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0022-538X/79/11-0606/08$02.00/0

Electron Microscopic Analysis of Partially

Replicated

Bacteriophage T7 DNA

KATHYBAUMAN

BURCK,1

DOUGLAS G. SCRABA,2ANDROBERT C. MILLER,JR.`* Department ofMicrobiology, University ofBritishColumbia, Vancouver,BritishColumbia, Canada V6T

1W5,1andDepartment of Biochemistry, University of Alberta, Edmonton, Alberta,Canada2

Received forpublication30May1979

Partiallyreplicated bacteriophage T7 DNAwasisolated from Escherichia coli

infected with UV-irradiated T7 bacteriophage and was analyzed by electron

microscopy. The analysis determined thedistribution ofeyeforms and forks in

thepartially replicated molecules. Eyeformsand forks in unitlength molecules

werealigned with respect tothe left end of the T7 genome,and segmentswere

scored forreplicationin eachmolecule. Theresulting histogramshowed thatonly the left 25 to 30% of the moleculeswas replicated. Several different origins of

DNAreplicationwereusedtoinitiate replication in the UV-irradiatedmolecules.

The resultsareinexcellentagreementwith those of hybridization experiments in

which 32P-labeled progeny DNA from UV-irradiated phage was annealed with

orderedrestrictionfragmentsof T7 DNA(K.B.Burck and R. C. Miller, Jr.,Proc.

Natl. Acad. Sci. U.S.A. 75:6144-6148, 1978). Both analysessupportpartial-replica

hypotheses (N. A. Barricelli and A. H. Doermann, Virology 13:460-476, 1961;

Doermann etal.,J. Cell.Comp. Physiol.45[Suppl.]:51-74, 1955)as anexplanation

for the distribution ofmarkerrescue frequencies during cross-reactivation; i.e.,

replication proceeds in a bidirectional manner from an origin to a site of UV

damage, and those regions ofthe genome which replicate most efficiently are

rescued most efficiently by a coinfecting phage. In addition, photoreactivation

studies supportthe hypothesis thatthymine dimers are themajor UV damage

blockingcross-reactivation in therightend oftheT7genome.

Cross-reactivation refersto aprocesswhereby

genetic markersare rescued from a UV-irradi-ated, wild-type phage by a coinfecting mutant phage.Theprobabilityofaspecific markerbeing rescuedduringT4orT7phage infection depends

onthe mappositionof the marker(3,23).During

T7 infection, onlymarkers toward the left end

of the molecules are rescued efficiently.

Evi-dence has been presentedwhichindicates that

thosemarkerswhicharerescuedefficiently are

markerswhich replicate efficiently (3);i.e., 32P_ labeledprogenyDNAsynthesized after infection

by a UV-irradiated T7+ phage hybridizes

pre-dominantly with restriction fragments of T7+

DNAknowntocarrymarkers rescued efficiently

duringcross-reactivation. The effect is dose de-pendent:thehigher the dose of UV irradiation, the fewer the markers which are rescued effi-cientlyand the smaller thearea of the genome

whichreplicatesefficiently.

One idea which correlates the efficiency of

replicationwith theefficiency of marker rescue

is thepartial-replicahypothesis (1, 6). This

the-ory postulates that replication of a

UV-irradi-ated genomestartsfromaspecific origin(s) and

proceedsin abidirectional manner to UV lesions

which block further

replication.

Subsequently,

those regions of the genome which have

repli-cated most efficiently are rescued most effi-ciently byacoinfecting phage.Thehybridization data mentioned above are in agreement with thishypothesis, thedata

clearly indicating

the

presenceof

partial

replicas

of UV-irradiated

ge-nomesafter infection.

This report presents the results ofanelectron

microscopicanalysis ofpartially replicated, UV-irradiated T7+ DNA. It describes the distribu-tion of replicated regions,

growing forks,

and

bubbles inreplicating, UV-irradiated T7+ DNA

isolated from infected cells. Inaddition, a

pho-toreactivation experiment is

reported

here

which supports the

hypothesis

that

thymine

di-mers normally block cross-reactivation in the

right end of theT7map. All of the results agree

with and extend our previous conclusions and

strongly supportthe

partial-replica hypothesis.

MATERIALS AND METHODS

Bacterial and phage strains. Escherichia coli B23 (sup°) wasused asthe nonpermissive host for

cross-reactivation experiments. After adaptation for

growth in density medium (see below), B23wasused asthe host for theisolationofpartially replicated T7+ 606

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DNAfor electronmicroscopy.E.coli011'(supE)was usedasthepermissive host during cross-reactivation experiments.

All phagestrainsoriginallywereprovided byF.W. Studier.OurT7+(wild-type)strain was determined to befree of deletionsandlike theoriginal Studierstrain byrestriction endonuclease(HpaIandMboI)analysis (12).Namxis the general designation for amber mu-tants,where N is thegenenumber, andxis thespecific mutant.Themutantsused in thisstudywere:lam193, lam323, lam342a, 2am64, 3am29, 4am208, 5am28, 6am233, 8amll, 11am37, 14am140, 16am194, 17am290,andl9amlO(20).

Chemicals and isotopes. Thymidine was pur-chasedfromWorthington BiochemicalsCorp. Uracil, 5-fluorodeoxyuridine, and cytochromec(type V)were fromSigma Chemical Co. The density isotopes 2H20, '5NH4Cl, and deuterated algal whole hydrolysatewere from Merck Sharp & Dohme. [methyl-3H]thymidine and32PasH332PO4werepurchased fromNew England Nuclear Corp. Formamidewasfrom Matheson, Cole-manandBell andwasdeionizedbeforeuseby treat-mentwith Bio-Rad AG 501-X8 mixed-bed resin.

Media and buffers. Density medium contained thefollowing (per liter of2H20):7.0gofNa2HPO4,3.0 gofKH2PO4,1.0gof'5NH4Cl,0.5gofNaCl, and0.03 ml of0.1 M FeCli. After autoclaving, the following wereadded:1.0 ml of1MMgSO4,0.1 ml of1 MCaCl2, 12.5mlof 20% glucosein2H20,and1.12mlof deuter-atedalgal wholehydrolysate (22).

Tris-NaCl-EDTA buffer (TNE) contained0.01 M Tris-hydrochloride,0.15MNaCl,and 0.015 M EDTA (pH 7.4). Tris-EDTA buffer contained 0.5 M Tris-hydrochlorideand 0.05 MEDTA(pH 7.0). Lysis buffer contained 0.1 M NaCl,0.02 MEDTA, 0.01M KCN, 0.01 Miodoacetate, and0.1 MTris (pH7.4) (22).T7 Trissaltwas1MNaCl and0.05MTris-hydrochloride

(pH7.4).

UVirradiation ofphage.T7+ phagewerediluted

to10"bacteriophageperml in T7 Tris salt andplaced

inaplasticpetridishonice. Irradiationwasfor 20sat

adistance of 30cmfromaGeneral Electric G14T8

15-Wgermicidal lamp. At thisdose, T7 phage received approximately 7.3 lethal hits per particle. Lethal eventswerequantitated by plotting asurvivalcurve of theirradiatedphage.

Cross-reactivation and photoreactivation. Cross-reactivation experiments were performed as

previously described(3).Whenphotoreactivationwas

desired, cross-reactivation wasperformed under flu-orescent-light illumination, and infectivecenterswere incubated beneath General Electric F40 fluorescent lights for15h atroomtemperature.

Electron microscopy. Selected fractions from CsClgradientsweredialyzed overnight (4°C) against 0.1 M Tris-10 mM EDTA (pH 7.0) before being mountedfor electronmicroscopicexaminationbythe 40%-10%formamide spreadingprocedure of Daviset

al. (4). Partially replicated molecules were photo-graphedat amagnificationofx44,000withaPhillips EM300 electron microscope operated at 60 kV and with a 30-nm-objective aperture. Molecular lengths were determined withamapmeasuringdevice from tracingsofprints enlargedthree times. All

measure-ments werenormalizedtounitlengthT7 DNA(found

tobe12.0+0.5

[Lm

intheseexperiments).

Isolation of partially replicated molecules after infection of E. coli B23 byUV-irradiated T7+ phage.E.coli B23wasadapted for growth in the densitymedium by serialpassage through 20, 40, 60, 75, 90, 95, and 100% substituted medium (22). The adaptedcells hadageneration timeof 75 to 80minat 37°C. Non-irradiated T7+ phage followed a normal single-step growthcurve at30°C in the density-labeled cells, giving a burst of120phage percell by 45min after infection. A 50-ml culture ofdensity-labeled E. coli B23wasgrowntoapproximately3x108cellsper ml at37°C inheavy mediumcontaining5

jig

of thy-midineperml,5, gof5-fluorodeoxyuridineperml,25 ,ugofuracilperml, and500,uCiof [methyl-'H]thymi-dine and then shiftedto30°C for15min. This proce-dure allowed labeling of the bacterial cells for HH reference(both strandslabeled)inCsClgradients. The cells thenwereinfectedat amultiplicity of infection of2 with UV-irradiated, 32P-labeled LL (no density label in either strand) T7+ containing7.3lethalevents pergenome.The32plabelwas at aspecific activity of 2mCi/mg, which leads tothe incorporation ofa32p atomin two-fifths of the phageparticles. The multi-plicity of infectionwasdeterminedbymonitoring sur-viving bacteria. Since noattempt was made in this experimentto overcome superinfection exclusion(2), a calculated multiplicity of infection of 2 does not necessarilymeanthatanycellsreceived injectedDNA frommorethan onephage. Infectivecentersand back-ground phage alsoweremonitored. UV-irradiated T7 phage are unable to conduct a productive infection: infective centers aretypically only0.1 to0.2% of the control bacteria. Non-irradiated T7+ produceinfective centers on 90% of the control bacteria. At 25

nmin

postinfection, the culturewaschilled by pipetting it into2volumes ofice-cold TNEplus1volume oflysis buffer. The infectedcellsweresedimented and resus-pendedataconcentrationof1.5x 109 bacteriaperml inlysis buffer. Cells then were lysed with lysozyme (400,ug/ml,0°C,45min)followed by detergent (0.1% sodiumlauryl sarcosinate, 65°C,20min). Thelysate wasdeproteinized bytreatmentwithself-digested pro-nase(1 mg/ml,37°C,12h).Samplesof thelysatewere

mixed1:4withsaturated CsCl(in distilled water) and centrifuged inanSB283rotorinaB60 International centrifuge at 30,000 rpm for 72 h at 12°C. Approxi-mately70fractionswerecollectedfromthe bottom of the tube and assayed for;'H (HH E. coliDNA) and 32p(T7DNA).Fractionsbandingontheheavy side of the T7 (P2P) peakwere pooled and centrifuged in a

Beckmantype 65rotor at32,000rpmfor72h at12°C. Specific fractions from the second gradient were

pooledfor electronmicroscopy.Beforecentrifugation,

thepolyallomertubesweretreated for1hwith 10%

bovineserumalbumin.

Othermethods.Preparationof'32P-labeledT7 bac-teriophage and determination of label uptake into acid-insolublematerialhave beendescribedpreviously (13).

RESULTS

Isolation ofpartially replicated T7+DNA.

Partially replicated T7 DNA molecules were

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608 BURCK, SCRABA, AND MILLER

isolated from density-labeled E. coli infected

with UV-irradiated phage as described above.

The DNA was sedimented to equilibrium in

CsCl density gradients, and the distribution of

radioactivity wasdetermined.Asexpected,T7+

irradiated to 7.3 phage lethal events did not

undergo even one round of replication as no

parental DNA banded at the hybrid location.

Material banding on the heavy side of the T7 DNA peak was pooled and resedimented in a second CsCl gradient (Fig. 1).The fractions of thisgradientindicated bybrackets werepooled andanalyzed byelectronmicroscopy.Thisfigure

shows that the T7 DNA was wellseparated from

contaminatingE. coliDNA.

Electron microscopy of partially repli-catedT7+ DNA. Partially replicated, UV-irra-diatedT7+DNAisolated from aCsCl

gradient

wasexamined by electron microscopy. Four cat-egories of partially replicated molecules ofT7

size DNA were distinguished: (i) eyeforms, (ii)

moleculescontaining forks withtwo

equal

arms, (iii) mrolecules containing both eye forms and forks, and (iv) molecules containing forkswith arms of unequal

length,

where two of the branches together were of T7 length; class iv moleculeswereassumed to be eye formsbroken because of the

fragility

of

single-strand

regions

atthefork.

Examples of severalpartially replicated mol-eculesareshown inFig.2.Figure2ashows a T7 length molecule with an internal eye form of

13.7%. Internally replicated regions spanning 1

to25% of unit T7lengthwerefound. Figure2b

is amolecule with alarge equal-armedfork. In additiontolarge forks spanningup to38% ofthe molecule, small forks occurringat oneend were found (Fig. 2c). A small number ofmolecules containingboth a fork at one end and an inter-nally duplicatedregion at the same end (Fig. 2d) were observed. Sixty-four partially replicated molecules were photographed and measured. Only molecules measuring 12.0 ± 0.5 ym (T7 unitlength underourcondition) wereincluded inthe analysis. Moleculeswerenormalizedto a

scale of 100 units and aligned such that all

partiallyreplicated regions occurred in thesame end (Fig. 3). We feel that it is reasonable to

assumethat theseregionsareall located at one

end,thegeneticleft end of themolecule,for the following reasons. (i) Progeny DNA produced

byinfection with UV-irradiated T7+ phage

con-taining six to seven phage lethal events per

genome hybridized to restriction fragmentsfrom

the left portion of the T7+ molecule butnot to

fragments from the right side (3). (ii) Wolfson et

al. (22) and Dressler et al. (7) examinedpartial

denaturation maps ofpartially replicated

non-irradiated T7+ DNA and found that Y and eye

I=1

Z

L!

C-Cl,

FRACTION NUMBER

FIG. 1. CsCldensity gradient analysis ofpartially replicated T7DNA. E. coli weregrown in density-labeled medium andinfectedwithUV-irradiated T7 asdescribed in thetext.Intracellular DNA was ex-tracted and banded inaCsCldensity gradient.DNA displacedfrom theunreplicated, parental T7 DNA toward the heavy location of the gradient was iso-lated and rebanded inasecondgradient. This figure shows the distribution of the DNA in the second gradient. The material in thefractions from the sec-ondgradient indicated by brackets waspooledfor electron microscopy. Symbols: (L-F) 3H counts

perminutex10-3(E.coliDNA);(- -0) 32pcounts perminutex 10-3(T7DNA).

forms werelocatedontheleftside of the

mole-cule.They mappedanorigin ofT7 DNA

repli-cation at a lorepli-cation17%from theleftend of the

T7 genome.(iii)Fourmoleculescontaining both

afork andaneyeformwereobserved,and these regionswerebothlocatedinthesameend of the molecule (ii) Nomoleculescontainingpartially replicated regionsatboth ends were seen.

It is evident from the distributionof eyeforms overtheleft 20% ofthemolecule thatno single origin of replication was utilized (Fig. 3). If

rep-lication isassumed to proceed

bidirectionally

at

the sameratefrom the originofreplicationof a

molecule,the distribution oforiginsamong our

eye forms (including unequal forks and eyes in

multiple structures) is givenin Table 1. In ad-dition,there areeightmolecules withforks

span-ning 0 to 5% of the molecule and an additional

sixspanning0 to10%ofthegenome.Therefore,

initiation can proceed from apoint at or near

theleft endofaUV-irradiated T7 DNA

mole-cule,aswellasfrom severallocationsalongthe left 30%of the genome.

The summed total ofpartially replicated

re-J. VIROL.

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a

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

b~~~~~~~~~~~~~~~~~~l

Q~~~~~~1 ~~

2~~~~~~~~~~~~~~~~~~~~~~~~~~~~ir~~~~~~~~~~~~~~~~7

-n SN

~~~~~~~~~~~~~~~C

J~ ~ ~ ~ --. .

7L-&.1V;Z0

Wf.. ~~~~.~~

FIG2Prtallreliate T7DN moecues(a)T7lenth olcul cotanin aney fom () oleul

cnanng

Sagk rnhTesmo h ogbac lseihro h hr rnhsi 7ui egh(2

+5im()7 egt mleul cnaiin ashrtbanh d)T7legh olcue onanig ot ashr

branchandaneyeform ~ ~ ~ ~ ~ Z~:-cal

gions observedin our molecules is given by the

histogram in Fig. 4; 75% of the partially

repli-cated areaslietothe left of 17%, whereasonly

25% are to the right of that point. This curve closely resemblesthat obtained for marker res-cue experiments, using T7+ irradiated with a similar dose (3) (Fig. 4). The similarity ofthe

twocurvesimpliesthatpartial replicationof the

left end of a UV-irradiated T7+ genome can

account for the distribution of marker rescue

efficiency andsupportsourestimationsof

partial

replication as determined by DNA hybridiza-tion.

Effect of

photoreactivation

on marker

rescue.The formation of pyrimidine dimers is

animportanttypeof UV radiation-induced

dam-agetoDNA (16,17,19). Several lines of evidence

have established that pyrimidine dimers are

blockstoDNAsynthesis(9, 11, 15). Illumination

ofUV-irradiated bacteria with photoreactiving

light resultsin the removal ofthymine dimers,

theresumption ofblocked DNAsynthesis, and the ultimate recovery of the UV-irradiated cells

(15, 16, 18). Our interpretation of

cross-reacti-vation experiments with bacteriophage T7 is

thatthyminedimerstotherightof theoriginof

T7DNAreplicationblock furtherreplicationto the right, thereby inhibiting marker rescue in therightend of theT7genome. The distribution ofpartially replicated regionsdescribed in this

paper and previously (3), therefore, reflect the

chance thatthymine dimers interrupt the

prog-ressof the replicationfork toward theright end

of the T7 DNA molecule. Consequently, one

should detect a large difference in the marker

rescue

patterns

resultingfrom cross-reactivation

experimentsperformedindimlightversusthose performed in photoreactivating light. Notonly

should the absolute level ofwild-type infective

centers increase, but also the overall pattern

should broaden toward the

right

end of the

genetic map as

thymine

dimers are removed, thus allowing replication to proceed further to

theright.

To test the hypothesis outlined above, we

coinfected E. coliwithUV-irradiatedT7+ phage

and one ofa series of T7 amber mutants. The

infectivecenters weredividedintotwo

portions;

onepartwashandled

continuously

in dimorno

light, andonepartwasilluminated withGeneral

Electric F40 fluorescent lights. The results of

theexperimentareshown inFig.5.As

expected,

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610 BURCK, SCRABA, AND MILLER

-0

n

- e

r-o

0 50 100

=C

0 50 100

PERCENT LENGTH T7

FIG. 3. Linediagramsofpartially replicatedT7 molecules.Partially replicatedmoleculesweremeasured

andnormalizedtounitlength.Allofthe branchesofthe molecules indicated in thesedrawingsweredouble stranded, butwedidnot undertakea comprehensiveanalysis ofvery smallsingle-strandedregions (a few hundrednucleotides),possibleatforks, forexample.

TABLE 1. Distributionof eye form centers"

Segment of T7DNA(% No. ofeyemidpoints

length)

0-5 6

5-10 9

10-15 10

15-20 9

>20 7

The midpoints of the eye forms represented by

thelinedrawingsofFig.3weremeasured.This table

lists the number of timesan eyeform midpointfalls within a particularsegment of T7 DNA among the moleculesdiagrammedinFig.3.

removing thymine dimers from the DNA by photoreactivation

changed

the overall pattern of cross-reactivation;

regions

toward the

right

end

ofthe mapwererescued witha greaterrelative

efficiency. Theabsolute levels of markerrescue

increasedslightlyfor allportionsofthegenome

afterphotoreactivation,but thedramaticchange

occurredin the pattern of markerrescuetoward

the right end of the map. The results of this

experimentwereconsistent withourhypothesis thatthymine dimers

normally

blockreplication

to the right. Asthymine dimers were removed,

replication proceeded to the right and more

markerrescue occurredtotheright.

DISCUSSION

In thispaper,wepresentdirect visual evidence

for theexistence ofpartial replicas of UV-irra-,

40-* I

0

20

0

'a-E zo

50 Percent T7Length

e -100 3

-50 a

e0

-v-o XL1 100

FIG. 4. Histogram of partially replicated regions from UV-irradiated T7DNA. Lengths of T7+ DNA in 1% incrementswerescoredfor replication as rep-resented in the linedrawings ofFig.3.The histogram shows the number oftimesaparticular segment of DNA was replicated in thepool of molecules dia-grammed inFig.3.The open circles represent marker rescuefrequencies normalized tothemaximum res-cueand distributedalongthegeneticmap of T7 as a functionofapercentlength of genome (3).

diated T7+ DNA. Partial replicas of

bacterio-phage DNA originally were postulated on

ge-netic grounds to account for the patterns of

cross-reactivation and multiplicity reactivation

observedwith bacteriophage T4 (1, 6). Several

linesof evidenceareconsistentwith the

hypoth-esis.In mixedinfectionby UV-irradiated T4D+

phageand differentmembers ofa setofdefined

T4Dmutants, theabilityofaparticular mutant

to rescue the damaged T4D+ genome was

de-pendentonthe mapposition of the marker (23).

Four distinctpeakswereobtained in this

exper-io

0 100

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and the

ability

ofa particular segment of the

genome tobe

efficiently

rescued.

Several features of bacteriophage T7 have facilitated an examination of the relationships

amongmarkerrescue,partialreplicationof

UV-irradiatedDNA,andorigins ofDNAreplication. An origin of replication was mapped for T7+ DNA by electron microscopic analysis of

par-tially replicated

(non-irradiated) T7+ molecules

(7). Restriction fragments of the T7 genome

0.3

1

C

13

1

5 8 I

14

1 7 werealigned withspecificregions of the genetic

0.7

1lb

2 4 6 11 16 19 map (12). We showed that partialreplicasof T7

DNAareproduced inE.coli infected with UV-MAPPOSITION

irradiated bacteriophage

by hybridizing

32P-la-beled progeny DNA from such an infection to ordered T7 restriction fragments. We showed further that the segments ofthe UV-irradiated

I I l l | T7 DNA which

replicated

efficiently

werethose

0 20 40 60 80 10 which rescued efficiently during

cross-reactiva-tion

experiments

(3). Partial

replicas

of

UV-ir-PERCENT LENGTHOFGENOME radiated DNA were thought to result from the

i. Effect of photoreactivationon marker res- blockage of

replication

at UV-damaged regions.

oli

011'(supE)wasgrownto 3x

108

bacteria

Replication originally

was thought to be

initi-in H-broth at 300C. Chloramphenicol was ated 17% from the left end of the irradiated T7 ct100pg/mlto the culture toinhibitsuperin- genome and to proceed bidirectionally at the exclusion (2).At 1-minintervals, samples of samerate tothe ends of the molecule ortothe

s were coinfected with UV-irradiated T7' est sites

tof

edamage.

UV

to

the

^eceiving10.1phagelethalevents)andoneof nearest sites of UV damage. On this basis,

ofT7amberphage. Themultiplicity of infec- markers around 17% on the genome should be achtypewasclosetofivephageperbacterium rescued most

efficiently. However,

wefound that monitoredby platingthesurviving bacteria. at

higher

radiation

doses,

markerstothe left of ng bacteria were monitored inparallelcul- 17% were rescued with consistently greater effi-fected with UV-irradiated T7+ alone and ciency than were markers to the right of 17% (3). mutants alone.)At10minafterinfection,a Analysis of the progeny DNA produced by UV-gftheinfectedcells wasdiluted into T7 anti- irradiated

T7+

parental phage

indicated that ncubatedfor5minat37°C,diluted further,

irradite

of

parenta

th

at

tedfor infectivecenters on E. coliB23(sup). segments of the genome to the left of 17% also

-center production is not significantly influ- were

replicated with

greater efficiency than were

yincubation inchloramphenicol forupto 30 those to the right. However, the location and osequential experiments usingthesameUV- size of the restriction

fragments

used in the edphagewereperformed.Onewasperformed

analysis

weresuch thatafine-structuremapping 'ark, andplateswereincubated in the dark. of the partial

replicas

was not possible. The erwasperformedwith the room(fluorescent) electron microscopic data presented here show

z,

andplateswereincubatedfor15 h under that more DNA to the left of 17% is replicated

entlamps.Allofthe amberphage represented than to the right (Fig.

4),

as expected from

curve wereexamined inasingle experiment. markerrescuedata.

ndIcstand

for

amberslamI93, lam323,and One limitation of the

previous

studiesonthe x, respectively. Symbols:

(O---O-)

no photo- Oelaino h rvossuiso h

tion;

(e-

)

with photoreactivation.

extent of partial replication (3)wasthe inability

todiscriminatebetween DNA

synthesis

due to

anumber consistent with thenumberof

replication

and that due to

repair.

32P-labeled

thought

tooperate

during

T4

replication

deoxynucleoside triphosphates

would have been

Rayssiguier

and

Vigier (14) analyzed

the

incorporated

into UV-irradiated DNA

by

both

inant clone size distribution ofprogeny processes.The

incorporation

dueto

repair

syn-produced

by multiplicity

reactivation of thesis

certainly

would have beenmorerandom

Ldiated

genetically

marked

parental

and,

consequently,

would have contributed to

Their

analysis

was consistent with the radioactive DNA

annealing

along

the whole of

at

partial replicas

of the

damaged

ge- the T7 genome. In

fact,

estimates of theextent

reassociate

by

recombining primarily

at of

replication

indicated that

portions

of the

right

atremities.

These

experiments suggested

end of the UV-irradiated molecules

might

be Lationbetween

origins

of DNA

replication

labeledto a greaterextent than

they

were

res-w

n

uC

w

LU

1.0

-IC

4

FIG. 5

cue.E. c per ml, addeda fection e the cell phage (r aseries tionofec andwas (Survivi? tures in, with T7 samplec

serum, ij andplat Infective enced

by

min. Tw irradiat in the d The otht lightsor fluoresc oneach la, Ib,a lam342c reactiva,

iment,;

origins

(5, 10). recomb phage I UV-irra phage.

idea th

nomes: their es

acorrel

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(7)

612 BURCK, SCRABA, AND MILLER

cued during cross-reactivation studies. This

could have been due to the presence of some repair synthesis. The direct visualization of par-tially replicated regions in an electron

micro-scope allows one to score forreplicatedregions,

as opposed toregions ofrepair synthesis. The excellent correlation between the histogram of replicated regionsand the distribution of marker

rescue frequencies supports very well our

pre-viousconclusions (Fig.4andreference 3).

One implicationofourpreviousresults is that the replication of UV-irradiated T7+ DNA is

initiated at anoriginororiginstothe leftof 17%.

Atthe sametime,additionalinterpretationsalso

are possible. Since the primary mechanism of UVradiation damagetoDNAisthe formation ofpyrimidine,andespecially thymidine, dimers

(16, 17, 19),regions of theDNArichin

adenine-thymine basepairswould beexpectedtosustain more lethal damage than would regions where guanine-cytosine base pairs predominate. Ex-amination ofthepartialdenaturation map of T7 (8) shows thatadenine-thymine-rich regionsare not uniformly distributed along the T7 mole-cules. The region from -10 to 30% containing thepresumedDNAreplication originsiteat17%

is very rich in adenine-thymine base pairs,

whereas the region from around 5 to 10% is

practically devoidof sitessusceptible topartial denaturation. With this consideration, the

marker rescue andhybridization datacould be

consistentwithbidirectionalreplication froman origin located around 17%from theleftenduntil asiteofUVdamage isreached.Suchsiteswould

bemore

probable

totheright thantothe left of

the origin. However, analysis of partially repli-cated T7+ molecules with an electron

micro-scope supports the conclusion that

UV-irradi-ated T7moleculesareable to initiatereplication

at a number of siteswithin the leftend ofthe

molecule (Fig. 3), mostof whichare locatedto

the left of 17% (Table 1). The timing of the

experiment allowed replication to proceed as far

as possible, so one cannot say exactly where

alongan eyereplication started since replication

stops at a UV lesion on either side of the

initia-tionsite; that is, replicationdidnotnecessarily

initiate at the center of the eye. Unirradiated

molecules may not behave inthe same way; i.e.,

itisentirely possible that normal molecules

ini-tiateprimarily at the 17% origin, but that

UV-irradiatedmolecules utilizesecondary sites along

the left end when damage is sustained in the

17%region.

An examination ofthe distribution of

repli-cated regions reveals that UV-irradiated

mole-cules arereplicated primarily to the left of 17%

(Fig. 4), since 75% of the

partially

replicated

regions observed were to the left of 17% whereas

only 25% were to theright. Itis not surprising

that most of thepartially replicated regionslie

to the left of 17%since, on a probability basis,

UVdamagewilloccur to agreater extent in the

adenine-thymine-rich region around andtothe

right of 17%. The photoreactivation data

pre-sented here (Fig. 5) support this view. As thy-mine dimers are removed from UV-irradiated

T7 DNA,thepatternof markers rescued

broad-ens toward the right end ofthe genome; when photoreactivation occurs, markers to the right

of 17% arerescuedwith ahigher efficiency. This

isconsistent with ourhypothesisthatthymine dimersnormally block replication to theright, thusinhibiting marker rescue in this direction. Inconclusion,the electronmicrographic anal-ysis confirms and extends ourprevious results

(3). There is an excellent correlation between

the areas of the genome which replicate

effi-ciently and those which are rescued efficiently during cross-reactivation. Furthermore, origins other than thatat 17%areusedtoreplicate UV-irradiatedDNA.Onthe other hand, the marker

rescue patterns obtained during

cross-reactiva-tion experiments probably best represent the

final extent ofpartialreplication rather than the

exactlocation oforiginsof DNAreplication.

ACKNOWLEDGMENTS

Thisresearchwassupported bygrants from theNational Research Council and the Medical Research Councilof Can-ada. K.B.B. wassupported byfellowships from the Killam Foundation and the Medical Research CouncilofCanada.

We thank W.F.Studierforthebacteriophage usedinthis study, A. H. Doermann and C. C. Richardson for helpful

suggestions, andRogerBradley,DeborahTaylor,and Helen Smith for technical assistance.

LITERATURE CITED

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Superinfection exclusion and lack of conservative

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4. Davis,R.W.,M.Simon,and N. Davidson. 1971. Elec-tron microscope heteroduplex methods for mapping

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8. Gomez,B., and D. Lang. 1972. Denaturation mapof bacteriophage T7 DNA and direction of DNA transcrip-tion. J. Mol. Biol. 70:239-251.

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formbyextractsofEscherichia coli. Mol. Gen. Genet. 149:335-345.

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Figure

FIG.1.perpergradient.replicateddisplacedondelectronshowsastractedlabeledlatedtoward described CsCl density gradient analysis ofpartially T7 DNA
FIG. 3.stranded,hundredand Line diagrams ofpartially replicated T7 molecules. Partially replicated molecules were measured normalized to unit length
FIG.onandencedandwithaddedationcue.(Survivi?irradiatinlights in a single photo- experiment.la,the lamI93, lam323,lam342cThe series the each (O---O-) no photoreactivation

References

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