Electron microscope localization of a protein bound near the origin of simian virus 40 DNA replication.

Copyright0 1975 AmericanSociety for Microbiology Printed inU.SA.

Electron

Microscope Localization

of

a

Protein

Bound Near the

Origin

of

Simian

Virus 40

DNA Replication

JACK GRIFFITH,* MARIANNE DIECKMANN, AND PAUL BERG

Department of Biochemistry, Stanford University MedicalCenter, Stanford, California94305 Receivedforpublication3September1974

Asalt-stablecomplex of protein and viral DNA obtainedfromSimianvirus 40

(SV40)-infected

monkey cells or mature

SV40

virions has a novel structure.

When viewed by high

resolution

electron microscopy, the circular

SV40

DNA molecule has boundtoitonetothree globular protein "knobs".UsingecoRIand

hpal restriction endonucleases, each of which cancleave

SV40

DNAonceata

known location (10, 11, 12, 14), the bound proteincanbelocalized at0.7 i 0.05 on the

SV40

DNA physical map

(SV40

fractional length, clockwise from the

ecoRI endonuclease-cleavage site).

Infection

of

permissive

monkey cell cultures

with simian

virus 40

(SV40)

results in the

accumulation

of

covalently closed viral

DNA,

and concommitantly,

maturevirions (16). As is

the

case

with polyoma

virus infection of mouse

cells (4),

most of the nonvirion

SV40

DNA occurs as a

nucleoprotein

complex (6, 17, and R.

Perez, J. Griffith, M. Dieckmann,

and P. Berg,

manuscript

in

preparation). The SV40

nucleo-protein

complex

is a

condensed, chromatin-like

structure

containing

a nearly equal mass of

DNA and

cellular histones

(6, 9, Perez et al.

manuscript

in

preparation;

W.

Meinke, M.

Hall, and D.

Goldstein, personal

communica-tion).

While

characterizing the chemical, physical,

and biologic

properties of

SV40 chromatin,

we

observed that the

bulk of the protein contained

in

the SV40 chromatin

is

dissociated

from the

DNA

after exposure to 1

M

NaCl (Perez

et

al.,

manuscript

in

preparation). The resulting

prod-uct, a

high

salt-stable protein DNA complex

(SSP-DNA complex),

sediments

slightly

faster

than

SV40 form

1

DNA

in a

neutral

sucrose

gradient;

after treatment of

the

complex with

sodium dodecyl sulfate

(SDS),

free

SV40

form 1

DNA is

released. The SSP-DNA complex,

ob-tained from either

SV40-infected cells

or ma-ture

SV40 virus, has

a

novel

structure

when

viewed

by high

resolution electron

microscopy; thecircular

SV40

DNAmolecule has

bound

to it a

single "knob"

of

protein.

Using

ecoRI and

hpaII

restriction

endonucleases, each

of

which

can

cleave SV40

DNA once at a knownlocation (10, 11, 12, 14), the

bound

protein can be

localized

at 0.7 i 0.05 on

the

SV40

DNA

physical

map

(SV40 fractional length, clockwise

from the

ecoRI endonuclease-cleavage

site).

MATERIALS AND METHODS

SV40 chromatin.

Monkey

kidney

cell cultures

(CV-1) were infected with SV40virus (strainRh

911)

ataninputmultiplicityof 30

PFU/cell and,

after42to 45 h

(QH]thymidine

[5 jACi/ml] was present during

the last 8 h of theincubation),theSV40

nucleoprotein

complex (SV40 chromatin) was extracted from the infected cells as described by Green et al. (4) and White and Eason (17); a portion of the resulting

Triton X-100extract (0.2 ml) wassedimented in a4 ml, 5to 20%sucrosegradient containing0.25%Triton

X-100, 100mMNaCl,10mM

Tris-hydrochloride (pH

7.9) and 1mM EDTA

(TTEN

buffer) at55,000rpm for 65 minat 4C in the SW-56rotor.SV40chromatin sedimented as asinglepeak between 45 and 55S rela-tiveto a 32P-labeled SV40 form I marker (seeFig.1in Eason and White [17

]).

Salt-extracted SV40chromatin

complex.

Pooled

fractions ofSV40 chromatin obtained either as de-scribed above or the analogous fractions recovered

from aTriton X-100 extract ofpurifiedinfected-cell nuclei(Perezet al., manuscript in preparation) were incubated in

TTEN

buffer with 1MNaCl for 20 min at 20C and then centrifuged for 4 h in a sucrose gradient containing 1 M NaCl. This treatment dis-sociated therapidlysedimenting form and caused the viral DNA to sediment justslightly faster than the

SV40 form 1 DNA marker. The same salt-stable protein-DNA complex (SSP-DNA) wasalsoprepared

from isolated SV40 chromatin afterequilibrium cen-trifugation in a CsCl gradient: SV40 chromatin (2.5

ml in

TTEN

buffer) was mixed with 3.0 ml of saturated CsCl containing 0.25% Triton X-100, 10 mMTris, pH7.8,and1 mMEDTA,andcentrifuged to equilibrium at 42,000 rpm (15 C) intheSW 50.1 rotor. Buoyant densities of the fractions were deter-minedby their refractive index.

The SSP-DNA complex was also obtained from

purified

SV40 virions as follows: a preparation of nuclei from cells infected as indicated above was mixed sequentially withTriton X-100 (to0.5%) and 167

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GRIFFITH, DIECKMANN, AND BERG

NaCl (to 1M) andincubated for 20 minat20C. The celllysatewascentrifugedat15,000xgfor20minto remove the cell DNA. The supernatantwas layered overtwo3-mllayersofCsClwith densities of 1.30 and 1.40g/ml and centrifugedfor 3 hat25,000rpm(4 C)

inan SW27rotor.Mature virus particles andempty capsids formedtwodistinctbands and couldreadily be separated. A viral suspension containing 1012 particles was dialyzed for 24 h at 4C in 0.05 M carbonatebuffer, pH 10.5,toobtain theSV40 DNA-proteinHcomplexdescribed by Huangetal(7); this

material was then centrifuged to equilibrium inthe CsCl gradient described above to yield the virion SSP-DNA complex. Triton X-100 (0.25%) was

in-cludedinall of thestoragebufferstominimize loss of the variouscomplexes.

Deproteinization ofthecomplexes.SV40 chroma-tinand SSP-DNA complexeswereincubated with 1% SDS for5minat65 Corwithpronase(50Asg/mlinthe

presenceof 5mMMgCl2)for 1 hat37 C.

Electron microscopy. Samples were mounted on

ultra-thin carbon films activated by glow discharge for 5 min, washed and dehydrated by immersion through graded ethanol solutions (0, 20, 50, 75, 100%), and stained by tungsten evaporation at 10-6 torr, using a technique described recently (5). A Philips EM 300 was used at 60 kV with a50-jim objective aperature.X-raytreatment(14)wasusedtonick and therebyrelax thesupercoiledDNA.

Cleavageof SSP-DNAcomplexeswith EcoRIor

HpaII restriction endonuclease. Exonuclease-free ecoRI endonuclease, prepared accordingtoYoshimora (R. Yoshimora,Ph.D.thesis,Univ. ofCalifornia,San Francisco, 1971), was a gift of M. Thomas and D. Glover; hpaII endonuclease was purified by J. E.

Mertz and J. F. MorrowaccordingtoSharpetal.(14). Thespecific conditions usedto cleave thecomplexes with eachenzymeis indicated in thelegendtoFig.3.

RESULTS

Throughout the infection of monkey kidney cell cultures by SV40 virus, most of the viral DNA exists as a non-encapsulated,

nucleo-protein complex (hereafter referred to as SV40

chromatin) (17, Perez et al., manuscript in preparation). Thiscomplex, obtained from

Tri-ton X-100 extracts of infected cells, sediments

at45to55S ina sucrosegradient containing 0.2

M NaCl (4, 6, 17); however, after brief incuba-tion of the same extract with 1 M NaCl, the SV40 chromatin complex is altered and the DNA sediments slightly faster than free SV40 form 1 DNA in asucrose gradient containing 1 M NaCl (Perez et al., manuscript in

prepara-tion). This new complex, the high salt-stable

protein-DNA complex (SSP-DNA complex), when examined by high resolution electron microscopy, without added cytochrome c (5),

resembles thetightly twisted rods characteristic ofSV40 form 1 DNA mounted under thesame

conditions (Fig. 1A).However, after nicking the

DNA by briefX-ray treatment, circular struc-tures(contour length 1.5

,m),

containing on the average one

"knob"

per DNA

molecule,

arethe predominant molecular species (Fig. 1B, D, and Table 1, line 1). If the SSP-DNA complex is

treated with

SDS

orpronase

(see Methods),

the circular DNA molecules no

longer

have detecta-ble

"knobs"

(Fig. 1C and Table 1, lines 2 and 3). The SSP-DNA complex was also obtained from Triton X-100 extracts of infected cells

centrifuged

to equilibrium in a

CsCl

gradient

(see Materials

and

Methods). The DNA,

which banded at a

buoyant

density

of 1.71

(Fig.

2), was

circular

and

about half

of

the molecules

contained a single "knob" (photographs not

shown

but

indistinguishable

from

Fig.

1

E,

F,

and

G; Table

1, line

-4).

As

before,

treatmentof

the

SSP-DNA

complex with SDS

or pronase

eliminated the "knobs"

(data

not

shown).

To determine if these structures occur in mature

SV40 virions, purified virus particles

(see

Materials and Methods)

were

disrupted

at

pH

10.5

according

to

Huang

et

al. (7) and the

resulting "cores"

were

banded

in a

CsCl

gradi-ent

(Fig. 2).

Examination of

the material

taken

from

the peak fractions

by high resolution

electron

microscopy (5) revealed

structures

that

closely resembled the SSP-DNA complexes

re-covered

directly from the infected cells (Fig. 1E,

F, and G, and Table

1,

line 5).

SV40 DNA

map

location

of the protein

"knobs"

of the SSP-DNA complex. A

deter-mination of

whether the protein "knobs"

are

bound

to

the

circular

SV40 DNA

molecules

at a

specific location

can

be made with restriction

endonucleases that cleave SV40 DNA

once at a

unique site (10,

11). Accordingly,

the DNA

in

the

SSP-DNA

complex

was

cleaved with ecoRI

endonuclease

to

yield unit

length linear

mole-cules. Measurement

of

the

distance from the

center of

the

"knobs"

to

the ends of the DNA

showed

that the "knobs"

occur

predominantly

at

0.65

to

0.75

SV40

fractional

lengths

from

the

farther end

(Fig.

3A). The

SSP-DNA

complex

obtained

from

mature

virions

also

yields linear

molecules with a "knob" at about the same position after

cleavage with ecoRl endonuclease

(Fig. 3B).

Because the

two

ends

ofthe

ecoRI

endonu-clease-generated linear molecules

are

indistin-guishable,

it is not

possible,

from

the above

experiments

alone,

to

assign

a

unique

map

location

to the protein

"knob".

This can be

done, however, by cleaving the SSP-DNA

com-plex

with hpaIIendonuclease, an enzyme which

cleaves SV40

DNA once at a site 0.735

SV40

fractional

length,

clockwise,

from the

ecoRI

endonuclease

restriction site (0.735 map

units)

168

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/1

Protein

Complex

Bound

to

the

Origin

of DNA

Replication

in

SV40

D*

1b -

*pv>:;

Complex isolated from

S;*tbi;*,.=j;.;

.*;.,>..

infected African green monkey cells.

r. *-.

,o'*' **''-,* l. *,'.' .''. t''.

eAojS

lEN,-wt.L

I;--

..

,t','.'*..

3 ;' Pb " >..;,. ;,.,,,.-.,;,,t, *... ;|'4 s..*' ,.q;. b"

Fr

,e

Complex

isolated fro

purified virions.

Work

of

Griffith,

Dieckmann, and Berg, J. Virol. 15:167-172,1975.

FIG. 1. High resolution electron micrographs of salt-stable protein-DNA (SSP-DNA) complexes. (A) SSP-DNA comptexmounted as described in Materials and Methods; (B) SSP-DNA complex (NaCI)exposed briefly to X-rays; (C) SSP-DNA complex treated with SDS (or pronase); (D) magnified protein "knobs" in SSP-DNA complexes (NaCI and CsCI); (E and F) SSP-DNA complexes (from virions) purified by CsCI gradient centrifugation and relaxed by X-ray treatment; (G) magnified protein "knobs" from SSP-DNA complexes fromvirions.

169

61

,

/7-17

A4

.L

3

--.

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GRIFFITH, DIECKMANN, ANDBERG

TABLE 1. Thehigh salt SV40 chromatin complex ispredominantly a circularDNA witha single protein "knob"a

Sample No. ofmolecules No.of"Knobs"percircular molecule

counted 0 1 2 3 4or more

1SSP-DNA (NaCl) 100 6 70 19 3 2

100 3 67 23 6 1

2SSP-DNA(NaCl) 100 0 0 0 0 0

Treated withSDS

3SSP-DNA(NaCl) 100 0 0 0 0 0

Treated with pronase

4SSP-DNA(CsCl) 100 49 48 3 0 0

5 VirionSSP-DNA 100 23 65 10 1 1

(CsCl)

aSalt-stable protein-DNA complex (NaCl) (1); aftertreatment with SDS (2) and pronase (3); salt-stable

protein-DNA complex (CsCl) (4) and the salt-stable protein-DNA complex (CsCl) obtained from virions (5) were prepared as described in Materials and Methods. X-ray irradiation just before mounting for high resolution electron microscopy wassufficient to relax 90% of the supercoiled molecules. Examples of molecules scoredashaving "knobs"areshowninFig. 1.

b-i P=171

z

o x

z 0

I5 IC 20

[image:4.504.47.445.103.229.2]

FRACTION

FIG. 2. Sedimentation of SSP-DNA complex to equilibriuminCsCl.(0) [3H]SV40 DNA present in a Triton X-100 extract of SV40 infected cells

cen-trifuged to equilibrium in a CsCl gradient. (0) [3H]SV40 DNA obtained from disrupted virions by treatment atpH10.5after centrifugation to equilib-rium inaCsCl gradient.

(11, 14); in more than 70% of the linear DNA

molecules,

the "knobs" were found at one end

(Fig.

3C). Since the "knob" is close to, but does not protect

the

hpall endonuclease

restriction site

against

cleavage,

we conclude that the

"knobs"

probably

occur at 0.70

±

0.05 on the

SV40

DNA map.

DISCUSSION

Progeny SV40 DNA

exists in

SV40-infected

cell nuclei

in

combination with

an

equal weight

of

proteins

in a

chromatin-like

structure

(6,

17, Perez et

al., manuscript

in

preparation and J.

Griffith,

Science,

in

press).

In

high

salt

(1

M

NaCl

or 5

M

CsCl)

this structure is

disrupted

and the

bulk

of

the

protein

isremoved from the DNA.

By

high resolution electron

microscopy

the

remaining

protein-DNA

com-plex (SSP-DNA comcom-plex)

appears to

consist

of two or three

globular protein units bound

side

by side,

generally,

to one

region

of

the

SV40

DNA. A

protein-SV40 DNA

structure,

closely

resembling the

one

obtained

from

infected

cells,

is

also

present in mature virions.

The

location, function, and

genetic

origin

of the

stably bound protein

on

SV40

DNA is

especially

intriguing.

Danna

and Nathans

(2)

and Fareed

etal.

(3)

have

mapped

the

origin

of

SV40

DNA

replication

at0.67 map

units.

Quite

possibly

the

protein(s) bound

tothe DNA

plays

a

role

in

initiating

DNA

replication.

Since

the

protein(s)

is

also

present in themature

virion,

it mayaccompany

the

DNA

during

the early phase of infection

and

block

or promote some

essen-tial

early

step in

expression. Robb and Martin

(13) and Chou and

Martin

(1) have

described

temperature-sensitive

mutants of

SV40

(D

cis-tron

mutants) which

are

unable

to express any

early

functions

at

the restrictive

temperature.

Though the

mutant

function

is

expressed early

after

infection,

itappears tobe

synthesized

late (1,

13).

A

logical

inference from

this

fact is

that

FIG. 3. TheSV40 DNA map location of the protein "knobs" in SSP-DNA complexes. (A) Histogram of the position of theprotein "knobs"after cleavage of SSP-DNA (CsCl) complexes with ecoRI endonuclease. An example ofthe cleavedcomplex is shown in the inset. The reaction mixture (50Mliters) contained10Ag ofDNA perml,100mMTris-hydrochloride(pH 7.5),5mMMgCl2 andwasincubatedat37Cfor20minwith enough ecoRI endonucleasetoconvert morethan

90%o

of the circular DNAtofull length linear molecules (assayed by

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[image:4.504.52.242.288.434.2]

* F . '-.A

I

<7%>.

ECO

I1-

CUT

HIGH SALT COMPLEX

nflF-4

'I

ECo

R1-CUT

HIGH SALT

COMPLEX

FRoM VIlRIONs

En~~~~~~~~~~~~~~~~~~~~~

HPA iT-

CUT

HIGH

SALT

COMPLEX

Mn

-A

50

75

iOO

DISTANCE FROM

FAR

END

OF RESTRICTED

DNA

(%)

EM.). The sample was diluted 20-fold in 1 MNaCl, 10 mM EDTA (pH 7.5), 0.25% Triton X-100, and incubatedatroom temperaturefor20min before mounting forelectronmicroscopy. (B)Sameasin(A) except with the SSP-DNA (CsCl) complexobtainedfromvirions. The digestionandmountingwasperformedas

de-scribed in(A). (C) Histogram oftheposition oftheprotein"knobs"after cleavage oftheSSP-DNA(CsC()

com-plexwithhpaIIendonuclease. The reaction mixture(20 uliters) contained 50ggofDNA/ml,10 mMTris-(pH 7.5),5 mMMgCl2,0.25% Triton X-100 withsufficienthpaIIendonucleasetoconvert greaterthan 75%ofthe circlestofull lengthlinear moleculesafter3 hat37C.Sampleswerediluted100-fold, incubated,and examined

asin(A).Asample ofacleavedcomplexisshownin the inset.

Measurementsforthesehistogramsweretaken with thePhilipsEM 300 in theStandardizedMagnification

mode described in thePhilips operationmanual. Themicroscopewascalibratedagainstastandarddiffraction

gratingand checkedagainst objectswithknownspacings.Moleculeswhoselengthdeviatedbymorethan 10%/o fromthe meanSV40 length (1.48gm) werenotincluded in the distributions.

171

A

12k-

6k-LU

w

-I

(3

LU

.1

0

I

IUL

0

Lu

z

40

B

C

20k_

-IF

I

I

EMCE

APO_f~- J% -At001

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GRIFFITH, DIECKMANN, AND BERG

the D cistron gene

product,

presumably

a

pro-tein,

is in the virus

particle

(1). Conceivably,

it

is the protein component in the SSP-DNA

complex

described here.

Thus,

the D cistron proteinmay

function

as a

"pilot"

protein

(8)

in

that it accompanies the SV40 DNA to the

nucleus

and

participates

in

initiating

its

expres-sion or

replication.

ACKNOWLEDGMENTS

This work was supported by grant GB 43576 from the

National ScienceFoundation.

LITERATURE CITED

1. Chou, J. Y., and R. Martin. 1974. DNAinfectivity and theinduction of hostDNAsynthesiswith temperature

sensitivemutantsofSV40. ColdSpringHarborSymp. Quant.Biol. Vol. 39.

2.Danna, K. J., and D. Nathans. 1972. Bidirectional

replicationof simianvirus40DNA.Proc. Nat.Acad. Sci. U.S.A.69:3097-3100.

3. Fareed, G. C., C. F. Garon, and N. P. Salzman. 1972.

Origin and directionofSimian virus 40 deoxyribonu-cleic acidreplication. J.Virol.10:484-491.

4. Green,M.,H. I.Miller,andS. Hendler. 1971. Isolation of

apolyoma nucleoprotein complexfrom infectedmouse

cell cultures. Proc. Nat. Acad. Sci. U.S.A.

68:1032-10.36.

5. Griffith, J.D.1973. Electronmicroscopicvisualization of

DNA in association with cellular components, p.

129-145. In D. M. Prescott (ed.), Methods in cell

biology,vol. 7.Academic PressInc.,New York. 6. Hall, M. R., W. Meinke, and D. A. Goldstein. 1973.

Nucleoprotein complexes containing replicatingsimian

virus 40 DNA: comparison with polyomanucleoprotein complexes. J. Virol. 12:901-908.

7. Huang, E. S., M. K. Estes, and J. S. Pagano. 1972. Structure and function of the polypeptides in simian

virus 40. I. Existence of subviral

deoxyribonucleo-proteincomplexes. J. Virol.9:923-929.

8. Kornberg, A. 1974. Replication ofDNA viruses, p. 240. In DNA Synthesis. W.H.Freeman, San Francisco. 9. Lake, R. S., S. Barban, and N. P. Salzman. 1973.

Resolution and identification of thecore

deoxynucleo-proteinsof the simian virus40.Biochem. Biophys.Res.

Comm. 54:640-647.

10. Morrow, J. F., and P. Berg.1972.Cleavageofsimian virus 40 DNA at a unique site by a bacterial restriction

enzyme.Proc. Nat. Acad. Sci. U.S.A.69:3365-3369.

11. Morrow, J. F., and P. Berg.1973.LocationoftheT4gene

32 protein binding siteon simian virus 40 DNA. J.

Virol. 12:1631-1632.

12. Mulder, C., and H. Delius.1972.Specificityofthe break

produced by restriction endonuclease RI inSV40 DNA

asrevealedby partialdenaturation. Proc. Nat.Acad.

Sci. U.S.A.69:3215-3219.

13. Robb, J. A., and R. G. Martin.1972.Genetic analysisof

simianvirus40.III.Characterization ofa

temperature-sensitivemutant blockedatanearlystageof

produc-tiveinfectioninmonkey cells. J. Virol.9:956-968. 14. Sharp, P. A., B.Sugden, and J. Sambrook. 1973.

Detec-tion,oftworestriction endonuclease activitiesin

Hae-mophilus parainfluenzae using analytical

agarose-ethidium bromide electrophoresis. Biochemistry 12:3055-3063.

15. Tai, H.T., C. A.Smith, P.A.Sharp, and J. Vinograd.

1972.Sequenceheterogeneity in closed simianvirus 40

deoxyribonucleic acid. J. Virol.9:317-325.

16. Tooze, J.1973.The molecular biologyoftumourviruses.

ColdSpring Harbor Laboratory, New York.

17. White,M., and R. Eason.1971.Nucleoproteincomplexes insimian virus40-infectedcells. J. Virol.8:363-371.

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