Two cellular single-strand-specific DNA-binding proteins interact with two regions of the bovine papillomavirus type 1 genome, including the origin of DNA replication.

0022-538X/92/105988-11$02.00/0

Copyright © 1992,American Societyfor Microbiology

Two

Cellular

Single-Strand-Specific

DNA-Binding Proteins Interact

with

Two

Regions of the Bovine Papillomavirus

Type

1

Genome,

Including the Origin of DNA Replication

CORNELIAHABIGER, GERTRAUD STELZER, URSULA SCHWARZ,

ANDERNST-LUDWIGWINNACKER*

Institutfiir Biochemie der Universitat Munchen im Max-Planck-Institut fiir Biochemie, AmKlopferspitz 18a, D-8033Martinsried bei Munchen, Germany

Received 28 April1992/Accepted 1 July 1992

We haveidentified andpurified to nearhomogeneitytwospecific single-stranded DNA-binding factors (SPSF Iand II) with molecular masses of 42 and39 kDa,

respectively,

fromcalf thymus. Gel retentionanalysis and competition experiments demonstrate that the ubiquitous proteins SPSF I and II specifically interact with single-strandedDNAderivedfromtheminimalin vitroorigin of replicationof bovinepapillomavirustype 1 and a regionoftheviral genome proposed to be involved in plasmid maintenance. Bovine papillomavirus type 1 proteins do not interfere with DNA binding of SPSF I and II. Theexact locationofthebinding domainsof SPSF IandIIonthe DNA has been determined by methylation interference and T4 DNA polymerase footprinting. A potential cellular binding site for SPSF I and II is the

major

promoter (P2) of the human c-myc gene.

The replication of the circular double-stranded bovine papillomavirustype 1 (BPV-1)genomeissubjecttocomplex control mechanisms. They guarantee the maintenance of about200copies per cell, all of which arereplicated extra-chromosomally once per cell cycle (5, 13). Unlike other DNAviruses, BPV-1 does notoverride cellular replication controls. Its bidirectional mode of replication hence pro-vides an excellent system with which to study the mecha-nismspreventing the reinitiation ofreplication from an origin that hasalready beenengaged in this process.

The minimal origin of BPV-1 DNA replication has been mapped recently to aregion between nucleotides 7900 and 50, containingan18-bppalindrome andanAT-rich sequence (39, 46). Earlier studies of the replicative intermediates of BPV-1 and DNase I-hypersensitive sites had suggested a

region between nucleotides 6600 and 7300 (30, 43). This region also contains one of the two plasmid maintenance sequences (PMS1; domain 1 and 2), which have been sug-gestedtofunctionascis-activeregulatoryelements support-ing extrachromosomal replication ofrecombinant plasmids in the presence of viral proteinsprovided in trans (18, 20). Other studies had shownastretch aroundbp

7730,

+100bp, designatedthe 2D-origin, tobe active as anorigin for viral plasmidreplication (45). A 69% subgenomic fragment(from nucleotides 6958 to 4450), which divides the BPV-1 early regions, andaportion of the long control region from the late regions and the other part of the long control region is sufficient forcellulartransformation and extrachromosomal replication oftheviral DNA. However, the transformation and replication efficiencies are reproducibly lower (25 to 50%) than those observed for the full-length BPV-1 genome

(15,

17,

32).

Of all viralproteins, two, designated El (68 kDa) and E2 (48 kDa), have been shown to be both necessary and sufficient forBPV-1replication (19,38). Phosphoprotein El forms stable complexes with E2 (21, 23). Clusters of E2 binding sitesarelocatedin the vicinity of the important early

* Corresponding author.

viral promoters. These sites normallyfunctionas transcrip-tional enhancers, either alone or in cooperation with other binding sites for cellularfactors, suchas SP1(14).

An exactinterpretation ofexperiments aimedat elucidat-ing the functions of El and E2 is complicated by the observation that an El fusion protein (RecA-El) interacts with the functionaloriginofreplication(nucleotides 7900to

50)and also withafragment containingthe2D-originandan E2-responsive element 1 (E2RE1) contained therein (42). Moreover, the E1-E2complexalso binds to a BPV-1 frag-mentcontaining E2-responsiveelements(E2bindingsites 1 and2, distinct fromE2RE1)andtoPMS1, aswellas tothe fragment containing the2D-origin andE2RE1 (21, 23).

Inthis report,wedescribe the identification,isolation, and characterization oftwocellularproteins, designatedSPSF I (42kDa)and II(39 kDa), forspecific single-stranded DNA-binding factors Iand II. These proteins, isolated from calf thymus, interact specificallywithsingle-stranded DNA de-rived from theminimal in vitrooriginofreplicationand also with sequences of domain I of PMS1. Theexpressionof viral proteinsdoesnotinterfere withprotein binding.Thesites of SPSFIandIIinteraction withDNAhave beenmapped by methylationinterferenceand T4DNApolymerase footprint-ing. SPSFIand IIarethefirstexamplesofcellularproteins involved in interactions with DNA regions required for BPV-1DNAreplication. Theyareexpressedinavarietyof cell types and in different

species

andarethereforegeneral factors inhighereukaryotes.

MATERIALSANDMETHODS

Cells.HeLa, NIH 3T3, C127, and CB1 cellswere grown asmonolayersin Dulbecco's modifiedEagle'smedium sup-plemented with 5% fetal calfserum. CB1 cells are mouse C127 cells which we obtained as a stably transformed cell line after calcium phosphate coprecipitation with plasmid pMLBPV(19).The BPV-1 genome ismaintained extrachro-mosomallyinaconstantnumber ofapproximately100to200 copies per cell, as proven by hybridization of the BPV-1 genomeagainstHirtextracts(9)fromcloned andpropagated

5988

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CELLULAR DNA-BINDING PROTEINS INTERACT WITH BPV-1 5989 CB1 foci.

F126,

an Epstein-Barrvirus (EBV)-negative

Bur-kitt's lymophoma cell line, and IARC 549, an EBV-trans-formed human lymphoblastoid B-cell line, were maintained assuspensioncultures in RPMI 1640 medium containing8% fetal calfserum.

Preparation of cell extracts. Nuclear and cytoplasmic ex-tractswerepreparedfrom subconfluent cells by themethod of

Dignam

etal.

(4)

with someimprovements. Specifically, Nonidet P-40

(0.5%)

was added tobuffer A, which caused the

cells,

in particular NIH 3T3, C127, and CB1, to swell more readily. Dounce homogenization thus could be

avoided,

and thenucleicould bereleased by vortexing. The nuclear extraction bufferCcontained 0.35 M instead of 0.43 MNaCl.

Oligonucleotides. Thesynthetic oligonucleotides (u, upper

strand; 1,

lower strand) representing the PMS1 domain 1

region

(P-u,

P-i,

and

Pk3-i)

and the BPV-1 origin of replica-tion

(ORI-u

and ORI-l) are shown in Fig. 1A. Oligonucleo-tideBE-i correspondstothe lower strandof the BPV-1 distal enhancer from nucleotides 4390 to 4440 (44). PyE-l repre-sents the lower strand of the B enhancer of polyomavirus from nucleotides 5160 to 5210 (27). Oligonucleotide EF2 contains an

NFl-binding

site from adenovirus FL, nucleo-tides 166to 151

(5'-GATCAACACAACACAGTIGGCACA

GTGCCAAAAGCCCCG-3'). Oligonucleotide

lex2 repre-sents the lower strand of the promoter region of the lexA gene of

Saccharomyces

cerevisiae (5'-CTCGAGTACTGTA TGTACATACAGTACTCGACTCGA-3'). All oligonucleo-tides were

purified

on denaturing urea-polyacrylamide gels before use.

Gel retention and competition experiments. Gel retention assayswere

performed

asdescribedbySchneider et al. (34).

Briefly,

a reaction mix of 30

j.l

typically contained 5 to 20 fmolof

single-stranded

oligonucleotides 5'-endlabeled with

[-y-32P]ATP by

T4 polynucleotide kinase, 1 to 2 ,ug of

poly[d(I-C)],

and up to 6 ,ug ofprotein. Thereaction buffer contained 25 mM HEPES

(N-2-hydroxyethylpiperazine-N'-2-ethanesulfonicacid, pH 7.9),5 mMdithiothreitol(DTT), 1 mMEDTA, 10% glycerol, and 150 mM NaCl. The reaction mixturewas

adjusted

to10 mMMgCl2and incubated on ice for 5 to 10 min. Competition analyseswere usually carried outwitha50- to100-fold molarexcessofcompetitor DNA.

DNA-protein complexes

wereseparated from free DNA on 8%

polyacrylamide

gelsandvisualized by autoradiography of thedriedgels.

Methylationinterferencefootprinting. 5'-end-labeled oligo-nucleotides were methylated with dimethyl sulfoxide as described

by

Maxam and Gilbert (22) and incubated with

partially

purified C127 extracts as for the gel retention assays. DNA-protein complexes and free oligonucleotides werevisualizedby autoradiographyand cut out from the gel. DNA was eluted from the gel by isotachophoresis (26), treated with

phenol-chloroform,

and ethanol precipitated. Aftertreatmentwith piperidin, theproducts were analyzed

on7to 10%urea denaturing sequencing gels.

T4DNA polymerase footprinting. 5'-end-labeled

oligonu-cleotides, protein

extract(asindicated in Results), T4 DNA

polymerase

buffer [16.6 mM (NH4)2SO4, 67 mM Tris-HCI

(pH 8.8),

6.7 mM

MgCl2,

10mM2-mercaptoethanol, 6.7 ,uM

EDTA,

1 to2 ,ug of

poly[d(A-T)],

and1 to2 ,ug of

poly[d(I-C),

and

competitor

DNA,ifneeded,wereincubated on ice for 10 min. Aftercompletionof thebinding reaction, 10 U of T4 DNApolymerase (Biolabs)was added, and the reaction mixturewasincubatedatroomtemperaturefor 15 min. The

proteins

were removedbyphenol extraction, and the DNA

was ethanol precipitated. The reaction products were ana-lyzed on 10% polyacrylamide sequencinggels.

Purification of SPSF I and SPSFII from calfthymus. All purification steps were carried out at

4°C.

Whole-cell ex-tracts from calf thymus were prepared by cutting 500 g of tissue into pieces, which were then suspended in 1 liter of buffer I(0.01%Nonidet P-40, 25 mM HEPES[pH

7.9],

5mM MgCl2, 50 mMNaCl, 250 mM sucrose, 5 mMDPT, 0.5 mM protease inhibitor phenylmethylsulfonyl fluoride [PMSF], 1 ,uMleupeptin, 1% Trasylol) and treated in a Waring blender (adjusted to low, for 2min). The crude extract was cleared by centrifugation at 8,000 rpm for 10min. The supernatant was filtered through cotton cloths and added to DEAE-Sepharose CL-6B(Pharmacia) equilibrated with buffer 11 (25 mM HEPES [pH 7.9], 1 mM EDTA, 5 mM DTT, 10% glycerol), with the addition of 50 mM NaCI. This mixture wasstirred slowly for 6 h. Unbound proteins were removed by filtration, and the resin with the bound proteins (including SPSF I and II) was washed with 5 volumes of buffer II-50 mM NaCl. The washed DEAE-Sepharose was added to a column, and the proteins were eluted in a single step with 300 mM NaCl in bufferII. Fractions containing SPSF I and II were monitored by gel retention analysis. Active fractions were dialyzed against buffer II with 50 mMNaCland loaded onto a MonoQ column (HR 16/10; Pharmacia) equilibrated with buffer II-50 mM NaCI in a fast protein liquid chroma-tography(FPLC) system. After the column was washed with 10 bed volumes of bufferII-50 mMNaCl, the proteins were elutedby a linear gradient (50 mM to 1 M) ofNaCI. SPSF I-and II-containing fractions (centered at 550 mM NaCI)were pooled and dialyzed against buffer II with 150 mMNaCI or diluted with buffer II without salt to obtain a final concen-tration of 150 mMNaCl.

DNA affinity chromatography columns were prepared by coupling the single-stranded oligonucleotide P-1 to CNBr-activated Sepharose (Pharmacia) following the instructions of the manufacturer. The column was connected to the FPLC system and equilibrated with buffer II containing 10 mMMgCl2 and 150 mM NaCl. The active MonoQ fractions, adjusted to 150 mM NaCl, were mixed with MgCl2 (end concentration, 10 mM) and with 1 ,ugof poly[d(I-C)] per ml and loaded onto the DNA affinity column. Washing with 10 volumes of buffer II-10 mM MgC12-150 mM NaCl was followed by elution with 5 bed volumes of a linear gradient (150 mM to 1 M) of NaCl. This protocol did not allow separation of the two factors because SPSF I eluted at about 950 mMNaCl and SPSF II eluted at about 850 mM NaCl. The pooled active fractions could be stored at

4°C

for up to 4 months without losing their binding activity. Aliquots were stored at -80°C.

UVcross-linking. Binding reactions were performed essen-tially as described for gel retention except that the amount of proteinextract was scaled up 5- to 10-fold and the amount of 5'-end-labeled radioactive oligonucleotides was scaled up 3-to5-fold. The reaction mixture was placed on a transillumi-nator (254 nm, 8,000 p.W/cm2), irradiated at room tempera-turefor 4min, mixed with Laemmli buffer and separated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electro-phoresis (12) together with suitable protein size markers. Themolecular masses of the free oligonucleotides and the UV-cross-linked DNA-protein complexes were determined by comparison of Coomassie-stained dried gels and their autoradiographs (exposure to X-ray film [KodakXR5] with an intensifying screen at -80°C for 2 to 3 days).

Renaturation of proteins from

SDS-polyacrylamide

gels. Protein extract (2 to 3 mg) was loaded on a preparative 10% VOL. 66,1992

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A

7945/1

I I I

AOI

I I

6500 700 7500 500 1000

I I

II

I

polyA-L P7185 PL

I I

P7940 P89 P890

6760 6849

~ ~ ~ 1

|P-U '5 -AATTCCTGTGTCACATCTGCAAGGACTTATGCsC TCTGTGCTTGAAAATTGGGAAATAGGTGTGCAGCCTCCTACCTCATCGATATTAGAGGACAG-3 ...

IP-l

3'-CGGACACAGTGTAGACGT TCCTGAATACGGGAGACACGAACTTTTAACCCTT M[SCdAC eTC ; rXddYdii~e--KYyitc

CTA_T

A-A'CCTTTA

P1~~~~~~ ~ ~ ~

~~~~~~~~~~~~~~~~k-[ORI-U S'-GCTCACCGAAACCGGTAAGTAAAGACTATGTATTTTTTCCCAGTGAATAATTGT TAATCACACCATCACCGTTTTTTCAAGCGGGAAAAAA-3T ORll| 3'-CGAGTGGCTTTGGCCATTCATTTCTGATACATAAAAAAGGGTCACTTATTAAC TATTAGTGTGGTAGTGGCAAAAAAGTTCGCCCTTTTTT-5'

I I

I %RW a AfT-rich region

LIZ]

*lDNasel-hypersensitive

site

EM-origin

1v\!

PMS1(domain

1and

2)

2D-origin

1minimaloriginof

replication

*4

18bp palindrome

B

|I'-tL/l'-1| (kS ss 1

I2 3 4 5 6, 7 8

Am.. ~ ~ ..

F-1"~

~~if-HpaI

FIG. 1. Cellular proteins interacting with single-strandedDNA. (A) Relevant portions of theBPV-1 genome.Openboxesatthetop indicate the positions of the late (L) and early (E) open reading frames Li, E6, E7, and El. The noncoding long control region located between the late and early coding regions carries the transcriptionalpromoters(P)andthepolyadenylation signal utilized by the late messages (polyA-L). The patterned boxes (bottom) indicateregions important for BPV-1DNAreplication(seetextfor details). The sequencesof the synthetic oligonucleotides derived from BPV-1 DNA (u, upper strand; 1, lower strand) used in the present studyarewrittenbelow.Their original locations are indi-catedbyboxes PandORI.(B) Gel retentionassays.Upon incuba-tion with nuclear extracts (5 p.g) from CB1, stable DNA-protein complexes (A and B1/B2, indicated by arrows) are formed with single-stranded oligonucleotides P-u and P-l (lanes 5 to 8). Only weak interactionsareobservedwiththe double-stranded(ds) oligo-nucleotideP-u/P-l (reassociated single strandsP-uandP-1)derived from the PMSIregion of BPV-1 (lanes 1 to 4). To obtain single-stranded(ss) DNA, thedouble strandsP-u/P-lwereheat-denatured (100°C for3min). Binding reactionswerecarriedout asdescribedin Materialsand Methods. Five femtomoles(105cpm)of each 3'-end-labeled(lanes1,2, 5, and6)and5'-end-labeled(lanes3, 4, 7, and8) oligonucleotidewasincubated with5 p,g ofCB1nuclearextract,and theresulting complexeswereseparatedon anative8% polyacryl-amide gel. Experiments displayed in lanes 1, 3, 5, and 7 were

performedin the absence ofprotein,and those in lanes2, 4, 6,and 8 wereperformed in the presenceof protein. F, free, nonbound DNA.

polyacrylamide-SDS gel, parallel to one lane of marker

proteins. Following electrophoresis, the marker lane was

separatedfrom thegeland stained with Coomassie blue. The rest of the gel was cut into 18 equally spaced horizontal slices, with individual molecular weight fractions being indicated by the molecular weight standards in the Coomassie-stained markerlane. Each slice was suspended inelution buffer(50 mM Tris-HCl [pH 7.9], 0.1% SDS, 0.1

mg ofbovine serum albumin per ml, 1 mM DTT, 0.2 mM EDTA,0.1 mMPMSF, 2.5% glycerol) and eluted by rotation overnight at4°C.Afterremoval of thegel pieces by centrif-ugation, the solution was precipitated with

methanol-ace-tone (1:1) at -80°C for atleast 2 h or overnight. Proteins werepelleted by centrifugation at 15,000rpm and 4°C. The

air-driedproteinsweresolubilizedfirst in 3p,lof renaturation bufferI(0.1MTris-HCl[pH 7.5],8 Murea)and then diluted in125,ulof renaturation bufferII(20mMTris-HCl [pH 7.6], 10 mMKCl,2 mMDTT, 10 p.MPMSF). Renaturation of the

proteins requiredatleast 18 hat4°C.From15to20,ul of the protein solution was used for gel retention assays. The molecularweightsof SPSF I and II in activegelsliceswere calculated by comparison with the Coomassie-stained pro-tein marker lane.

RESULTS

Identification ofproteinsthat interact withsingle-stranded oligonucleotidesfrom BPV-1 DNA.Ourapproachtostudying and

identifying

putativecellular factors requiredfor BPV-1 DNAreplicationwas to search forproteins binding to the origin.The targetsofourefforts thus became domain1(D1) of the plasmid maintenance region PMS1, originally de-scribed by Lusky and Botchan (18, 20), and the minimal origin regionbetweenpositions 7911 and22,morerecently showntoberequiredfor BPV-1replicationin vivo(39)and in vitro(46). Thesetworegionsaredepicted inFig. 1Aand will be referredto asPMS1 forthePMS1Dlregionand ORI

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[image:3.612.71.556.58.513.2]

CELLULAR DNA-BINDING PROTEINS INTERACT WITH BPV-1 5991 for the minimal

origin region.

The PMS1 D2regionwasnot

studied.

Inaninitial

experiment,

32P-labeleddouble-stranded DNA

fragments (Fig.

1B, lanes 1to 4)from the PMS1 region and their heat-denatured counterparts (lanes 5 to 8) were incu-bated with anuclear extract from BPV-1-transformed C127 mouse cells

(CB1 cells).

Onlyaweaknonspecific band was observed with the double-strandedfragments(Fig. 1B, lanes 2 and

4),

while a strong triplet of retarded bands was observed with the denaturedfragments ingel retention gels. This indicated that only single-stranded DNA-specific fac-tors bind to this

particular

region of the viral genome (Fig.

1B,

lanes 6 and

8).

The triplet bands (which can be recog-nized much better in

Fig.

2 and which subsequently were shown to result from the binding of two different proteins

[see

below])

have been

designated

A (for the upper band) and

B1/B2

(for

the lower

doublet).

The sequence

specificity

oftheseinteractionswasstudied

[image:4.612.323.550.79.498.2]

by

competition analysis. Single-stranded

oligonucleotides from the lower and upper strands of the PMS1 region (P-u

[upper]

and P-l

[lower];

96 nucleotides each; Fig. 1A), the

origin region

(ORI-u

and ORI-l; 100nucleotides each; Fig.

1A),

and a number of control oligonucleotides were incu-bated witha

partially purified

extractfrom calfthymus(see

below).

The

competing oligonucleotides

were derived from the PMS1

region (P-u

and

P-l),

theorigin region (ORI-l), the upperstrand ofa

region

of the c-myc P2 promoter(MYC-u), the lower strand ofa

region

from the polyomavirus genome

(PyE-l;

seeMaterials and

Methods),

and the lower strand of the distal enhancer of BPV-1

(BE-l;

see Materials and

Methods).

In the case of the PMSI region (Fig. 2A), the lowerstrand

(P-l)

displays

astrongretardationsignal (lane1) whichcanbeinhibited

by

itself

(lane

2), thecomplementary upperstrand

(P-u), ORI-l,

andMYC-u. Nocompetition was observed with

oligonucleotides

PyE-l and BE-l. The upper strand

(P-u) (Fig.

2B) displays

a

signal

comparabletothat of the lower strand

(P-l,

Fig. 2A) in strength, position, and

competition

behavior. Bothcomplementarysingle strands of this

region

thus possessbinding activity.

The presence of bandsin

Fig.

2A,lane3,andFig. 2B, lane

2,

which have a lower

mobility

thanfree DNA (marked F)

might

be taken as evidence for double-strand formation between P-u and P-l.

Indeed,

the reactionconditions of the

competition

experiments involving

complementary single strands would allow the formation of double strands. Since double-stranded

fragments

donotpossessbindingactivity, it is

possible

that the observed absence ofashiftedsignalis not due to true

competition

but rather the result of the inability of the

newly

formed double-strandedfragmenttobind pro-tein

(Fig.

1B).

This

interpretation

canbe ruled out, however, because

protein-binding

single-stranded oligonucleotides such as ORI-l and MYC-u, under theseconditions, are not inhibited

by

their

corresponding

complementary strands

lacking

a

protein-binding

site

(data

not shown).

In the case of the ORI

region,

only the oligonucleotide derived from the lower strand

(Fig.

2C) displays a retarda-tion

signal which, again,

behaves like the oligonucleotides from thePMSl

region

describedabove, i.e.,it competes with itself and with

oligonucleotides P-l,

P-u,andMYC-u but not with

PyE-l

orBE-l. Thecorresponding oligonucleotidefrom the upper strand of the ORIregionisinactive and does not compete with its

complementary

strand for the binding of

protein (not

shown).

Similarbehavior is observed witholigonucleotidesderived from the c-myc P2 promoter. In thiscase(Fig. 2D), only the upper strand leads to the formation of shifted bands. The

A

F

I

com;tit=r- r_ =

conmpetitor -

='

-s, -c -. F:

^p

c

F -I *_F

1 2 3 4 5 6 7 1 2 3 4 5 6 7

B

c}mpetitsr

P-

YC-u

^'7

\ -- - = -,=

B-

~

~

I

D

>

.-,R

-FF 1 2 3 4 5 6 7 1 2 3 4 5 6 7

FIG. 2. Three differentoligonucleotides arerecognized specifi-cally bySPSFI andII.Competitionanalysisrevealed highlyspecific interactionsofSPSFIand IIwiththe single-stranded oligonucleo-tides P-1 (A), P-u (B), ORI-I (C), and MYC-u (D). Each of the oligonucleotides (5fmol, 105cpm) was incubated with an SPSF I-andII-enriched MonoQ fraction (4 zg of protein) in the absence (lanes 1) orinthepresence(lanes 2 to 7) of a 100-fold molar excess of competitor oligonucleotide.Inlanes2to 5,only the SPSF I- and II-complexingoligonucleotides(asmarked on top of the gel) were used as competitors,whereastwounrelatedoligonucleotides were added to thebindingreactionmixture in lanes 6(BE-1,representing the BPV-1 distal enhancer) and7(PyE-1, representing the polyoma-virus Benhancer).TheSPSFI- andII-DNAcomplexes A, Bi, and B2 are indicated byarrows. F indicatesthepositionof free, unbound oligonucleotide.

upperstrand is inhibited by itself (lane 5),ORI-I (lane 2),P-l (lane 3), and P-u (lane 4) but not byBE-1(lane 6) andPyE-1 (lane 7). The complementary oligonucleotide is inactive and does not compete (not shown). The c-myc-derived oligonu-cleotidewastheonlynon-BPVfragmentamong avariety of promoter- and origin-derivedfragments which wesuspected would interactwith SPSF I andII.These includedfragments VOL.66, 1992

00-B B- r.

.44 A

::I=BI

B

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A

_A

r

P-i

.-i. F -

-_F;

'

wfl

k11B

si

4--0

:~~~~~~~~~~.*F

B

_ =

.r, ;t

--, _-

r--,I _; IIa Ii =

;, _ -

_-- ---A

_ ~~~~~~~-4TW131132

.- F

1 2 3 4 5 6 7 1 23 4 5 6 7

FIG. 3. SPSFI-andII-bindingactivities occur in extracts of cells from different sources. Oligonucleotides P-I(A) and MYC-u (B) were incubated, asdescribed in Materials and Methods, with different nuclear extracts (2 to 4 ,ug): human B cell lines F126 (lane 1) and IARC 549 (lane2); HeLa cells (lane 3);CB1 cells, aBPV-1-transformed C127mouse cell line (lane 5); NIH 3T3 cells (lane 7); and nuclear (NE) and cytoplasmic (CP)extractsofC127cells.Protein-DNAcomplexes A and B1/B2 and free, unbound oligonucleotide (F) are indicated by arrows. Because ofproteolytic activitiesin thecytoplasmic C127 extract, only a weak complex A could be formed in this particular experiment (lane 6 in A andB). The additionallower band inpanelA,lane 6, may have beenformed from apartiallydegradedprotein, which isstillable to binditscognate DNA.

of thesimianvirus40(SV40) origin of replication,afragment

containing theautonomousreplicationsequence(ARS) con-sensus sequence ofS. cerevisiae, the lexA and actin gene

promotersof S. cerevisiae, amousecellular DNAfragment interfering withSV40 DNA replication (8), and manymore

enhancerandpromotersequences.Asanexample,

oligonu-cleotidesPyE-landBE-lfromthepolyomavirus enhancerB and the distal BPV-1 enhancer areincluded inallpanelsof Fig.2.Therelative binding strengths of proteins SPSFIand II to the different binding sites were assessed by titration

competition experiments with increasing oligonucleotide concentrations. No significant differences between the dif-ferent fragments could be observed (data not shown), al-though allfouroligonucleotides haveonlytheweak homol-ogies indicated inFig. 9.

Theretardation activity displayed bythe single-stranded oligonucleotidesispresent notonlyincalfthymusbutalso in

avariety of othersources. As shown in Fig.3 for

oligonu-cleotides P-l(Fig. 3A) and MYC-u(Fig. 3B), binding activ-ities can be identified in extracts from human B-cell lines F126 (EBV negative) and IARC 549 (EBV transformed), fromHeLa, NIH3T3,andBPV-1-transformedmouseC127 cells (CB1 cells), and in nuclear (lane 4) and cytoplasmic (lane 6) extracts ofC127 cells. Thebinding activitiesoccur

ubiquitouslyinextractsof mammaliancells.Nocomparable activity, however,wasobservedin extractsfromS. cerevi-siae (not shown). Acomparison of the BPV-1-transformed

mouse C127 cell line, CB1, and its virus-free parent line demonstrates thatSPSFIand IIinteractions with DNAare

innowayinfluencedbytheexpression of viral proteins. Purificationof SPSF I and II.Since calf thymusappeared

tobethemostabundantsourceofthematerial,weinitiated apurificationofSPSFI and IIinvolvingthree majorsteps, anion exchange chromatography on DEAE-Sepharose and on MonoQaswellasDNAaffinitychromatography. Single-stranded DNA-Sepharose columnswere prepared by

cou-pling the single-stranded oligonucleotide P-l to

CNBr-acti-vatedSepharose. Thepurityandcomposition of the proteins were assessed by SDS gel electrophoresis (Fig. 4). Only three major bands are detectable following DNA affinity chromatography. The upper band (Fig. 4, marked by an asterisk) is an associated impurity which can be seen in fractions from all purification steps. The other two bands, which we designated SPSF I and II (for specific single-stranded DNA-bindingfactor), displaymolecular masses of 42and 39kDa, respectively.

SPSFIandIIbindspecificallytosingle-stranded DNA. Two typesofexperimentswereperformedtoshow that thesetwo

proteins represent single-stranded DNA-binding activities, UVcross-linking and reactivationfrom SDS gels. For UV cross-linking, extracts from various stages of the purifica-tion, all of whichwere active ingel retention assays, were incubated with

32P-labeled

oligonucleotide P-l, irradiated with UVlight of 254 nm, andsubjectedto SDSgel electro-phoresis. While crude and DEAE-Sepharose extracts

yielded complex and broad-banding patterns, the MonoQ-and the DNAaffinity-purifiedextractsrevealedtwodistinct bands (Fig. 5A, lanes 3 and 5). The DNA affinity column flowthrough was inactive (lane 4). The two bands ran at

positions correspondingto 73 to 78 kDa,which represents approximately thesumofthemolecularmassesofeither of thetwoproteinsand thesingle-strandedoligonucleotide (97 bases; molecular mass, 31.7kDa). Inseparateexperiments,

wehave demonstrated that themobility ofoligonucleotides inthese SDSgels corresponds tothemobilityofdenatured proteins with similar molecular weights (not

shown).

The high specificity of these interactions was proven by UV cross-linkingof SPSFIandII tooligonucleotidePk3-l in the presence ofhomologous (Fig. SB,lanes 2 to

6)

and heterol-ogous(lanes7to12) competitorDNA.Oligonucleotide Pk3-l represents the 5' portion ofoligonucleotide

P-l, containing

one SPSF I and II binding site (Fig. 1A; see Fig. 9). As expected, no cross-linking products were detected in the presence ofa100-fold molarexcessoffragmentscontaining

on November 9, 2019 by guest

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[image:5.612.175.449.77.269.2]

CELLULAR DNA-BINDING PROTEINS INTERACT WITH BPV-1 5993

1 2 3 4 5 6 7 8 NI

97.4

* _66.2

4_5.0

"-~~~~~-.*SPSFI - ~ ---SPSFII

[image:6.612.75.289.76.221.2]

--- - - ~~~~~~31.0

FIG. 4. Analysis of purification of SPSF I and II from calf thymus bySDS-polyacrylamide gelelectrophoresis. Lane 1 shows theprotein compositionof the initial whole-cell extract. Lanes 2 and 3 represent flowthrough and a DNA-binding fraction of the DEAE-Sepharose column, respectively.Lane 4is a sample from the pooled active DEAE-Sepharose fractions loaded onto the MonoQ column. Activity peaksoftheMonoQ columns (one of them is shown in lane 5)werepooled and applied to DNA affinity chromatography (lane 6). The flow-through of this column is shown in lane 7. Lane 8 representspurified SPSFI andII,asindicated by arrows. The upper band, marked byanasterisk, isanassociated impurity,which can be seen in fractions from all purification steps. The samples were analyzed on a 10% polyacrylamide-SDS gel and stained with Coomassieblue.Lane M,proteinsizemarkers (in kilodaltons).

SPSF I and II binding sites (Fig. 5B, lanes 2 to 6). The presenceofheterologous competitors (lanes 7 to 12) did not disturb theformation of the expected cross-linked products. In a second set ofexperiments, the two bands from the

A

I~-

,,

-C ;j a ;F <

SDS gelin Fig.4 and materialfrom other regions of the gel were eluted separately from a preparative gel, renatured, incubatedwith 32P-labeled fragment P-l, and subjected to gel retardation analysis. As shown in Fig. 6 (lanes 5 and 6), only the two fractions, corresponding to 40 to 45 and 35 to 39 kDa, displayed binding activity. The higher-molecular-weight material from the SDS gel yielded a shifted band of lowermobility (designated band A), while the lower-molec-ular-weight material yielded a higher-mobility signal desig-nated B1/B2. These twobands are identical in mobility to the signals observed with different DNA affinity-purified frac-tions(Fig.6,lanes 1 to4) as well as to those observed in Fig. 1 and 2. Competition analysis proved the highly specific binding capacities of the renatured proteins (data not shown). We conclude that two activeproteinsof 39 and 42 kDa present incalf thymus extracts are responsible for the observedsingle-strandedDNA-binding activities.

Points ofcontact between SPSFIandIIandsingle-stranded oligonucleotides. The binding specificities of the two proteins were established in footprint experiments. For a T4 DNA polymerase footprint, the 5'-end-labeled oligonucleotide P-u wasincubatedwith a DNA affinity-purified preparation (Fig. 7A) and subsequently exposed to T4 DNA polymerase. Digestion with its endogneous 3'--5' exonuclease activity displaystwostopscentered around positions 6819 and 6812 in the centerof the PMSI region (Fig. 7A, lanes 1, 2, and 3). Incompetition experimentswith the SPSF I-and II-binding oligonucleotides (Fig. 7A, lanes 5 and 6), the stops disap-pear,whiletwounrelatedoligonucleotides (Fig. 7A, lanes4 and7)donotinfluence theprotectionpattern. Inlane 4, the band iscompressed because too little material was applied to thegel.Methylation interference footprints (Fig. 7A, lanes 9 to11)wereperformedwithaDEAE-Sepharoseextractfrom C127 cells. In the free DNAportion, all Gresidues remain

B

0:

0__-]- Pk3-1

Comp..oitor / - Aa- 7A - ,. 97.4

97.4

7*

-o-SPSt

SPSF

I e

6(.2

U

'SPSFII

SPSFII-=*-415.11

131.F 1 2 3 4

--.. 2=|i.;011|E

:,.>'f;_31_

,e, t:' w

.S.z.

^/><J;> SeSs<e

66.2

45.0

31.0

1 2 3 4 .5 6 7 8 9 10 11 12

FIG. 5. Twodistinctpolypeptides are cross-linked tooligonucleotides P-1and Pk3-1 byUVlightirradiation. (A) To perform theUVcross-linking experiment, calfthymus protein extracts from various purificationsteps were incubated with 20 fmol (4 x 105cpm) ofoligonucleotide P-1. DNA-boundproteinswerecross-linked to the labeledoligonucleotidebyirradiation withUVlight (254 nm, 8,000pLW/cm2)for 4min. The reaction productsweresubjectedtoelectrophoresison a10%polyacrylamide-SDSgel, stained withCoomassie blue to visualize the protein markers, dried, andexposedtoX-rayfilms.In lanes 1 and2,proteinsfrom a crude extract (CE) and from a DEAE-Sepharose fraction (DS) wereUVcross-linked

toP-1. Lane 3shows the reactionproductsof an SPSF I- and II-enriched MonoQ fraction(MQ)with P-1.Lane 4 representsflowthrough,and lane 5 shows a sample ofpooled activity peaks from DNA affinity chromatography columns. The cross-linking products of SPSF I and II with oligonucleotideP-1areindicatedby arrows in lane 3 (MonoQ fraction) and in lane 5 (DNA affinity-purified proteins). (B) UVcross-linkingofan

SPSF I- and II-enriched MonoQ fraction to oligonucleotide Pk3-1 in the presence of a 100-fold molar excess of SPSF I- and II-binding oligonucleotidesashomologouscompetitors (lanes2to6,asmarkedontopof thegel)andavarietyofheterologouscompetitors(lanes7to12). Thecross-linking productsbetween Pk3-1 and SPSF I and IIareindicatedby arrows. Lane M, size markers(inkilodaltons).

.-.

"ff&lARd-VOL.66, 1992

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[image:6.612.114.498.430.622.2]

I 2 3 45

*

I

-*11B/B2

FIG. 6. Renaturation of purified SPSF I and II from SDS-polyacrylamide gels. Size-fractionated, renatured proteins (de-scribed in Materials andMethods)fromanSPSFI- andII-enriched MonoQ fractionwereassayed for theirDNA-bindingactivity. The twoactive fractions, bindingto oligonucleotide P-l, areshown in comparison tothemobilityof SPSF I and II from different DNA affinitychromatography purifications (lanes 1 to4). Lane5 repre-sentsafractionof renaturedproteincorrespondingto40to45kDa, which forms DNA-protein complex A with oligonucleotide P-1 (indicatedbyanarrow). ComplexesB1/B2(indicated byanarrow) in lane6 arethe results of interaction of the 35-to39-kDafraction of renaturedproteins with P-l. FreeDNAis indicatedbyF.

unprotected,while incomplexed DNA,the four G residues indicated by arrows are protected. One of these (position

6807)

resides closetooneofthe

regions

protectedin the T4 DNA polymerase footprint, while the other three G's are located upto 15bases upstream(5').

Similar

experiments

were

performed

with the

complemen-tarystrand P-1. Two T4 stopswere observed, around posi-tion 6783(nearthestartof thegel)and aroundposition 6823 (Fig. 7B, lanes 1 to 3). Correspondingly, five G residues (indicated by arrows)werefoundtobeprotectedin methyl-ation interferenceexperiments. Theyaresituated inaregion close tothe upper T4 stop, while the G residues around the lower T4 stop are not protected. In a methylation interfer-ence experiment with the shorter fragment Pk3-1, four G residues (indicated by open arrowheads in Fig.

9)

were

protected (not shown). We conclude that the T4 DNA polymerase andthe methylation interference data coincide and overlap and that two binding sites for SPSF I and II within the PMS1 region thusare located between positions 6780 and 6825

(see Fig. 9).

As

already

indicated

by

the UV cross-linking experimentshownin

Fig.

5B,one

binding site,

locatedon oligonucleotide Pk3-1,issufficientforinteracting with SPSFIand II.Thus,thetwoseparateprotected

regions

of theT4

footprint analysis

eachrepresentaseparate

binding

site.

Asecondbindingsite forproteinsSPSF I and IIonBPV-1 DNAlocated around the minimaloriginof BPV-1replication

was analyzed by T4 DNA polymerase footprinting with oligonucleotide ORI-1, derived from the lower strand of this region.Asdiscussedabove,thecomplementary oligonucle-otide ORI-u does not bind. The footprint (Fig. 8, lane 4) shows two T4 DNA polymerase stop regions, one around

position

7925 andone around position11. InFig. 8, lanes 2 and 3, a control experiment with a flowthrough from the DNA

affinity

column, which is devoid of DNA-binding activity, is shown. Lanes 5to 10 ofFig.8 showcompetition

experiments

with homologous oligonucleotides (lanes 5 to

8),

whichremoveboth T4 stops, and with nonhomologous

oligonucleotides (lanes9and 10).The dataaresummarized in Fig. 9 and show that thebinding site for SPSFI and II mapsbetween positions 7925 and 11, around anHpaI site locatedwithin theminimaloriginof BPV-1 DNAreplication.

DISCUSSION

Wehave

purified

two

single-strand-specific DNA-binding

proteinsfromcalf

thymus.

The

proteins, designated

SPSFI and SPSF II, have molecular masses of approximately42 and39kDa,

respectively,

asshownby

SDS-polyacrylamide

gel

electrophoresis

of the DNA

affinity

chromatography-purified proteins

(Fig. 4),

byUV

cross-linking experiments

(Fig. 5), and bythe behavior of thetwo renatured

proteins

reisolatedfrom

denaturing gels (Fig.

6).

Both

proteins

canbe isolated fromavarietyof othersources

(Fig. 3)

and appearto be

ubiquitous

inextractsof mammalian cells.

SPSF I and II

specifically

bindto

single-stranded

oligonu-cleotides

containing

sequences derived from one strand of the BPV-1 minimal

origin

of

replication,

from either strand of domain1 of the

plasmid

maintenance sequence

(PMS)

of BPV-1, and fromonestrand ofthe P2 promoterofthe human c-myc gene. We have

performed gel

retention

analysis,

methylation interference,and T4 DNApolymerase

footprint

experiments

to determine the

precise

points

of contact between thetwo

proteins

andtheir DNA targets.

AsshowninFig. 2,SPSF I-andII-enrichedfractions

give

rise to three

specific DNA-protein complexes

(A

and a

doublet,

B1/B2)

in

gel

retention assayswith

oligonucleotides

containing

binding

sequencesfor SPSF I and II. Because of their similar elution

profiles (see

Materials and

Methods),

the two

proteins

couldnotberesolved

properly during

column

chromatography.

In orderto

assign

each

protein

to one of the observed

complexes,

wehave therefore used renatured SPSF I and II

preparations

obtained fromSDS

gels

(Fig. 4).

Ascanbeseenin

Fig.

6,this

experiment

allowedus to

assign

the upper

complex

A

(seen

in

Fig.

2)

tothe 42-kDa

protein

SPSF I and the doublet

B1/B2

(seen

in

Fig. 2)

tothe39-kDa SPSF II. The association of SPSF II with the doublet complex B1/B2is observed

throughout

all

purification

steps, including DNA

affinity chromatography.

This observation rulesoutthe presence of

proteins

other than SPSF I and II and proves that both

proteins

bind

separately

to the same single-stranded

oligonucleotides.

We also know that both

proteins copurify

under allconditions tested and that com-plexesAandB1/B2behave

indistinguishably

in

competition

experiments.

These observations donotprovebut

strongly

indicate thattheyare

structurally

relatedtoeach other. The doublet retention

signal

observed with SPSF II may be due to a

posttranslational species

of SPSF II.

In a first step to

gaining

information about the

binding

specificities

of SPSF I and II, we

performed competition

experimentswith different

oligonucleotides

froma

variety

of sources, such as the

polyomavirus

enhancer

B,

the SV40 origin of DNA

replication,

a

synthetic

S. cerevisiae

origin

carrying

a consensusARS sequence, and various

eukaryotic

promoter

regions.

None of theseboundtoSPSF I and

II,

nor didtheycompetein double-strandedor

single-stranded

form with SPSF I- and SPSF

II-DNA complexes.

The

only

oligonucleotide

other than the BPV-1-derived

fragments

foundtointerferewith SPSF I- andII-DNA

complex

forma-tionwasthe upper strand ofthe P2 promoter from the human c-myc gene.As shown in

Fig. 2,

itwas

capable

of

competing

for DNA

binding

with any of theother three

oligonucleotides

containing

an SPSF I and II

binding

site. A sequence

comparison

of the DNA

regions containing

the

putative

on November 9, 2019 by guest

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[image:7.612.126.235.73.218.2]

CELLULAR DNA-BINDING PROTEINS INTERACT WITH BPV-1 5995

NI 1 23 4 56 7

A

6821j

6817

6813

681(. _

As

(;

4-A _~~~~.

A X1

G~~~~~~A.s G~~~

g roll

M +

_

_s

._. ..

i = _r,._

I ... W.

f s

A {; js *

C

S t?:

*

C h:o

T .4,

C .

C Rea C ,,^ C .

C C

(; * _'

^..s:7e.:.

C .s _

(s *

_H#--T =; _;S_

(; t

(; _ _

A w

1' X tZ

A .6 . ;e

A,.^

_;;

1. _e _

(; w

{;

r w _>

T b

A t.

A t*'

.

A g

_t-T *

T ?/

c .*.

_

; *.} _

Y

_

{i

*@jeb

Ni 1 2 3 4 5 6 7

B

6781 7Z8

(682(I

6824 I

(

4'

.X. *

t- .

rtl: rII

I O

I _

1 . 4

_..mf

I OMM _tw

afowt

( = _

M910 11

_4 _

_

; ^.

A( .

FIG. 7. T4 DNA polymerase and methylation interference footprinting experiments with SPSF I and II. For T4 DNA polymerase

footprinting (lanes1to7 inpanelsAandB), differentamountsofprotein(approximately 50to100pg, asestimated from gel retention assays)

ofaDNAaffinity-purified SPSFI and IIpreparationwereincubatedwith5'-end-labeled oligonucleotide P-u (A)orP-1 (B) inabinding reaction

as described in Materials and Methods. The products of the subsequent T4 DNA polymerase treatment were analyzed on 10% polyacrylamide-SDS-ureagels. Lanes 1to3(AandB)showtheT4DNApolymerase cleavagepatternofSPSFI-andII-boundDNA inthe

absence ofcompetitorDNA. A 100-foldmolarexcessofhomologous competitorwasaddedtothereaction mixtures inlanes5(A, P-u; B, P-i) and 6 (A and B, ORI-I). The reaction products obtained in thepresenceofheterologous competitorsareshown in lanes 4 (A and B, BE-I)

and 7 (A and B, PyE-l). Nucleotides shown in white letters against black indicate the exact positions of T4 DNA polymerase stops.

MethylationinterferencestudieswerecarriedoutwithanactiveDEAE-Sepharoseextractfrom C127 cells. Inlanes 9 and 10 (A, P-u; B, P-i),

thepiperidinecleavagepatternoffree, unboundDNAisshown, whereas in lane11, DNA from complexes A andB11/B2wasanalyzed. Arrows

indicatethepositions of G residues which,ifmethylated,preventproteinbinding. Gsequencesizemarkers for oligonucleotides P-u (A) and

P-i (B)are shown in lanes M.

SPSF I- and II-binding regions in the four known active

fragmentsidentified certainsequence homologieswhichare

subdivided into short homology boxes (Fig. 9). We even

attempted to define a consensus sequence. Its functional significance,apartfromservingas aDNAtargetforproteins SPSF I andII,remains unknown.

Thepresenceof cellularprotein-binding sites around viral origins of DNA replication is not uncommon. In many

instances, they represent binding sites for transcription factors(3). Some elements involved in transcriptional

regu-lationarelocatedclosetotheSPSF I- andII-bindingsite in the BPV-1 PMSI region: the polyadenylation signalforlate BPV-1 gene expression, the promoters P7185 and PL, and protein E2 recognition element E2RE2. A constitutive

en-hancer sequence which is negatively regulated by the E2

repressor through competitive binding for a cellular factor was identified between nucleotides 7162 and 7275 (40).

Recently, this element was reported to be necessary for transformation andreplication functions(41). The constitu-tive enhancer appears to be a master regulator of BPV-1 transcription and is required for maintenance of BPV-1 replication in latently infected cells, although Ustav and Stenlund (38)have reported that it is not requiredin

tran-sient-replication assays. cis-Acting regulatory elements close to the BPV-1 origin of replication are E2RE1, E2-binding sites 11 and 12, and the promoters P7940 and P89. Whetherand how someoftheseelements, inparticular the constitutive enhancer, which is positioned closest to the VOL. 66, 1992

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[image:8.612.70.549.73.451.2]

A

.1.

'1.

A A

A

A A

A

1311

C

*1

.1.

;

C

FIG. 8. T4 DNA polymerase footprinting with SPSF I- and

II-complexed oligonucleotide ORI-1. The experiment was carried outas described in the legend to Fig. 7. Lane 1 shows T4 DNA

polymerase-cleaved oligonucleotide without protein as a control. Different amounts of a complexformed between the flowthrough

material fromaDNAaffinity chromatographycolumn andfragment ORI-Iweredigestedwith T4 DNApolymerase (lanes2and3).Lane 4shows the reactionproductsof SPSF I- andII-complexed ORI-Iin the absence ofcompetitors. Lanes5(ORI-1),6 (P-u),7(P-l), and 8

(MYC-u) show the results obtainedwitha100-fold molarexcessof

oligonucleotides that were able to bind SPSF I and II upon gel retention.Inlane9, oligonucleotide BE-1wasusedas aheterologous

competitor, while lane 10 shows the result obtained with

oligonu-cleotidePyE-las aheterologous competitor.ThesequenceofORI-I

is written frombottomtotopalonglaneM,which indicates the G

sequencesize markers revealedbyMaxam andGilbertsequencing. Nucleotides showninwhitelettersagainstblack indicate theexact

positionsof T4 DNApolymerasestops.

bindingsite inPMSl,interact with SPSF I andIIwill haveto

beinvestigatedinthe future.

Data about therole ofPMS sequencesseemtobe contra-dictory. Earlier resultsconcerningthe role of PMS and the function of El (18-20) were difficult if not impossible to reproduce (38).Weattemptedtodetermine therequirements

forPMSI domains 1 and 2by performing transient-replica-tionassays bythe method of Ustav and Stenlund(38),with mutantplasmidsdeleted for domain 1and/ordomain 2(44a). Without domain 1, the plasmid was able to replicate as efficientlyaswild-type BPV-1,whereas the domain 2 dele-tion mutant failed toreplicate.In accordance with the results of Vande Pol andHowley (41)and Ustav and Stenlund(38), we assume that deletion of domain 2, which encompasses regions E2RE2, PL,

P7185.

and the constitutive enhancer, destroys important transcriptionalelements. Unless BPV-1 proteins El and E2 are provided in trans, this deletion prevents extrachromosomal

replication

of the transfected BPV-1

plasmid.

These data would thus indicate that PMSI domain 1

plays

norole in

plasmid

maintenance and

replica-tion.Interaction ofSPSF I and IIwiththis

region might

have otherfunctions than an involvement in maintenance of the extrachromosomal

plasmid

state. Inthis context, it may be relevant thatonlythe full BPV-1 genome is able to

display

100% transformationandreplication efficiency

(15,

17,

32).

The elements which contributeto 100% activity of the full genomehavenotyetbeenidentified and may include SPSF I andIIbindingsites.

SPSF I and II were isolated as

single-stranded

DNA-specific proteins. However, they can also bind with low affinitytoveryshortdouble-stranded

oligonucleotides

(c50

bp)

containing

their

specific binding

sites. The

affinity

to double-stranded

fragments strongly

depends

on

fragment

length. Double-strandedfragments longerthan about 100

bp

are nolongerrecognized bySPSF Iand II. Other

sequence-specific single-stranded DNA-binding proteins,

with strong indications that

they

are involved in initiation of DNA synthesis,have also been shownto

display

weakaffinitiesto double-stranded DNA sequences. The S. cerevisiae

proteins

ACBP

(10)

and ssARS-T

(33),

which appear to be indistin-guishable, interact

specifically

with the T-rich strand of the ARS core sequence. Both groups reported low

affinity

for their

respective proteins

todouble-stranded ARS sequences. Additionally, an SV40 origin-binding protein,

purified

from primate cells,alsointeracts withsingle-anddouble-stranded DNA

(37).

Recently, a proteinfrom HeLa

cells,

Pur, was described

(1)

to bind

specifically

to

single-stranded

se-quencesin

5'-flanking regions

of the c-myc gene

(1.6

kb from thestartof

transcription

of

P1)

and alsotothe

corresponding

double-stranded sequences. The

binding

site of Pur is lo-cated inanuclease

Si-sensitive

region

nearDNase

I-hyper-sensitive site

II,

(35). Interestingly,

the SPSF I and II

binding

sites in the P2 promoter of the c-myc geneaswellas thebindingsites in thePMSI

region

of BPV-1

(30)

also reside in the direct

vicinity

of DNase

I-hypersensitive

sites.

We have wondered how a

single-stranded

DNA-specific

protein

might

find

binding

sites inadouble-stranded

plasmid.

Sincethiscanbe

readily explained

via

secondary

structural

features,

we subcloned a

fragment

from nucleotides 6685

(AccI)

to 6945

(BglII) containing

the SPSF I- and II-PMSI binding sites into a pGEM4Zvector and treated this con-struct and the control

plasmid

with endonuclease VII from

phage

T4

(11).

This enzyme

specifically recognizes

second-ary structures in

single-

and double-stranded DNA. The

preliminary

results of this

experiment (unpublished

data)

indicated that indeed these DNA sequences form stable

secondary

structures, which may include

partially

single-stranded DNA

regions,

and thus could represent efficient

binding

sites for SPSF I and II.

Afinal

point

regarding

theaction ofSPSFIand II relates to the observed presence of two

binding

sites within a distanceof 800

bp.

Ifthese

represented

twopartsofan

origin

;.dowf

-z'

N-1 1 2 3 4 5 6 7 8 9 11)

G

7924 7927

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[image:9.612.103.251.73.468.2]

CELLULAR DNA-BINDING PROTEINS INTERACT WITH BPV-1 5997

P- 5'-TGTGTCACATCTGCAAGGACTTATGCCCT'

6760

G

_T

_T

_G

_{CCTCCTACCTCATC TATTA CA-3'}

6849

5ITAccATA

ACACg

T^TTACCT"TTGCAGTGTGACAC 3

6859 6849 6760

4'-TTTTTTC5 T AAlTTTATTCACT-rirCeAgnTC7TTT89TTCCTTTC1QTCC,

2414

consensus sequence

2451i

5t

--3A

YSS KA

FIG. 9. Contact pointsofSPSF I andII.White letters in black boxes in the four oligonucleotides binding to SPSF I and II indicate T4 DNA polymerase stops. Arrows above thesequences indicate the positions of protected G residues, as determined by methylation interference footprinting. Numbers below the sequences refer to positions in the original DNAs (BPV-1 DNA and myc). The underlined sequence in oligonucleotideMYC-u representsthe original DNA sequence from nucleotides 2414 to 2451 of the humanc-mycgene; the flanking sequences arerandom.Sequencehomologies, identifiedby comparison of thefour SPSF I- andII-bindingoligonucleotidesequences, are subdivided into fourshort homology boxes. A proposed consensus sequence is written below.

ofreplication,this wouldhardly be uncommon. The origin of replication of EBV, another DNA virus replicating as an extrachromosomal plasmid in the latent state (16, 29), and the prokaryotic origin of plasmid R6K (25) consist of two partswhichare separated by more than 1 kb.

Inaddition to a putative role in DNA replication, SPSF I and IImight also be involved in the regulation of transcrip-tion. Several single-stranded DNA-binding proteins have beenreported to be involved in transcriptional regulation. A nuclear protein fromDrosophila melanogaster specifically interacts with the mRNA-like strand of the long terminal repeat of retrotransposon 1731,which plays a role in regu-lation of 1731 expression (6). CNBP, which sequence-spe-cificallybindssingle-stranded DNA,recognizes the noncod-ing strand of the sterol regulatory element found in the HMG-coenzyme A reductase promoter (28). Factor SRE-BF, isolated from Chinese hamster ovary nuclear extracts, interacts with double- and single-stranded DNA sequences from acis-acting element,designatedSRE-1,whichhas been identified in several promoters of genes necessary forsterol regulation (36). A protein from skeletal muscle extracts, designated muscle factor 3, interacts with single-stranded

DNA regions from three different muscle gene DNA ele-ments: the MCAT gene sequence motif, the skeletal actin muscle regulatory element, and acreatine kinase enhancer probe (31). The single-stranded DNA-binding stimulatory factor DBSF,purified fromS. cerevisiae, enables the puri-fied human estrogen receptor to bind as a dimer to its cognate palindromic DNA sequence and thus to activate genetranscription (24).Protein H16 from nuclearextractsof noninfected monkey cells binds a unique single-stranded

DNAsequence in theearly promoter of SV40 and has been shown to stimulate transcription invitro bypurified RNA polymerase II (7). Aprotein purified from rat liver nuclei, called PYBP, bindsonlyto single-strandedpyrimidine-rich DNA, present, for example, in the human transferrin gene promoter (2). A direct involvement of SPSF I and II in transcriptional regulation by bindingdirectlyto RNAcould beruledout.Invitro-transcribedRNAfrom the SPSFI-and II-PMSlbinding sites did notinteract with purified SPSF I andII (datanotshown).

Insummary,ourdata indicate thatSPSFIandII,purified from calf thymus, belong to a class of single-stranded DNA-binding proteins,whichbind with sequencespecificity

tosequences mainly located close to or directlywithin an

origin of DNA replication. The precise functions of these proteins have yet to be elucidated. Several models exist which propose functions as helix-destabilizing proteins at theinitiationsite of DNAsynthesis(1)or asspecificinitiator proteins (10,33). Furtherinvestigations willbe necessaryto

obtain insightinto these interactions. ACKNOWLEDGMENTS

We thankB6rriesW.Kemper,University of Cologne, for provid-ingT4endonucleaseVII and HorstIbelgauftsforcritically reading themanuscript.

Thiswork wassupported byDeutsche Forschungsgemeinschaft grant Fa138/3-4.

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