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 V6T1W5,1andDepartment 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
thepresenceof
partial
replicas
of UV-irradiatedge-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,
andbubbles inreplicating, UV-irradiated T7+ DNA
isolated from infected cells. Inaddition, a
pho-toreactivation experiment is
reported
herewhich supports the
hypothesis
thatthymine
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 25nmin
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 thelysateweremixed1:4withsaturated 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 ofT7size 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 unequallength,
where two of the branches together were of T7 length; class iv moleculeswereassumed to be eye formsbroken because of thefragility
ofsingle-strand
regionsatthefork.
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
atthe 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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[image:3.507.267.461.60.287.2]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 markerrescue.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-reactivationexperimentsperformedindimlightversusthose 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 thegenetic map as
thymine
dimers are removed, thus allowing replication to proceed further totheright.
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 dimornolight, 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 theright
endofthe 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
blockreplicationto 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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[image:5.507.121.401.72.280.2] [image:5.507.263.461.334.425.2]and the
ability
ofa particular segment of thegenome 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
C13
1
5 8 I14
1 7 werealigned withspecificregions of the genetic0.7
1lb
2 4 6 11 16 19 map (12). We showed that partialreplicasof T7DNAareproduced 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-irradiatedI I l l | T7 DNA which
replicated
efficiently
werethose0 20 40 60 80 10 which rescued efficiently during
cross-reactiva-tion
experiments
(3). Partialreplicas
ofUV-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 3x108
bacteriaReplication originally
was thought to beiniti-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.
UVto
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. athigher
radiationdoses,
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- irradiatedT7+
parental phage
indicated that ncubatedfor5minat37°C,diluted further,irradite
ofparenta
that
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 wereyincubation inchloramphenicol forupto 30 those to the right. However, the location and osequential experiments usingthesameUV- size of the restriction
fragments
used in the edphagewereperformed.Onewasperformedanalysis
weresuch thatafine-structuremapping 'ark, andplateswereincubated in the dark. of the partialreplicas
was not possible. The erwasperformedwith the room(fluorescent) electron microscopic data presented here showz,
andplateswereincubatedfor15 h under that more DNA to the left of 17% is replicatedentlamps.Allofthe amberphage represented than to the right (Fig.
4),
as expected fromcurve wereexamined inasingle experiment. markerrescuedata.
ndIcstand
for
amberslamI93, lam323,and One limitation of theprevious
studiesonthe x, respectively. Symbols:(O---O-)
no photo- Oelaino h rvossuiso htion;
(e-
)with photoreactivation.
extent of partial replication (3)wasthe inabilitytodiscriminatebetween DNA
synthesis
due toanumber consistent with thenumberof
replication
and that due torepair.
32P-labeled
thought
tooperateduring
T4replication
deoxynucleoside triphosphates
would have beenRayssiguier
andVigier (14) analyzed
theincorporated
into UV-irradiated DNAby
bothinant clone size distribution ofprogeny processes.The
incorporation
duetorepair
syn-produced
by multiplicity
reactivation of thesiscertainly
would have beenmorerandomLdiated
genetically
markedparental
and,
consequently,
would have contributed toTheir
analysis
was consistent with the radioactive DNAannealing
along
the whole ofat
partial replicas
of thedamaged
ge- the T7 genome. Infact,
estimates of theextentreassociate
by
recombining primarily
at ofreplication
indicated thatportions
of theright
atremities.
Theseexperiments suggested
end of the UV-irradiated moleculesmight
be Lationbetweenorigins
of DNAreplication
labeledto a greaterextent thanthey
wereres-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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[image:6.507.52.247.62.279.2]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 ofthe 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
replicatedregions 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
1. Barricelli, N. A., and A.H.Doermann.1961. An
ana-lytical approachtotheproblems of phage recombina-tionandreproduction.III.Cross-reactivation.Virology 13:460-476.
2. Benbasat, J., K.B.Burck, andR.C.Miller,Jr. 1978.
Superinfection exclusion and lack of conservative
trans-fer ofbacteriophageT7 DNA.Virology87:164-171. 3. Burck,K.B., and R. C.Miller,Jr. 1978.Markerrescue
andpartial replicationofbacteriophageT7DNA. Proc. Natl. Acad. Sci. U.S.A.75:6144-6148.
4. Davis,R.W.,M.Simon,and N. Davidson. 1971. Elec-tron microscope heteroduplex methods for mapping
regions ofbase sequence homology in nucleic acids.
MethodsEnzymol.21:413-428.
5. Delius, H.,C.Howe,and A. W. Kozinski. 1971. Struc-ture of the replicatingDNA frombacteriophage T4. Proc. Natl. Acad. Sci.U.S.A.68:3049-3053.
6. Doermann,A.H.,M.Chase,and F. W.Stahl. 1955. Genetic recombination and replication in
bacterio-phage.J.Cell.Comp. Physiol. 45(Suppl.):51-74.
7.Dressler, D.,J.Wolfson,and M. Magazin.1972. Initi-ation and reinitiIniti-ation of DNA synthesis during replica-tion ofbacteriophageT7. Proc.Natl. Acad. Sci. U.S.A. 69:998-1002.
on November 10, 2019 by guest
http://jvi.asm.org/
8. Gomez,B., and D. Lang. 1972. Denaturation mapof bacteriophage T7 DNA and direction of DNA transcrip-tion. J. Mol. Biol. 70:239-251.
9. Hourcade, D., and D.Dressler.1978.Site-specific ini-tiation ofaDNAfragment. Proc. Natl. Acad. Sci. U.S.A.
75:1652-1656.
10.Howe, C. C.,P. J.Buckley,K.Carlson, andA. W. Kozinski. 1973. Multiple andspecificinitiationof T4 DNAreplication.J.Virol. 12:130-148.
11.Masamune, Y. 1976. Effectofultraviolet irradiation of bacteriophage Fl DNAonits conversiontoreplicative
formbyextractsofEscherichia coli. Mol. Gen. Genet. 149:335-345.
12.McDonnel,M.W., M. N.Simon, andF. W.Studier. 1977.Analysis of restriction fragments of T7 DNA and determination of molecularweights by electrophoresis in neutral and alkalinegels. J. Mol. Biol. 110:119-146. 13.Miller,R. C.Jr.,M.Lee,D. G.Scraba,and V. Paet-kau. 1976. The role ofbacteriophage T7 exonuclease (gene 6) in genetic recombination and production of concatemers.J. Mol. Biol. 101:223-234.
14. Rayssiguier, C., and P. R. R. Vigier. 1977. Genetic evidence for the existence of partial replicas of T4
genomes inactivated by irradiation under ultraviolet light. Virology 78:442-452.
15. Rupp, W., and P. Howard-Flanders. 1968.
Discontin-uitiesin the DNAsynthesizedinanexcision-defective
strainofEscherichia coli following ultraviolet irradia-tion.J. Mol. Biol. 31:291-304.
16. Setlow, J. K. 1966. The molecular basis of biological effects ofultraviolet radiation and photoreactivation. Curr.Top.Radiat.Res. 2:195-248.
17. Setlow, R. B. 1964. Physical changes and mutagenesis. J. Cell.Comp. Physiol.64(Suppl.1):51-68.
18. Setlow, R. B. 1968. The photochemistry, photobiology,
andrepair of polynucleotides. Prog. Nucleic Acid Res. Mol. Biol. 8:257-295.
19.Setlow, R. B., and W. L. Carrier. 1966. Pyrimidine
dimers inultraviolet-irradiated DNA. J. Mol.Biol.17:
237-254.
20. Studier, F. W. 1969. The genetics and physiology of bacteriophage T7. Virology 39:562-574.
21. Studier, F. W. 1973. Geneticanalysis ofnon-essential
bacteriophage T7genes.J.Mol. Biol.79:227-236. 22. Wolfson, J. D.,D.Dressler, and M. Magazin. 1972.
Bacteriophage T7 DNA replication:alinear replicating
intermediate. Proc. Natl. Acad. Sci.U.S.A. 69:499-504. 23. Womack, F. C. 1965. Cross-reactivation differences in
bacteriophage T4 D. Virology 26:758-761.