beenmapped by anextensive geneticanalysis (12-14, 16). It

consists of ancillary and essential core components.

T-antigen-binding site I at the early end of the origin and

promoter-enhancer elements at the late end of the origin

facilitatebutare notessential for replication. In contrast, a

central 64-base-pair (bp) core origin of replication is

abso-lutely required fortheinitiation ofDNAsynthesis.The core

origin consists ofat least three functional domains. Inthe

presenceofATP, Tantigenbindstofour recognition

penta-nucleotides inthecentral domain(4, 15)andtoanimperfect,

inverted repetition in adomainatthe earlyendofthe core

sequences (5). The binding of T antigen induces melting

within the inverted repeat domain and causes a

conforma-tionalchangeintheadenine-thymine(AT)domaininthe late

endofthecoreorigin(5).

Al

three domainsof the coreorigin

arerequired forDNAreplication invivo(12-14).

In vitro, purified Tantigen can melt only 10 to 20 bp of

origin DNA (5). Presumably, the opening of origin DNA

leadstopositivesupercoiling ofthecircularsubstrate DNA and prevents further unwinding of DNA by the intrinsic

helicase activity of T antigen (11). Transient cleavage by

topoisomeraseI(TopoI)orTopo IIrelievesthestressinthe

DNA and leads to extensive unwinding of the circular

molecule (11, 34). The mechanism by which cellular

topo-isomerases interact with SV40 DNA in vivo is not clear.

Topo I is preferentially associated with SV40 replicative intermediates and inducesbreaks atreplication forks (1, 9, 29). Furthermore, Porter and Champoux (28)have mapped TopoIsites inSV40-infected cells and havefoundthatmost

majorbreaksitesarefoundonthe strand that is thetemplate

for discontinuous DNA synthesis. To explain this

distribu-tionofsites, theysuggestthat Topo Iaction may bespatially

coordinated with thereplication complex. Thus, it is

impor-*Corresponding author.

tant tomappotentialTopo Isiteswithin theoriginregion,in which the siteswould be crucial for successfulinitiation of replication.

Inthisstudy,we haveusedcamptothecintofacilitatethe

mapping ofTopo I sites in the core origin and in flanking

ancillary regions ofDNA. Camptothecin appears toblock

therejoining stepof theTopoIreaction(24) andhaslittleor

noeffect onthebreakage specificity ofTopo I(9a, 25). We

found clustersofstrongTopoIsites available withinthecore

origin ofreplicationonthe strands thatbecome

templates

for

discontinuous DNA

synthesis

in both directions. These

clusters map within functional

replication

domains of the

core origin andmay contributetotheir

importance

in

repli-cation. We have also taken advantage ofmany

single

base

substitution mutations in the origin to define further the

nature ofTopo I

recognition

sites.

MATERIALS ANDMETHODS

Enzymes. Restriction enzymes (New England BioLabs, Inc.), T4polynucleotidekinase(BoehringerMannheim

Bio-chemicals), and the Klenow fragment of Escherichia coli

DNA polymerase I(Bethesda Research

Laboratories,

Inc.)

were usedaccordingto therecommendations ofthe

suppli-ers. Purified calfthymus Topo I was purchased from

Be-thesda Research Laboratories. Human Topo I that was

partially

purified

from 293cellsafter

extraction,

asdescribed

by

Dignam

etal.

(18),

was

kindly provided by

R.Richard and

D. Bogenhagen. Camptothecin was

purchased

from

Sigma

Chemical Co.

Plasmid constructions. The

pOR plasmids

have been

de-scribed

previously

(16). Plasmid

pORi

contains the SV40

coreoriginwithout

ancillary

origin regions. pOR2

consists of

the core originandT-antigen-binding

region

I. pOR4 has a

complete SV40

origin

of

replication,

which consists of

T-antigen-binding region I, the core

origin,

and the

early

promoter-enhancer

region.

Singlebase

pair

substitutions in

thecoreoriginwereobtainedby

misincorporation

mutagen-esisorby cassette

mutagenesis,

as described

by

Deb et al. 5175

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5176 TSUI ET AL.

Lower strand

_ + +

Upper strand

+ + Topol + + + +

+ + CPT + +

+ - Kinase - +

Marker

- *bI

- -.

35/39-~~~~~~~~~$SW

me

A/SQ-_ a

i

sp

i"

t,,8?8

-*

arn *

..

t

I

t

U

_

-. A

AM

Ac".

_..!M._

-~~~~~~~~~~~~.

':~

40,

IU~~~~~~~

_.e_

CUb -I

4ii ...

.U

'um

._.

a

ml

a

U

U.

ft

±

A

T

Si

-ale

-aa

-s

-a

0--:S

b

40.

aw

FIG. 1. Mapping ofTopo Isites in theSV40coreoriginof replication. Purified calf thymus Topo Iwasincubated with3'-end-labeledDNA,

containing T-antigen-binding region I and core origin DNA, under conditions designed for in vitro DNA replication. In some cases,

camptothecin (CPT)wasaddedtothe reactions. Someof the productswerephosphorylatedattheir5'endsattheTopo Icleavage sitesby theadditionof kinase. ThesameDNAsubstratewasusedin the Maxam and Gilbert reaction for G-A and T-Csequenceladders. Theproducts

wereresolved inan8%urea-denaturing polyacrylamide gel. Thepresence(+)orabsence(-)of variouscomponentsis indicatedatthetop ofthefigure. The structural landmarksshown in between the gel panels correspondtoreplication domains in theSV40coreorigin shownin

Fig. 3. Symbols: 9, inverted repeat domains;

t,

T-antigen recognition pentanucleotides; AT-rich domains. Nucleotide locations of the Topo I cutting sitesareshownatthesides of the figure.

_- +

G/A C/T

+

_-G/A C/T

5222

/23-522161t 7-52'4

15-5211/ 12-5208/O 9-52 t1/

07--5218/19

-11/1 2

-16/17

-20/21

-29/30

-30/31 -33/34

-40/41

NMMW

lll...

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TOPOISOMERASE I SITES IN SV40 ORIGIN OF REPLICATION 5177 (12-14). All the base substitution mutants are in a pOR1

background.Theplasmid DNAs were prepared and purified

by CsClequilibrium centrifugation as described by DeLucia

etal. (16).

3'-End labeling oftheDNA fragments containingtheSV40 origin.Plasmids were digested with eitherHindIIIorEcoRI. The 3' ends werelabeled with[32P]deoxynucleoside

triphos-phates and Klenow polymerase. EcoRI or HindIII was

added to make a second cut and to generate DNA fragments

labeled only on the 3' ends. The DNA fragments were

purified by polyacrylamide gel electrophoresis and were

electroeluted from the gels for Maxam and Gilbert DNA

sequencing reactions (27)andtopoisomerasecleavage.

Topoisomerase cleavage reactions. Topo I cleavage

reac-tions were carried out underconditions suitable for in vitro

DNAreplication (26, 30, 32) (30 mM

N-2-hydroxyethylpip-erazine-N'-2-ethanesulfonicacid [HEPES]-KOH [pH7.5], 1

mMdithioerythritol,0.1 mgof bovine serum albumin per ml,

7 mM MgCl2, 40 mM creatine phosphate-KOH [pH 7.5], 4

mM ATP [pH 7], and 2 ,ug of creatine phosphokinase per ml). The pH of the replication buffer at 37°C after the

addition of allcomponents to thereaction mixtureswas7.5.

PurifiedTopo I (10 U) was added to the end-labeled DNA

fragment(0.7 ng) in afinal volume of100 ,ul ofreplication

buffer. In some cases, 50 ,uM camptothecin (storedasa 10

mM stock solutionin dimethyl sulfoxide) wasadded to the

reaction mixtures. After incubation for 15 min at 37°C,

reactions were terminated by the addition of 1% sodium

dodecyl sulfate.

Phosphorylation of the 5' ends of topoisomerase cleavage sites. The Topo I cleavage products wereprecipitated with

ethanol and dissolved in 50 mM Tris hydrochloride (pH

7.5)-10

mM

MgCI2-5

mM dithiothreitol-50 ,ug of bovine

serum albumin per ml. Cleaved DNA products were heat

denaturedpriortothereactiontoincreasethe efficiency of

phosphorylation.Aftertheaddition of10 mM ATP and 10 U

ofT4 polynucleotide kinase, reactions were incubated at

37°C for 1h. Thekinase was inactivated

by

theadditionof

EDTA and by heating at 70°C for 10 min before

phenol-chloroform extraction andethanol

precipitation.

Sequencing gels.

Ethanol-precipitated

pellets were dried

andsuspendedin95% formamide bufferand heated to90°C

for3 minbeforebeing loadedontoa

sequencing

gel. Samples were resolvedbyelectrophoresis in aurea-denaturing

poly-acrylamide gel. After electrophoresis, gelswerefixedin10%

acetic acid, dried,and exposedtoX-ray film.

RESULTS

Mapping of TopoIsitesintheSV40coreorigin of replica-tion. We digested 3'-end-labeled duplex DNA with calf

thymusTopoI, underconditionsdesignedfor DNA

replica-tion in vitro, to map cleavage sites within the origin of

replication (Fig. 1).In somecases, camptothecinwasadded

to thereactions totrap the covalent Topo I-DNA

interme-diatesin acleavedstate(24).Thereactionswereterminated

with sodium dodecyl sulfate, and the products were

ana-lyzed by electophoresis through urea polyacrylamide gels

and compared with Maxam and Gilbert (27) sequence

lad-ders of the same DNAfragment. Topo Icleavagegenerates

single-strand breaks with the enzyme covalently linked to

the 3' phosphoryl end ofthe break and a free 5'

hydroxyl

group (6-8, 22). We

phosphorylated

the free 5'

hydroxyl

groups of the 3'-end-labeledDNA

fragments

withkinase for

precise comparison with the Maxam and Gilbert

fragments

that have 5'phosphategroups. The

phosphorylated products

had an increased mobility during gel electrophoresis. This

resultconfirmsthat theobserved scissions are characteristic ofTopo I cleavage.

In the absence of camptothecin, Topo Iappeared to cut

only scattered sites on boththe upper andlower strands of

the core origin. Twoofthe three sites onthe lowerstrand

Lower strand

topol

+CPT C/TA/G

w1r

_

. -|-- VW

0

a

do

0S

U

_9I

-a.

-hi

ii

w0

Upper strand

topoI

+CPT

-)

I

I

lI

a

a

FIG. 2. Mapping of TopoIsitesin thecomplete SV40originof replication. Purified calf thymus Topo Iand camptothecin (CPT) wereincubated under in vitro DNAreplication conditionswithan

end-labeled DNAfragment containing T-antigen-binding region I, thecoreorigin,the21-bprepeats of theearlypromoter, andmostof the enhancer. Conditions and procedures for the reaction are

describedinMaterials and Methods. TheTopoIcleavageproducts were run next to Maxam and Gilbert C-T and G-A sequencing laddersin aurea-denaturing polyacrylamidegel. Thepositions of Topo Icleavage sitesareidentified withSV40 nucleotide numbers. Mapping of siteswithquestionmarks isnotprecise.

VOL.63,1989

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5178 TSUI ET AL.

A. Topolsomerase I sites In T antigen binding reglon I

5'-AAGCTTTTTGCAAAAGCCTAGGCCTCCAAAAAAGCCTC

3'-TTCGAAAAACGTTTTCGGATCCGGAGGTTTTTTCGGAG

HindIII A A A

5197/98 5206/07 5208/09

B. Topolsomerase I sites In the core orlgin and their alignment with repiication domains

5218/19 11/12 16/17 20/21 33/34

-CTCACTACTTCTGGAATAGCTCAGAGGCCGAGGCGGCCTCGGCCTCTGCATAAATAAAAAAAATTAGTC-I1

3~4

4

AT

-GAGTGATGAAGACCTTATCGAGTCTCCGGCTCCGCCGGAGCCGGAGACGTATTTATTTTTTTTAATCAG-A AA A A AA A

5211/12 522V23 5231/32 16/17 17/18 31/32

a

a 0

-cm z

a

N

C. Topolsomerase I sites In the promoter-enhancer region

40/41 72/73 93/94

-AGCCATGGGGCGGAGAATGGGCGGAACTGGGCGGAGTTAGGGGCGGGATGGGCGGAGTTAGGGGCGGGACTA-

-TCGGTACCCCGCCTCTTACCCGCCTTGACCCGCCTCAATCCCCGCCCTACCCGCCTCAATCCCCGCCCTGAT-A A

38/39 58/59

111/12 122V23 127/28 143/44

-TGGTTGCTGACTAATTGAGATGCATGCTTTGCATACTTCTGCCTGCTGGGGAGCCTGAATTC-3' -ACCAACGACTGATTAACTCTACGTACGAAACGTATGAAGACGGACGACCCCTCGGACTTAAG-5'

[image:4.612.71.550.73.571.2]

A 138/39

FIG. 3. Correlation ofTopo I sites with functional elementsofthe SV40 originofreplication. The sequence ofthe entire originof replication is shown. (A) Topo Isites inT-antigen-binding region I. (B) Coreorigin.Thehistogram shows themajorreplicationdomainsin theSV40 coreorigin. These weremapped by comparingthereplicationefficiencyofcoreoriginswithsinglebase substitutionmutantswith theefficiency of the wild-typecoreorigin. (C)Topo Isitesin theSV40earlypromoterand enhancerregion.SixSPl-bindingsitesin the21-bp repeatsof thepromoter are shown. A partof theSV40 enhancer72-bprepeatisshownatthebottom. Symbols: A,TopoIcleavagesites; m.,T-antigen recognitionpentanucleotides;

E=[>,

earlyinverted repeats;

Fii,

AT-richsegment; 0,single-basesubstitutionmutants; _, Belement oftheenhancer(23); _, SP-1 binding site.

(betweennucleotides 31 and 32 and between 5231 and 5232)

were previously described by Edwards et al. (19). When

camptothecin was added to inhibit the rejoining reaction,

clustersofadditionalprominentcleavage siteswererevealed

onbothstrandsoforiginDNA. These clusterswerelocated in theinvertedrepeatdomainof the core origin on the lower DNAstrand and in the AT-rich domain on the upper strand.

Alongerexposureof theautoradiogramsinFig. 1showed

similar patterns of Topo I cleavage in the presence or

absence of camptothecin, although the drug increased or decreased cleavage at some sites more thanatother sites. Thesefindings are consistent withrecentreportsby others (25, 28). Thus, itis verylikelythatallof the sites shownin Fig. 1areauthenticTopoI sites, althoughitisnotpossible to determine which of thesitesare mostfrequentlyused in

vivo. Human Topo Ifrom 293 cells cleaved sites virtually

identical to those cut by calf thymus Topo I (data not shown). We also found thatTopoIcleavesatidentical sites J. VIROL.

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TOPOISOMERASE I SITES IN SV40 ORIGIN OF REPLICATION 5179

(A) INVERTED REPEAT- Lower Strand

,(AiL)4 - IF 1)4f;,

S'C T C A C T A C T T C T G G A A T A G C T C 3' 3lG A G T G A T G A A G A C C T T A T C G A GS5

A/T SEGMENT- Upper Strand (B)

0 [E!2 1 4I 13 25 22 24 2C 28 3O 32 34

5lT C T G C A T A A A T A A A A A A A A T T A G T C A3' 3'A G A C G T A T T T A T T T T T T T T A A T C A G T S

A G C C C TTC G G A G G C G

T C G G G A A G A C MUTATIONS C T C C G C

I // / / // / / W T -..-i | I / I \

_ _'m _no _ =0 w m _s= _ _ _ ~ _ -32 _

2/28

'----L- I; 4 -J

-_5,'2 2.f"3 -~~~~~~~~~~~~52~~~~~~-1E/.1'Ii"

-L64i

2~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

9:

C C C G T A

G G : G C A T

I

II

/1

/

hT

P-~~~~-*W ~ W-W~ ~ -W

ft

-m_

-~4 44 -n 4m -~mmml -~am - -w 4m- 1

-I 5 : 4

24 /25

-3 1'3z

a

O__a ___-lm _ _ _ _

am

FIG. 4. Effects of single base substitution mutations in the SV40 core origin of replication on Topo I cleavage sites. Topo I and

camptothecin were added to 3'-end-labeled DNAfragments containing mutant coreorigins asdescribed in Materials and Methods. The

productswererunnext toanA-G sequencing ladder inaurea-denaturing polyacrylamide gel. (A) Effects of base substitutionmutantsin the early invertedrepeatregion (arrows)onTopo I sites in the lowerstrand of DNA. Mutants with base substitutionsatpositions 5220, 5221, 5225, and 5231 werealsoanalyzed inaseparateexperiment. (B) Effects of single base substitutionmutantsinthe AT-rich domain (_)on

Topo I sites in theupperstrand of DNA. Positions of the basesubstitution mutationsareshownatthetop.Topo Icleavage sitesaremarked

(in nucleotides) atthesides of the gels.

underabroadrangeof conditions. Thepresence orabsence ofATP, MgCl2, and 100 mM KCI had little effect on the specificityofTopoIcleavage (datanotshown). Camptothe-cinalone didnotbreak DNA.

MappingofTopoI sites in the complete origin of replica-tion. Ancillary regions on either side ofthe core origin of replication increase DNA replication 10- to 20-fold over levelssupported by thecoreorigin alone (16). We wishedto determinethefrequency andstrength ofTopo I sites in the ancillary regions because strong sites in appropriate loca-tionsmayfacilitatetheefficiency ofDNAreplication. Figure 2 maps the stronger Topo I sites in the complete origin of replication. For this study, we used a DNA fragment that contains T-antigen DNA-binding regionI,thecoreorigin of

replication,the21-bprepeatsofthepromoter, andapartof the enhancer. The relative strength ofTopo I cutting sites withineither strand is evident, but thetwostrandscannotbe comparedwith oneanother. The strongest sites in the core

origin map from nucleotides 5208-5209 to 5222-5223 in the invertedrepeatregionof the lower strand andatnucleotides 20-21 in the AT segmentof theupper strand. These results confirmthose shown inFig. 1. Theremainingweaker sites in thecore regionwere seen onlongerexposure of the autora-diograms (data not shown). Outside the core origin, scat-tered sitesof intermediatestrengthwereevident in theupper

andlower strands of thepromoter region; very strong sites

were found between nucleotides 122-123 and 127-128in the

upperstrand of the enhancerregion.

Correlation ofTopoI sites with functionalelements of the SV40 regulatory region. The positions ofTopo I sites in the

Inverted Repeats Pentanucleotides AT Segment

r AT

33t

N

Replication Complexes

FIG. 5. Model for thespatial arrangement ofTopoIat replica-tion forksin thecoreorigin. TopoI,bound eithertoTantigenorto

acellularproteininareplicative complex,ispositionedin thecore origintotake advantage of the clusters ofTopoI sites in theAT segmentontheupperstrand and in the invertedrepeatregiononthe lower strandduringthe formation ofareplicationbubbleearlyinthe

initiationofSV40DNAreplication.

I--2'8.1 ..---j

-.-

mi-VOL.63, 1989

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[image:5.612.66.559.65.376.2] [image:5.612.322.554.531.658.2]

5180 TSUI ET AL.

A. SEQUENCES AND MUTATIONS AROUND ORIGIN TOPO I SITES:

5'- 10 9 * 7 6 5 4 3 2 i-V+4 2 3 4 5 6 7 S 9 10 - 3V

rico-vC (521/19

+/-CPT C T C A C T A C

+/-CIT C C T C G G C C

G C C T C T G C

a

&

+/-CIT C T G C A T A A

a

&

a

I

GC

&

A TA

aa@@

A A

0

ATA CA TA A A AT AA +/-CIT T A A A A A A A +/-CPT A A A A A A A A A A A A A T T A A G T C A G C C

+/-CP GA G G C G G A G

+/-CPT G G G C G G A G G A C T A T G G

T A T G G T T G

+/-CPT G G T T G C T G C T G A C T A A +/-CPT T A A T T G A G +/-CPT T T T G C A T A

*G G C C T C T G *C T C T G C A T

*T C T G C A T A *T G C A T A A A

*A A A A A A A T LOWER STRAND

G C A G A A G T A T C. C C G C C C A G T T t T C C G C C C C A T c

+/-C PT C A T G G C T G A C T T T T T T T A T T I

T T T T T T A T T T I

+/-CPT C C T C G G C C T C I

CCGCCCAGTT~~~~~~~~~~C

+/-CPT C T G A G C T A TCT T A T T C C A G A A c T T C C A G A A G TC

C A G A A G T A G T c

A A G T A G T G A G G T A G T G A G G A A G G C T T T T T T *C T A T T C C A G A I *C T C T G A G C T A B.FREQUENCY OF NUCLZOTXIDX8 AT ZACH POS

A 7 12 11 11 14 10 18 15 17 11 I

C 9 7 9 7 10 10 4 14 6 1 3

C 13 8 8 10 8 9 S 6 2 4

T 11 13 12 12 8 11 13 5 15 24

C. NUMBZR oir MUTATIONS THCT AGrACT TOPO

TOTAL 5 8 7 9 8 7 7 11 7 9 S

G C A A A G C A T G

C C G C C C A T T C G G C T G A C T A A

T A A T T T T T T T T A T G C A G A G G

A T G C A G A G G C

T G A G C T A T T C

C C A G A A G T A G

G T A G T G A G G A

A G T G A G G A G G

9)

10

G A G G A G G C T T

G A G G C T T T T T G G C T T T T T T G G G A G G C C T A G A G T A G T G A G G

T T C C A G A A G T

BITION:

13 17 17 12 17 15 14 17 11 12 14 11 11 14 7 10 9 8 13 12 4 6 6 6 8 8 6 3 2 ! 9 6 6 8 8 7 11 12 14 13

7 4 1 3 2 2 1 3 2 2 9 9 6 6 5 6 5 7 5 4 D. CONSENSUS: - - - A A A T a - - -

-T a T A

T T T C A T

0

A T

0

A A AA A A A

0

T T T G T A T TT T T T T C T A C T T A T C T C A A A A A T A T A

C T G T G C A A A A A

A A A TC,

A A A

A A A

A A A

T A G

A G T

A G G A G G G C T G A C T A A G A G G C A T C T T A A

A T A

T A A

A A A

G T C G A A A T A

T A A

A A A

A A A

A A T

0

A T T

@D0

T C A

C A G

C C A

G C G G G C G G C G A C T A A T T G A T G T G C G C C A T A

A A A

A A A

A A A

A G C

T A G C A A T A

A A A A

A A T TQ

aL

T T A G T A G T

0

Q

A G T C

aD

G C C A

C C A T

T G G G

G A G A G G G A G G G A T A A T T T G A A G A T C A T G T T T G T G C T A A A A

A A A A

A A A T

A T T A

C A T G

(5218/19) (11/12) (16/17) (20/21) (22/23) (23/24) (24/25) (29/30) (30/31) (33/34) (40/41) (72/73) (93/94) (111/112) (114/115) (117/118) (122/123) (127/128) (143/144) (15/16) (18/19) (19/20) (21/22) (31/32) (138/139) (58/59) (38/39) (31/32) (17/18) (16/17) (5231/32) (5222/23) (5216/17) (5214/15) (5211/12) (5208/09) (5206/07) (5197/98) (5217/18) (5224/25) J. VIROL. urrzxR 2 2 5 1

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TOPOISOMERASE I SITES IN SV40 ORIGIN OF REPLICATION 5181

SV40origin are summarized in Fig. 3. The positions of sites in the core origin are correlated with replication domains that have been mapped by using single base substitution mutations (12-14) (Fig. 3B). The great majority of the cleavagesites cluster in the important replication domains at

eitherendof the core origin. Most of the sites at theearly,

left end of the origin are in the lower strand of DNA and correspond to the major and minor domains of the inverted repeatregion. At the late or right end of the core origin, the Topo Isites map in the upper strand of the AT-rich domain. The correlation of these sites with replication domains is

consistent withtheidea thatTopo Isites may play functional

roles in the initiation ofDNA replication. This possibility

does not imply that Topo I sites are the only important

featuresofthesedomains.

There are fewer Topo I sites in ancillary regions of the

SV40origin of replication than in the core origin of replica-tion. Still,they tend to be located on the lower strand of the T-antigen-binding region I to the left of the core origin (Fig. 3A) and on the upperstrands of the promoter and enhancer regions (Fig. 3C). The strongest Topo I sites cluster in and around the B elementofthe enhancer(23).

Genetic analysis of Topo I sites. Theexistence ofnumerous

single base substitution mutations in the SV40 core origin

and the frequency of Topo I sites in the same region

presented an opportunity to investigate which nucleotide

positions can influence Topo I sites. Figure 4 shows the

effects of 24 different mutations on the clusters of Topo I

sitesatboth ends of thecoreorigin. Manymutationshad no

effect on Topo I sites trapped in an open position by

camptothecin, while some mutations either created

com-pletelynew sites orabolished existing sites.Other mutations

caused only subtle changes in the strength of the cutting

sites. Mostofthemutations thataffectedTopo Isiteswere

located quiteclose to thecuttingsite. For example, aT-to-G

substitution at position 30 in the upper strand of the AT

domain (Fig. 4B) drastically reduced sites 30-31 and 33-34

while enhancingthe 29-30 site.Remarkably,mutationsinthe

inverted repeat region modulated Topo I sites that were

separated from the mutations by significant distances (Fig.

4A). For example, a mutation at nucleotide 5214 reduced

cuttingatsite5231-5232, which isseparated fromitby17bp,

and nearby mutations at nucleotides 5215 and 5217 had

similar effectson the same site. Furthermore, mutations in

the ATsegment alsomodulated Topo I sites atdistances of up to 10bp.Acompleteanalysis ofthe consensussequences

ofthecutting sites andtheeffects of mutations on the sites is

presentedinthefollowing section.

DISCUSSION

We have mapped Topo I sites in the core and ancillary

regions oftheSV40origin of replicationinvitroaspartofa

complete analysis oforigin functions intheinitiation ofDNA

replication. Theuseofcamptothecinto arresttheclosing of

TopoI sites has ledtotheidentification ofadditionalstrong

cutting sites in origin DNA. All available evidence suggests that ourchoiceof various parameters for the topoisomerase assay is valid. Camptothecin has little effect on the

speci-ficityof Topo I sites in vitro (9a, 25). We (data not shown)

and others (19) havefound that Topo I purified from either calfthymus or human cells cuts at the same sites with similar

efficiencies. Furthermore,thelocations of the sites identified

underconditions designed for SV40 DNA replication were the same as those of sites found under conditionsdesigned

for optimal topoisomerase activity (2, 19; our data not

shown). We haveidentified clusters of Topo I sites at both endsofthe coreorigin of replication. These arearrangedbn the same strands that are cutpreferentially in vivo (28). In an invivoanalysis, Porter and Champoux (28) found some, but not all, of the sites that we have identified in vitro. These

differences mayreflectthe sensitivityof theirin vivo

map-pingapproach, whichreliedon primerextension over large regions ofDNA.

Wepresume that the actual use of potential Topo Isitesin vivo during DNA replication or transcription would depend

both on the intrinsic strength of the sites and on the

probabilityof theirexposure totopoisomerase. Exposure to

theenzyme could bedetermined eitherbychromatin struc-ture orby the use ofspecific mechanisms todeliver

topo-isomerase tointrinsic sites. Clearly,notallpotential cutting

sites in the cell are equally available for interactions with

Topo I. The enzyme associates preferentially with active genes(3, 10, 20, 21, 31, 35)andprefers cuttingsites onSV40 DNA strands that are templates for discontinuous DNA

replication (28). Furthermore, Topo I associates

preferen-tiallywithreplicating SV40 DNA(9)andcuts thereplicative

intermediates at or near replication forks (1, 29). These

findingssuggest that sites in the openchromatinregion ofthe

origin of replication mightbe moreavailable than other sites

earlyin DNAreplication and thatcomplexes ofreplication

proteins might guide topoisomeraseto onestrandofDNA at

replication forks.

Borowiec and Hurwitz (5) have shown that T antigen

inducesmelting ofDNA in the

origin

ofreplication. In the

absenceoftopoisomerase, however, melting is limitedto a

smallsegmentofDNAcorrespondingtotheinvertedrepeat

domain at the early end of the core origin. If T antigen

directlyorindirectly guidesTopo I toreplication forks along

withother replication proteins as suggested above, thenit

would be essential to have cutting sites within the core

origin. Figure5shows how Tantigenitselforotherproteins

bound to T antigen in a large

replication

complex might

position Topo I to takeadvantage ofthe clustered sites on

oppositestrandsatthetwo

replication

forks formedearlyin

the process ofreplication. In this study, we have

demon-stratedthatsuchTopoIsitesareavailablein abundanceon

theappropriate strands. Itis

unlikely

that loss ofTopoIsites

accountsforreplication defectscausedbybasesubstitutions inthe core

origin

becauseany

given

mutation affects

only

a

subset oftheclustered sites

(Fig.

4).

Indeed,

the

redundancy

FIG. 6. Analysisofrecognitionsequencesfor TopoIin thecompleteoriginofreplication. (A)Ten-base-pairsegmentsoneach side of40 sites for TopoIcleavage(V)arealigned forcomparison ofsequences. Thelocations ofthesitesin theSV40sequenceareindicatedonthe right. Sitesmarked+/-CPTaresitesthatareevidentineither the presenceorabsenceofcamptothecin;other sitesareseenonlyinthe presence ofcamptothecin. The sites marked with stars arepresentonlyinmutantorigins andthusare notshown in Fig. 3. Singlebase mutations withinthevarioussegmentsareshown below thewild-typesequences. Theeffects ofthesemutationsoncleavageby TopoIare

indicatedasfollows:0,decreasedcutting;O,increasedcutting;

-,

littleor nochangeincutting. (B)Frequencyof appearance of the four possible nucleotidesateach sequenceposition.(C)Numbersofmutations ineach ofthe nucleotidepositionsthatalter theefficiencyofcutting are tabulated. (D) Consensus sequence basedon frequency ofoccurrence ofnucleotides ateach position and onthe effects of specific mutationsinthe40Topo Isites.

VOL. 63,1989

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5182 TSUI ET AL.

ofpotential TopoI sites in the core origin may ensure the

efficiencyof theinteraction andrender it resistantto

muta-tional effects. The existence ofstrong Topo I sites in the

ancillarycomponents of the origin of

replication

may

con-tribute to their roles in

facilitating

DNA

replication

or

transcription.

We have identified 40 Topo I sites in the

wild-type

and

mutantorigins ofreplication; thesearealignedina5'-to-3'

orientation in

Fig.

6A, and the sitesareidentified

by

SV40

sequencenumbersatthe

right

ofthe

figure.

BecauseTopoI

sitesoccuratalimited numberof

locations, they clearly

are

not random in sequence. The frequency of nucleotide

oc-curence ateach ofthe 10

positions

toeithersideofthe break is tabulated in Fig. 6B. None ofthe sequence positions is

absolutely

conserved. With the exception ofthe

thymine

at

position

-1, it is often difficult to

judge

which of the sequences to include in a consensus sequence. In some cases,a

preference

for

purines

orforadenines and

thymines

rather thanforaspecificbase mightbepossible.

The

availability

ofmanysingle base substitution mutations

in the SV40

origin

of

replication

(12-14)

provided

uswithan

opportunitytoinvestigate further which nucleotide positions

determine the location of Topo I sites. Existing mutations

arelocated within 10 nucleotides of TopoI

cutting

sitesin

the core

origin

a total of 140 times. All

single

nucleotide

substitutions within10bp of TopoI sites and theeffects of

these mutations are shown in

Fig.

6A. The mutational

analysis

inFig. 6C summarizes the number of mutationsthat

havean effectonthe efficiency of TopoI cleavage and the

total number of mutations available at eachposition. This

analysis confirms that positions -4 to -1 and +1 are the

mostimportant positions in the TopoIcleavage reaction.

On the basisof the effects of

specific

basesubstitutionson

Topo I

efficiency,

we were sometimes able to

distinguish

which of the alternativeconsensus sequencessuggested by

the

frequency

analyses in

Fig.

6B are

preferred.

The

pre-ferred Topo I sequence is

5'-AIT-A/G-A/T-T-break-G/A-3'

(Fig.

6D). This sequence is in close agreement with the consensussequencereported

by

PorterandChampoux after

analysis ofinvivocutting sitesinSV40DNAin the presence

ofcamptothecin (28). Nevertheless, there are many

excep-tions to this idealized sequence. These may be determined

by complex

combinatorial factors within the 5-nucleotide

preferred recognition

segment or by effects of adjacent sequences. It is remarkable that base substitutions can

modulate Topo I sites at great distances. Twenty-seven

mutations outside the five nucleotides of thecoreconsensus

sequence modulate the

efficiency

ofTopo I cleavage (Fig. 6C).

Furthermore,

three mutants in the inverted repeat

region

affect

cleavage

as many as 17 basesaway(Fig. 4A).

Although these bases could interact with Topo I at a

dis-tance, it seems more likely that they act by altering the structureoforiginDNA. We have reason tosuspect that the core

origin

has an unusual structure. The inverted repeat

region

is highly susceptible to protein-induced melting (5), and the AT segment causes anomalous migration of DNA

fragments

during gel electrophoresis(13). The three domains of the core origin may coordinately encode a complex structure in

origin

DNA.

ACKNOWLEDGMENTS

This work was supported by Public Health Service grants CA-18808 and CA-28146 from the National CancerInstitute.

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