Replication process of the parvovirus H-1. VIII. Partial denaturation mapping and localization of the replication origin of H-1 replicative-form DNA with electron microscopy.

(1)

Replication Process of the Parvovirus H-1

VIII. Partial

Denaturation Mapping and Localization of the

Replication

Origin of H-1 Replicative-Form DNA with Electron

Microscopy

IRWIN I. SINGER AND SOLON L. RHODE III*

PutnamMemorialHospital Institute for MedicalResearch,Bennington, Vermont05201 Receivedforpublication 11June 1976

Partial denaturation mapping, restriction endonuclease digestion, and elec-tronmicroscopy were used to determinewhichendof the linearduplex replica-tive-form (RF) DNA molecule contains the origin of RF replication for the parvovirusH-1. Thisoriginwaslocalized withinapproximately 300basepairs of

the arbitrarily designated right end of the RF DNA, inthe EcoRI orHaeII-A

fragment. Based ondenaturationbehaviorinformamide,therightendwas also found to have a relatively high guanine plus cytosine content, whereas the regionadjacenttotheleft terminus oftheRFDNAmolecule was adenineplus thymine rich.

In the previous paper ofthis series (10), we The tsl mutant of the H-1 parvovirus was grownat presented electron microscopic evidence indi- the restrictive temperature (39.5°C) in serum-syn-catingthatreplication ofreplicative form (RF) chronized hamsterembryofibroblasts; RF DNA

rep-DNA, inthe nondefective parvovirus H-1 (tsl lication is enhanced, and progeny DNA synthesis

mutant), occursvia adouble-stranded (ds) lin- shows a temperature-dependent inhibition in this

muant),

s t

.

mutant. Infected cells were labeled with

ear Y-Shape rep

licatiove

ntermed

ate

(RIl,

[3H]bromodeoxyuridine

(Q3H]BUdR),

and the viral

1.55

.tm

inlength. Replicationappeared to

hi-

DNA was extracted by the Hirt method. Labeled

H-1

tiate near (within 15%o of the genome length) RF and RI DNA were purified by velocity sedimen-one end of the RF DNA molecule; a single tation in a sucrose gradient followed by isopycnic replication fork proceeded toward the other end centrifugation in a second gradient ofCs2SO4. The

ata uniformrate. Thepurpose ofthisstudy is yield of viral DNA fromonepreparationwas

suffi-to determine which end of the H-1 RF DNA cient for all of theexperimentsdescribed below

(ex-moleculecontainsthe initiation siteof RF repli- cept forone in which the label was [3H]thymidine

cation,viapartial denaturationmapping of in- instead of [3H]BUdR). RF DNA wasdigested with

tactRFDNA, RIDNA, andpurifiedEcoRIor EcoRI orHaeII (from

Haemophilus aegyptius)

re-tact

_NaeII

RFndonua,eRI

_{endonuclease-generated}

enA,and

purified Eo

_fragments_ofR_RFo striction_{completion of the digestion}endonuclease (9; unpublished data); the wasverified by

analyti-DNA. cal agarose-gel electrophoresis, and the fragments

Wefoundthat the origin of RF replication is were separated via velocity sedimentation through a

localized nearthe external end of the EcoRI or sucrose gradient. Partial denaturation of the viral

HaeII "A" RFfragment, which constitutes ap- DNA was achieved using high concentrations of proximately 80% of the H-1 genome length (9). formamide (Matheson Scientific Co.) in the

DNA-In addition, this terminus of the A fragment spreadingsolution;82%wasthe formamide

concen-hereafter termedthe right end of the RF mole- tration expected to initiate partial denaturation,

cule, has a relatively highguanine plus

cyto-whereas

90% formamide was anticipated to

com-cule, has a

relatively

whgh

guanine

plus cyto- pletely melt the DNA (13). Three microliters ofa

sine (G+C) content, whereas the small EcoRI

0.5-mg/ml

cytochrome c solution (horse

heart,

orHaeII"B" RF fragment (representing about Sigma, type III) in 0.5 M Tris(pH 8.5)-0.05 M EDTA 20% of H-1 genome), at the left end of the RF was thoroughly mixed with 1 ,lof purified (10)

H-1

DNA, has an elevated adenine plus thymine DNA (in 0.01 M

TrisipH

8.5]-0.001 M EDTA), 41 to

(A+T) composition. 45 ,ul offormamide, and 1 to 5

Al

of water to produce

50 ,ulofspreading solution possessing the desired MATERIALS AND METHODS formamide concentration. This was immediately spreadonto afreshly preparedhypophase of 1 mM The methodsof H-1 viruspropagation, RFDNA Tris(pH 8.5)-0.1 mM EDTA containing from 52 to labeling andpurification, and electron microscopy 60% formamide; a30%difference between the form-have already been describedindetail in the previous amideconcentrations ofthe spreading solution and papers ofthis series (9, 10);therefore, only a brief hypophase was alwaysmaintained toapproximate methodological summary will be presented here. isodenaturing conditions in both "phases" (3). All

724

on November 10, 2019 by guest

http://jvi.asm.org/

(2)

solutions were adjusted to 23°C before spreading, . . and were also membrane-filtered (Millipore Corp.)

(except for the DNA and formamide).Preparation of Eco Rl A

grids, DNA spreading, uranyl acetate staining, Pt-Pd rotary shadowing, electron microscopy, and measurement of the DNA contour length were per- 20 formed as previously detailed (10; 4).

RESULTS x

Evaluation of restriction endonuclease E

EcoRIB

digestions and purification of H-1 RF DNA u

fragments. Analytical gel electrophoresis was 10 _

used to determine whether all ofthe H-1 RF

DNA had been cleaved byEcoRI orHaeII. Fig-ure 1shows an agarose-gel profile for theEcoRI

digestion used in this study. All of the labeled A

H-1 RFwas cleaved into fragment A, represent- 0 _

ingapproximately 78% of the label, fragment B 1 10 20 30

representing about 22% of the label, and a Tube Number

small amount ofdimer-length B fragment, pre- FIG. 2. Preparative sucrose gradient

centrifuga-sumably derived from the digestion of dimer RF tion of restriction endonuclease EcoRI fragments A

DNAswhichare linked "tail-to-tail" at the left and B of tsl H-1RF DNA. The EcoRIdigestofH-1

ends of their component monomers (9). Similar RF DNAdescribed inFig.1 wassubjectedtovelocity

results were obtained with endonuclease HaeII sedimentation in agradientof5 to 20% sucrose in 50

(unpublished data).

The A andB H-1 RF frag- mM

Tris-hydrochloride

(pH 8.0), 1 M

NaCl,

1 mM mentswere

separated

using

velocity

sedimen- EDTA, and

0.2%Sarkosyl,

for 7 h at 42,000 rpm in a

tationthroughasucrosegradient(Fig. 2),orb SW50rotor at4°C.Fractionsof150 ,lwerecollected

tationtrgauory through the bottom of the tube, and

10-pi

aliquots

preparative gel electrophoresis. Electron micro- were used to determine the position of

radioactivity.

scopic

examination ofRF fragments prepared Fractions containing the EcoRI-A and EcoRI-B

frag-by either method showed slight interpeak con- ments were pooled as illustrated, and the DNA was

precipitatedwith 2.5 volumesofethanolat-20°C for

I 16h. Theprecipitates werecollected by

centrifuga-tion, washed once with 70% ethanol-30% 0.3 M

30 - EcoRIA NaCl-50 mM Tris(pH 7.5)-i mM EDTA,

recentri-fuged,andfinallydissolvedin10mMTris(pH8.5)-i mM EDTAfor electron microscopy. The directionof sedimentation isfromrighttoleft.

tamination, but thiswas of little consequence

20 since these fragments are readily

distinguish-ableonthe basis of

length.

Thesepreparations

x of H-1 RF A and B fragmentswereutilized in

E the experimentsoutlined below.

EcoRIB Partial denaturation mapping of

H-1

RF,

n10_ 2 _

RI,

andrestriction endonuclease

fragments

of

RF DNA. A highlypurifiedmixture ofH-1 ds

RF and RIDNA, combined with either82, 84,

dimer or 86% formamide in the presence of

cyto-Eco RI l chrome c and

immediately spread

onto an

iso-01 20 4060-

denaturing

hypophase

at

23°C,

exhibited a

120 40 60 unique partial denaturation pattern. One to

Fraction two loops, presumably formed by the preferen-FIG. 1. EcoRIdigestion ofthe[3H]BUdR-labeled tial melting of A+T-rich regions ofDNA

(5-7),

H-1 RF DNA preparation used throughout this were found at an end ofthe RF molecules

(Fig.

study. tsl H-1 RFDNA waspreparedaspreviously 3A-H). This A+T-containing region extended described(9). Analiquotwasdigestedwith EcoRI in fromaDNAend(often

forming

aterminal

loop)

a reaction mixtureof50pi for4h at37C,and a4-Pi al of e

genomlngth

to

loop)

portionwassubjectedtoelectrophoresisin acylindri- along 20% of the genome length into the

RF

calgel (0.6by15cm) ofl.4% agaroseat 25Vfor17h molecule in 76% of the partially denatured RFs;

at23°C (9).Thegelorigin is locatedtotheleft,and the looptraversed 20 to 50% of the RF length in

the anode is located totheright. the

remaining

DNAs

(Fig.

4A-C).The extent of

on November 10, 2019 by guest

http://jvi.asm.org/

[image:2.501.251.448.48.249.2] [image:2.501.48.244.369.566.2]

(3)

_ . , f s1,

A F

1K

:~~~~~~~~~~~:

FIG. 3. Electronmicrographs oftslH-i RF DNA, RIDNA, HaeII-B,and EcoRI-B fragments,partially

denatured with formamide. Bar = 0.5 pm. (A) RF DNA partially denatured with 82%formamide; the

hypophasecontained 52%formamide. (B-H)As(A),butpartiallymelted withaspreadingsolution

contain-ing84%formamideandahypophasewith54%formamide.(I-L) Uncleaved RI DNAincompletelymeltedas in(B-H).Thepercentageofgenomereplicationincreasesfrom lefttoright:I=12%, J=14%,K =36%,andL = 78%. (M-S)HaeII-Bfragments labeled with[3H]thymidineandpartially denaturedas in (B-H). (T-Z) [3H]BUdR-substitutedEcoRI-Bfragmentspartiallymeltedinan86%formamidespreadingsolution,usinga

hypophase containing56%formamide.

intramolecular

melting

increased with

rising

(Fig.

4F). DNA

fragments

isolated from restric-formamide concentrations above 82%: 14% of tion endonuclease HaeII

digests

of the H-i RF the

partially

denatured DNAs showed more described above were also

partially

denatured than 20%

melting

with 82% formamide, 19% with 84% formamide.

Incomplete

melting was

with 84% formamide, and 44% inthe presence observedinthe small B

fragments (Fig.

3M-S) of 86% formamide.

Partially

denatured RIs andwas

conspicuously

absent from the

larger

A

(Fig.

31-L) were also observed,

constituting

fragments.

Approximately

60% of these par-24% of the total number of

partially

denatured

tially

denatured B

fragments

apparently

were

molecules; a similar

frequency

of RIs was seen melted closetotheleftend of the RFDNA,thus

when this DNA was

prepared

under nondena-

exhibiting

terminal

loops,

whereas others

ap-turing conditions with 50% formamide.

Only

pearedtohave melted

through

theHaeII

cleav-the

unreplicated

regionsof theseRIDNAmole- age site,

forming Y-shaped fragments (Fig.

cules exhibited evidence of

partial melting.

4D). Similar results were obtained using

re-Thus, terminal denaturation

loops

were local- striction endonuclease EcoRI, whose

cleavage

ized atthe endof the molecule

opposite

tothat site islocated 0.016 of thegenome

length

tothe

containing

the

origin

of RF DNA

replication

right

of the HaeII siteonthe

physical

mapof

on November 10, 2019 by guest

http://jvi.asm.org/

[image:3.501.69.456.64.403.2]

(4)

___________________ 1, and thus produces B fragments equal to

_________________ ~ 21.6% of the RF DNA length (9; unpublished

A_

_ data). EcoRI-B fragmentsalso exhibitedeither

_______________ terminal

loops

or forked ends

(Fig.

3T-Z,

Fig.

--____________

4E), whereas the A fragments appeared un-melted upon exposure to 86% formamide. The initiation site for H-1 RF DNA replication is

________________

therefore located at the right end of the

mole--______________ ~ cule, within adomain comprised of 6% of the

- _______________ genome (the smallest fork size), whereas a

_ ______________ unique region of elevated A+T content,

local-E ized

by

reference to the

EcoRIIHaeII

cleavage

________________

sites, is atthe left end of theRFDNAmolecule.

-____________

=Localization of a G+C-rich region near the

--______________ initiation site of H-1 RF DNA

replication.

-________

During experiments conducted to verify that

___

_D

our preparation ofH-1 ds RF DNA was fully -

-=________

melted by90%formamide under the same

con-________

-ditions employed for partial denaturation, we

____,

____

observed a dramatic increase in RF contour

-

__________

- length(loops were no longerfound). Themean

C _ E

lengths

ofH-1 RFDNAdecreasedwith

increas--E

ing concentrations of formamide: 1.33 + 0.12

,um

(n = 55. +99% confidence interval)at82%

.9

formamide,

1.25 + 0.04

,um

(n = 154) for 84%,

HaI Ecofa and 1.13 ±0.08 ,um (n = 66) at 86%. We

there-FRACTIONAL LENGTH OF GENOME fore expected the

RF

length to decreasefurther when

fully

melted in 90%

formamide,

espe-ciallysincethelength ofsingle-stranded (ss)

H-___________________ 1 viralDNAwas33%shorter thanthatofds

H-___________________ 1 RF DNA in 50% formamide (10). However,

________________ the mean contour length ofH-1 RF spread in

______________________ 90%formamide was 1.40 + 0.02 ,um (n = 131), ____________________ whereas that of monomeric ss H-1 DNA

puri-=___________________

fied from virions was nearly halfthat value

F whenpreparedunder the same conditions: X =

0.71 ± 0.04 Am (n = 141) (Fig. 5A). These

____________________ resultssuggested that90% formamide had

en--__________________

tirely converted dsH-1RFto assDNA molecule

__-_- ofdimer length. Sincetreatment of dsH-1 RF

6di 63 0:i 07 dI

FRACTKNAL LENGTH OFGENOME [3H]thymidine-labeled HaeII-B fragments, 84% FIG. 4. Formamidepartialdenaturation maps of formamide;and(E) [3H]BUdR-substitutedEcoRI-B tsl H-1 DNA. (A-C) Intact RF DNA, (D) HaeII fragments, 86% formamide. In the case ofthe B endonuclease B fragments ofRFDNA, (E) EcoRI fragments, denaturation sitesrepresented byclosed endonuclease Bfragments ofRFDNA, (F)RIsofH-1 loopswereplacedattheleftendofthe map, andthose RFDNA.Contourlengths ofthe measured molecules melted regions that appeared forked (presumably

werenormalizedto1.0forthe RFs andRIs,or to0.2, meltedthroughthenucleasecleavage sites)were ori-the approximatefractional sizeoftheH-1 chromo- ented to the right. (F) Partially denaturedH-1 RI someforthe RF Bfragmentsgenerated byboth endo- DNAs obtained using 82, 84, or86% formamide. nucleases;arrowsindicate thepositionsoftheHaeII Branched (replicating) molecules whose lengths andEcoRIcleavage sites on theH-1 genome map. (sumoflengths of unreplicatedbaseplusonebranch) The location of denatured regions (indicated by werewithin thelengthrangeofRF DNAsprepared heavy lines)wasorientedtotheleftendofthe RF and with corresponding formamide concentrations, and RImolecules,andexpressedas afraction oftheH-1 which also contained denaturation loops, were

se-genome length. The lengths ofthese denatured ss lectedforthis map. Thelengthswerenormalized and regions were notcorrectedforincreasedshrinkage, thepositionsofthereplication forkswereplottedasa

relativetods areas, whichoccurswith theformamide fraction ofthe total chromosomal length; denatura-concentrations used here. (A)82%Formamide; (B) tionloopswerepositionedasin(A-C).These RIsare 84% formamide; (C) 86% formamide; (D) displayedinorderoftheir extentof replication.

on November 10, 2019 by guest

http://jvi.asm.org/

[image:4.501.72.212.60.489.2]

(5)

I I, 1 I I I II I I5II lI I I 1I 'I I I I 'I ' I '

A.90% C.90%

X.0.19

dimer 1

qds Eco RI-B

40

U~~~~~~~~~~~s.40r

40 ~~~monomer

,

x.0.71 dsRF

dimer

E~~~~~~~~~~~~~~~~~~~d Ecofr RI_1

30 _monom

Viral RF.w6

20

|

RFnom~

RF

bU5R

R

EcoRI

4) I I . 8 RI.A

0

~~~~~~~~I

I

0l

QS

1.I.

.

o0

B.96%

D. 96%

length

_E

of1.40,um,corremonomer

i-.8S6

z

40

monomer ia0.64

30

20

10 RF

~~~~~~~~~~~~~~~~Eco

RI-A

10R

00

05

1.0

1.5

2.0

0

0.5

1.0

1.5 Length

(m)

FIG. 5. Histograms ofthe contour lengthsofintact tslH-i RF DNA moleculesand theirendonuclease

EcoRI-generated fragments spread after "fully denaturing" (90%formamide,230C), or fhyperdenaturing"

(96%formamide,s300C)incubations.SinglesarrowsidepictmeanlengthsforssDNA,anddoublearrowsdenote

the meanlengthsofdsDNA. (A)H-iRE DNA meltedwith 90%formamideat230C (solidlines)hasamean

length of1.40 pm, correspondingtodimer-lengthssDNA, determinedby measuringssDNA extractedfrom

purifiedvirions (brokenlines; =(0.71 pm), underthesame denaturation andspreadingconditions. The meanlength ofdsREDNAmonomersmeasuredunder partiallymelting conditions (86% formamide,230C)

was1.13 pm(histogram notshown). (B) DistributionofH-i Re DNAlengthsobtainedbytreatmentunder

whypermeltingconditions"(96%formamide,300C)andimmediatespreadingin90%formamideat23mC.The

meanlengthis0.86 pm,and themajor histogrampeakcorrespondstothatofssDNAmonomers(ssviralDNA in[ADspread in thesamemanner.(C) Endonuclease EcoRI-A and-Bfragments ofH-i REDNApreparedin 90% formamide233C.atssBfragmentshadameanlengthof(0.19 m,which isshorterthanthatobservedfor monomerdsBfragmentsprepared underpartiallydenaturingconditions(86%formamidet,23tC;s 0f24=

pm,histogramnotshown).Afragmentsexhibitedtwomajorpeaks in90%23[C (solidformamideat lines),

withmeanscorrespondingtossdimerlength(~=1.15pmn),andssmonomerlength (* =0.61 pm).Themean

length of monomer ds EcoRI-A fragments prepared in 86% formamide at 239C (partially denaturing

conditions)was0.86 pm(histogramnotshown).(D)HistogramofEcoRI-Afragments ofH-iRE

'"hyperdena-tured" with 96%formamide at30"C, andspread as in (B). The DNA was completelydissociated into ss monomerAfragments (~=0.64 pm), whosecontourlength distribution coincides with thessAfragment

monomersobservedafterdenaturation with 90%formamide (shownin[C]).

on November 10, 2019 by guest

http://jvi.asm.org/

[image:5.501.64.460.51.465.2]

(6)

REPLICATION 729

withanalkalinesucrose

gradient

doesconvert

after

similar treatment of ds H-1 RF (Fig. 5A).

90% of it into monomer ss H-1 DNA

(9),

we

Incubation

of EcoRI-A

fragments

of H-1 RF

decided

tofurther

investigate

thenatureof the DNA in 96%

formamide

at 30°C for 30 min

linkagebetween the two ss monomers. ds H-1 converted them intossmonomers (£ = 0.64 +

RF DNA (in 10mM

Tris[pH

8.5]-1 mM

EDTA)

0.03

,um;

n = 107) (Fig. 5D). These data

collec-was placed in 96% formamide at

30°C

for 30

tively demonstrate

that

the

regionof

high

G+ C

min,followed

by

theaddition of sufficient cyto- content is

located close

to

the

right

endof

the

chromecsolution (in0.5M

Tris[pH

8.5]-0.05

M RF DNAmap.

EDTA)tolower the formamideconcentrationof

theresulting solutionto

90%,

and

immediately

DISCUSSION

spread onto an

isodenaturing

hypophase

con- Features ofthe

H-1

RF DNA monomer, as

taining 60%

formamide

at

23°C.

Weestimated determinedinthis and the twopreceding

stud-that the

melting

temperature

(Tm)

of the 90% ies in this series, are summarized

diagramati-formamide spreading solution was

approxi-

cally inFig. 6. Our results show that aregion

mately

300

below the

T,,

of

poly(deoxyguani-

susceptibletomeltingunder partialdenaturing

dylic

acid:deoxycytidylic

acid)[poly(dG

dC),

conditions existsadjacenttothe endo R-EcoRI

and that the Tm of the96%formamidemixture and HaeII cleavage sites, which are located

was

10.40C

above that of this

homopolymer.

approximately 20% of the genome lengthfrom These calculations are based on a Tm of

780C

the arbitrarily defined left end of the

H-1

RF

for dG dC in a solution with acation concen- DNA molecule (9). Partially denatured

inter-trationof6mM(3)andapH of7.0 (12), aswell mediates in

H-1

RFDNAreplication

(RIs)

simi-as a 0.6°C Tm reduction for each 1% increase larlyexhibited melting near the left end of the

informamide concentration (1). After

exposing

molecule, whereas the origin ofreplication,

de-ds H-1 RFDNA tothe96%formamide

solution,

termined by the position of early replication

the contour

length

decreasedto that ofss

H-1

forks, was localized close to the right end of this

DNAmonomer size (Fig.

5B):

X =

0.86

±

0.04

DNA. Based on the size of the smallest

daugh-um, n = 197,

indicating

that the

region

com- ter arms observed, the region containing the

prising theintermonomer

linkage

probably

has replication origin is located within 700 base

a

high

G+C contentandmorestable

hydrogen

pairs (10), but more probably within 300 base

bonding, as

opposed

to acovalent"turnaround" pairs(this study) of the right end of the

H-1

RF

ofribonucleotides.

We performed the above experiments on Left

Right

EcoRI restriction

endonuclease-generated

A A-T

Rich

G-C

Rich

and B fragments of dsH-1 RF DNA to deter- R c

mine which end of this molecule is G+C rich

C

and

responsible

for

linking

the termini of the

'E

monomers inthess

dimers

generated

with90%

HaeEL

EcoRI V Origin

formamide. Themean

length

of the ds A

frag-

t I I

ment in 86% formamide

(distribution

not 0 1 2 3 4 5

shown)

was 0.86 ±0.05 am

(n

=

72), represent-

Kilobase Pairs

ing78.2% of the genome, and that of the ds B

fragment

spread

under thesameconditionswas FIG. 6.

Schematic

representation

of

H-i

RFDNA 0.24 + 0.02 ,um (n =

75),

equal

to 21.8%of the

monomer in the

foldback

configuration

(9).

The

mol-RF length. These size measurements are in ecule is_tion _{endonuclease HaeII and EcoRI sites}4,900 base pairs in length, with the restric-_(arrows)

good agreement with data obtained from ana- located 900 and 1,000 base pairs,respectively, from

lytical

gel

electrophoresis

of

EcoRI-generated

the endcontainingthe covalent turnaround (based

H-1 RF DNA

fragments (9). When

the B

frag-

on molecular weights given in

[9D;

the large and

ments were

spread

inthepresenceof90%form- small RFfragmentsgenerated bythese

endonuclea-amide, their

mean

length

decreasedto 0.19 ± ses are

referred

to asA and B

(respectively).

V,

Viral 0.01

,tm

(n =

85),

indicating

that this

fragment

strand; C,

complementary

strand. Thezonein which

does not contain the

G+C-rich

linkage

group the initiation site forRF DNA replication is located is

under

study (Fig.

50).

Preparation

of the A delineated by the open bar and is composed of base

fragmentin 90%formamide

generated

twosub- pairs 4,600 through 4,900

(6%

of genome). The B

popultn(e

banalysis,

region is

relatively

richin A +T content(indicatedby

puion (revea led

by

hiAStor

a 3

X

crosshatchedbar),and the

right

end hasanelevated

(Fig. 50) with meanlengthsOf 0.61 ± 0.03 zm G+C composition (asterisk). More recent

experi-(n = 94) and 1.15 + 0.03 ,um

(n

=

108),

repre- ments have shown that the structure oftherightend

senting the ss A

fragment

monomer andss A is morecomplicated thanindicatedinthis

diagram

fragment

dimer,

respectively,

as was observed

(manuscript

in

preparation).

on November 10, 2019 by guest

http://jvi.asm.org/

[image:6.501.254.441.386.472.2]

(7)

DNAmolecule. The right terminus of the H-1 cause many of the undigested RF molecules chromosome also exhibits a region that is re- treated with 86% formamide exhibited

melting

fractory to melting in a solution containing 90% at thesecleavage sites. Furthermore,

complete

formamide [withan effectivemelting tempera- melting of the left terminus of RI molecules ture about 3°C below that of poly(dG dC)], re- subjected to partial denaturation would

sulting in the formation of ss dimers under have generated double Y-shaped molecules

these conditions. No knobs or branches were (> <), which were not observed. However,

detected at the midpoint of these ss dimers after we could not definitely exclude thepossibility

electron microscopic study of more than 200 thatmeltinghad occurredthroughthenatural molecules, indicating that this presumably ds left RF end because the resultant Y-shaped

linkage region isrelatively small.The observa- molecules would have been easily confused tions that it remains intact under conditions with unmelted early RIs and therefore would that completely melt the rest of the RF mole- not be included in oursample.

cule, and that exposing this DNA to an esti- It is alsopossible that the branchedportions

mated

T,,,

of10.4°C above the melting point of of the RIscontaining denaturationloopsin the

dG dC homopolymers converts the ss dimers B region could have been formed viamelting of

into ssmonomers, strongly suggested that this the right end the molecule instead of being linkage is due to a G+C-rich terminus rather intermediates of semiconservative RF DNA than tocovalentbonding withribonucleotides. replication. We believe this to be unlikely since

The renaturation properties of the EcoRI-A branched structures were never observed fragment, which contains the rightend, have amongmorethan 200EcoRI-Afragments mea-also ruledoutthepossibility ofacovalent link- sured with the electronmicroscope afterpartial

age at the right endof H-1 RF DNA (9). Cur- denaturation. Also, the number and

distribu-rentexperimentsindicate that the structure of tion of replication forks along the contour the rightendis morecomplex than indicated in lengths of incompletely melted H-1 RI mole-Fig. 6. Inaddition, theHaeII or EcoRI endonu- cules is very similar to that observed after clease B fragment, containing the left end of spreadinginnondenaturing conditions (10). the RF DNA, forms denaturation loops under The electron microscopic localization of the

partially denaturing conditions (82, 84, or86% initiation site forH-1RFDNA replication close

formamide), and therefore is A+T rich (5-7). to the right terminus of the RF molecule is

Most of the RF molecules observed had par- consistent with radiochemical experiments in

tially melted so close to the left end that a the accompanying manuscript (9), monitoring

terminal loopwasgenerated. Sinceonly 10% of the incorporation of [3H]thymidine intoEcoRI

H-1 RFmoleculesexhibited evidence of a cova- fragmentsof H-1 RF DNA after short labeling lently closed turnaround at the left end (9), it periods, which indicated that the origin of RF seems unlikely that covalent bonds were re- DNA synthesis is located somewhere within

sponsible forstabilizing the apex of this end in theEcoRI-A fragment. The latter experiments

the other90% of themolecules under partially have also shown that the initiation point for

denaturingconditions. It is more probable that progeny ssDNA synthesis probably has a

simi-this B apex isstabilizedby adG dC enrichment lar location. These data collectively indicate similar to that present atthe right terminus, that the H-1 RF DNA is a structurally and

but exhibiting a lower melting temperature, metabolically polarized molecule, with RF and

since EcoRI-B fragments (with the left end) probably progeny DNA replication initiating

werecompletely dissociatedby90%formamide, nearthe dG+dC-rich right end, and then

pro-whereas the A fragments (with the right end) ceedingtothe leftterminus(10). This left end is were not. Theexpected10%of the B fragments, adjacent to a region with a high dA+dT con-which should have been dimer length in 90% tent, as demonstrated by partial denaturation formamide due to the presence of a covalently mapping.Italso exhibitsatleast two structural closedturnaround, were not demonstrated with forms thatdifferby50to70base pairs in length anycertainty usingelectron microscopic meth- (9).Theself-cohesiveproperty of this left termi-ods. We also observed that incomplete melting nus implies that it contains an inverted self-conditions gave rise to Y-shaped B fragments in complementary sequence, which forms a cova-addition to loops located at either the natural

lently

closed turnaround in some molecules. left terminus, or near the middle of this frag- This structural model for the left end of H-1 RF ment. We postulate that these Y-shaped frag- DNAissimilartothat recently propounded for mentsresulted from melting through theEcoRI both DNA ends of adenovirus-associated virus

and HaeII cleavage sites, rather than from

(AAV),

a defective parvovirus, only capable of

melting of the natural left end of the RF, be-

replication

in cells coinfected with adenovirus

on November 10, 2019 by guest

http://jvi.asm.org/

(8)

VOL.

(2). AAV ss viral DNA molecules are either adenovirus for adenovirus-associatedvirus

multipli-plus or minus strands containing self-comple- cation.Nature(London)New Biol. 244:71-73.

mentarytemnlsep 3. Davis, R. W., and R. W. Hyman. 1971. A study in

mentary terminal sequences capable ofintra- evolution: the DNAbasesequence homology between

molecular annealing with theconsequentgen- coliphages T7 and T3. J. Mol. Biol. 62:287-301.

eration ofss circles orconcatamers (8). It has 4. Davis,R. W., M.Simon, and N. Davidson. 1971. Elec-beenproposed that both ends of duplex AAV RF tronmicroscope heteroduplex methods for mapping

DNAcontainaself-complementaryfoldbackse1 regionsofbasesequencehomologyinnucleicacids,p.

D)NA

containaself-complementary foldback se-

413-428.

In L.Grossman and K.Moldave (ed.),

Meth-quencewhose 3' hydroxyl terminus may serve ods in enzymology, vol. XXI. Academic Press Inc.,

as a primer for both RF and progeny DNA NewYork.

replication at either end of the RF molecule 5. Goldstein,L., M.Thomas, and R. W.Davis. 1975.Eco

(1 Since the initiation site

of.

H.1 RF and T TRIendonuclease cleavagemap ofbacteriophage

P4-(11). Since the

initiation site of H-i

RF

and

DNA. Virology 66:420-427.

presumably progeny DNA synthesis is at or 6. Inman, R. B., andM. Schnos. 1970. Partial

denatura-near the G+C-rich right end of the H-1 RF tion of thymidine- and 5-bromouracil-containing A

molecule,

andacovalentturnaroundislocated DNA in alkali. J. Mol. Biol. 49:93-98.

ontheleft,

end,(9,

this inverted

self-comple,

7. Jacob, R. J., J. Lebowitz, and A. K. Kleinschmidt.

On the left end (9), this inverted self-comple- 1974. Locating interrupted hydrogen bonding in the

mentary terminus is notinvolvedin theinitia- secondary structure of PM2 circular DNA by compar-tion of H-1 DNA replication. It is of further ativedenaturation mapping. J. Virol. 13:1176-1185. interest thatunlike

AAV,

H-1 ss progeny DNA 8. Koczot, F., B. J.Carter, C. F. Garon, and J. A.Rose.

consists

ofonlonefthetwocmplemnta1973.

Self-complementarity of terminal sequences

consists of only one of the two complementary within plus or minus strands of

adenovirus-associ-strands, whichmaybe relatedtotheasymmet- ated virus DNA. Proc. Natl. Acad. Sci. U.S.A.

ricinitiationof DNAsynthesis atonlytheright 70:215-219.

end ofthe H-1 RF DNAmolecule. 9. Rhode,S. L. 1977.Replication process oftheparvovirus

H-1. VI.Characterization of a replication terminus of H-1 replicative-form DNA. J. Virol. 21:694-712. ACKNOWLEDGMENTS 10. Singer, I. I., and S. L. Rhode III. 1977. Replication This work wassupportedby PublicHealthService grant processof theparvovirusH-1.VII. Electron micros-* CA-07826-11 from the National Cancer Institute, and a copy of replicative-form DNA synthesis. J. Virol.

generousgiftfromThe Given Foundation. 21:713-723.

Wegratefully appreciate the excellent technical assist- 11. Strauss, S. E., E. D. Sebring, and J. A. Rose. 1976. anceofRobertCostantino and JessicaBratton,and thank Concatemers of alternating plus and minus strands Kay A. 0.EllemandHelene Toolan forcritically reading are intermediates in adenovirus-associated virus thismanuscript, andVirginiaHaas and Janeen Pratt for DNA synthesis. Proc. Natl. Acad. Sci. U.S.A.

73:742-secretarialduties. 746.

12. Szybalski,W.1967.Effectsofelevatedtemperatures on LITERATURE CITED DNA andon somepolynucleotides:denaturation,

re-naturationand cleavage of glycosidic and phosphate 1. Bluthmann, H.,D.Bruck,L.Hubner,and A.Schoff- esterbonds, p. 73-122. In A. H. Rose (ed.),

Thermo-ski. 1973. Reassociation of nucleicacidsinsolutions biology. Academic Press Inc., London.

containingformamide. Biochem. Biophys. Res. Com- 13. Wolfson, J., D.Dressier, and M. Magazin. 1971. Bacte-mun.50:91-97. riophage T7 DNA replication: a linear replicating 2. Carter,B.J., F. J.Koczot,J.Garrison,R.Dolin,and J. intermediate. Proc. Natl. Acad. Sci. U.S.A.

69:499-A.Rose. 1973. Separatehelper functionsprovided by 504.