Effects of UV irradiation on the fate of 5-bromodeoxyuridine-substituted bacteriophage T4 DNA.

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0022-538X/83/070151-20$02.00/0

Effects of

UV

Irradiation

on

the Fate

of

5-Bromodeoxyuridine-Substituted

Bacteriophage

T4

DNA

LINDAL.RESTIFO, HELEN H. VOGELBACKER, THOMAS MADARA, SHUI-KUENLING, AND

ANDRZEJ W. KOZINSKI*

DepartmentofHumanGenetics, Universityof Pennsylvania Schoolof Medicine, Philadelphia, Pennsylvania 19104

Received 28 December1982/Accepted 12 April 1983

We have carried out a seriesof experiments designed to characterize the impact

of UV irradiation (260 nm)on5-bromodeoxyuridine-labeled (heavy)T4

bacterio-phage,both before and after infection of Escherichia coli.Inmany respects, these

effects differ greatly from those previously described for non-density-labeled

(light)phage. Moreover, our results have ledus to propose amodel foranovel

mechanism of host-mediated repairsynthesis,inwhich excision of UV-damaged

areasis followed by initiation ofreplication, stranddisplacement,and a

consider-ableamountofDNAreplication. UV irradiation of 5-bromodeoxyuridine-labeled

phage results insingle-stranded breaksin alinear, dose-dependentmanner(1.3to

1.5breaks per genomic strand per lethal hit). This damage does not interfere with

injection of the phagegenome,butsomeoftheUV-irradiated heavy phage DNA

undergoes additional intracellular breakdown(also dosedependent). However,a

minority (25%) of the injected parental DNA is protected, maintaining its

preinjection size. This protected moiety is associatedwith areplicative complex

ofDNAandproteins,and ismoreefficiently replicatedthanis theparentalDNA

not so associated. Mostofthe progeny DNA is alsofound with the replicative

complex. The5-bromodeoxyuridine of heavy phageDNAisdebrominatedbyUV

irradiation,resulting in uracil which is removed by host uracil glycosylase. Unlike

the simple gap-filling repair synthesis after infection with UV-irradiated light

phage, the repair replication of UV-irradiated heavy phage is extensive as

determined by density shift ofthe parental label in CsC1 gradients. The newly

synthesized segments are covalently attached to the parental fragments. The

repair replication takes placeevenin the presenceofchloramphenicol, aprotein

synthesisinhibitor, suggesting it is host mediated. Furthermore, theextentofthe

repair replication isgreater athigherdosesofUVirradiationappliedtotheheavy

phage. This abundant synthesis results ultimately in dispersion of the parental

sequencesas shortstretches inthemidstoflongsegmentsofnewly synthesized

progeny DNA. Together, the extensivereplication and the resultingdistribution

pattern of parental sequences,withoutsignificantsolubilization ofparental label,

are mostconsistent withamodelofrepair synthesisin whichthe leadingstrand

displaces, rather thanligates to, theencountered5' end.

Since thispaperdescribes theeventsensuing

from UV irradiation (260 nm) of

5-bromodeox-yuridine (BUdR)-substituted (heavy) T4

bacte-riophage, it will be useful to review the structur-al and functional consequences of irradiating

non-density-labeled (light) phage. Studies from

thislaboratoryhave shown (13) that UV

irradia-tionoflight phagecauses, in addition tothymine

dimerformation, cross-linking ofthe DNA,

ap-proximately0.1 cross-linkper genome per lethal

hit. Note, however, that before infection no

single- or double-stranded breakscan be found

in the irradiated phage DNA (13). Upon

infec-tion with UV-irradiatedlight phage,theparental

DNAacquires approximatelyone

single-strand-ed break per lethal hit (16). These breaks are

believed to be introduced adjacent to the

thy-mine dimersby thephage-coded v gene product

sincesingle-strandedcutting isnotobserved in v

mutants(13).

The nicking ofthe light DNA isfollowed by

excision ofthe damage and repair synthesis to

fill in the gap, resulting in a majority ofunit

length molecules as evidenced by alkaline

su-crose gradient analysis (13). Of importance to the data we will present below is the fact that these gaps must be shortsincetherepair

synthe-sis,when carried out indensity-labeled bacteria, 151

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152 RESTIFO ET AL.

causes nodensity shiftof parental label in CsCl gradients (21; E. Shahn, Ph.D. thesis, Universi-ty ofPennsylvania, Philadelphia, 1965).

Thegap-filling repair replication of

UV-irradi-ated light phage occurs even in thepresence of

chloramphenicol, suggesting arole of

host-cod-ed functions. In particular, it requires DNA

polymeraseI since host strains defective in this

enzyme show poor repairof UV-induced dam-age to light phage DNA (2). Upon removal of

chloramphenicol from the medium and further

incubation, the repaired DNAis highly

degrad-ed, probably byphage-codedenzymes

recogniz-ing the unmodified cytosine residues inserted

duringhost-mediated repair(2).

Inhostcellsmultiplyinfectedwith

UV-irradi-ated light phage, complete phage genomes can

bereconstituted,resulting inaproductive

infec-tion. We have shown that this phenomenon,

multiplicity reactivation, occurs by repeated

partialreplication of undamaged areasfollowed

by recombination ofthose partial replicas (21)

and not by interparental recombination as had

previously been supposed. The level of DNA

synthesis underthese conditionsisgreater than

canbeaccounted forbymultiplicityreactivation

alone. Theparentalcontributiontothe progeny

DNA molecule was shown tobein the form of

short, semi-conservatively replicated subunits,

covalently linkedtothe progeny DNA(21).The

lengthof these parental subunits became smaller

with increasingdoses ofUVirradiation(21; E.

Shahn, Ph.D. thesis, University of

Pennsylva-nia,Philadelphia, 1965).Therefore,the presence

oftheparentalDNAistheprogenyphageisnot

due to the scavenging of thedamaged parental

nucleotides or to the ligation of conservative

(unreplicated) double-strandedparentalsubunits

totheprogeny DNA.

Thispaper is the secondinaseriesexamining

the consequencesofUVirradiationonheavyT4

bacteriophage.Inanearlierpaper(15),we

docu-mented a number ofproperties ofthe progeny

DNA synthesized after infection with such

phage. Inparticular, it was shown (15) that the

gene representation of the progeny DNA was

biased in favor ofthe genetic areas containing

the origins of DNA replication (3), reflecting

amplification of these areas. Over the course of

the infection, the size of such progeny DNA

increased while the genetic bias decreased,

probably reflectingrecombination among

small-er progeny segments. CsCl gradient analysis

indicatedmostof thenewlysynthesizedprogeny

DNA was not covalentlyjoined to the heavy

parentalDNA (15).

Theexperimentsdescribed belowfocus

atten-tion on the fate of theheavy parentalDNAafter

UV irradiation and subsequent infection. We will first document the damagesinflicted directly on the irradiated phage DNA. Our data will

further show that the events after infection are the resultof a competition between two oppos-ing forces which either protect against or initiate furtherbreakdown of the already damaged irra-diated DNA. Thisprotection appears to allow an extensive repair replication which is host medi-ated. It results in a scattering of very short segments of parental sequences among long stretches ofprogeny DNA towhich theformer are covalently joined. The balance (or imbal-ance) between the degradative and protective processes mayprovideanexplanation for differ-ences we have observedinthelevel of multiplic-ity reactivation between heavy and light UV-irradiated phage.

More important, these results have led us to

propose a newmodel of repair replication which differs significantly from classical gap-filling ex-cision repair. In the latter case, the maximal progeny contribution to the repaired molecule can beonly50%. The model described below is

reminiscent of replication late inT4infectionat

which time recombinational intersections can

act assites of initiation of DNA synthesis(4).

(This researchispartially infulfillmentof the

requirements for a Ph.D. thesis of Helen H.

Vogelbacker.)

MATERIALS ANDMETHODS

Escherichia coli strain B-23was usedin all of the experiments. Thebacteriophage usedwastheosmotic shock-resistant strainT4B01r.

The growth medium was TCG (14). The isotope labeling, the heavymedium, and the phage containing BUdRwerepreparedaspreviously described (8). The

percentsubstitution of BUdR for thymidine in heavy phagewasapproximately95%. Theheavy phage(we

will designate the DNAofsuch phage as HH DNA)

used in our experiments were between 80 and 95% viableasdetermined by twoindependent criteria: (i) calculating the ratio of plating titertothe

concentra-tion of bacteria-killing particlesand(ii) comparing the totalamountof phageDNA totheplating titer ofthe

phage. When infections werecarriedoutin the pres-ence of chloramphenicol, it was added to a final

concentration of200,ug/ml. Test of inhibition of

pro-teinsynthesiswasperformed previouslyby measuring the uptakeof[3H]leucine. The observeduptake was

lessthan1%ofthatin theuntreated control.

When usedforlabelingtheprogeny DNA, [3H]thy-midine was includedinapackage, whichupondilution into the experimental medium resulted in 5 ,ug of thymidineperml, an adequateamountof

[3H]thymi-dineto assurethe desiredspecific activity,5 p.gof

5-fluorodeoxyuridineperml,5,ugof adenineperml, and

10,ugof uracil perml. All experiments werecarried

out at37°C.

Inpreparationfor DNAextraction, samples of

sus-pensions of infected cellsweredilutedtwofold in ice-cold LTL-EDTA (0.15 M NaCl, 0.01 M Tris-hydro-chloride,0.015 MEDTA, pH 7.4).Thecellswerethen sedimentedandsuspendedin 1 mlofLTL-EDTA. For

CsClgradient analysis,the DNAwasextractedbythe J. VIROL.

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sodium dodecyl sulfate-pronase-phenol method (11).

The efficiency ofphage DNArecovery upon extrac-tion is 100%. For alkaline sucrosegradient analysis,

theDNAwasreleased from the cellsaccordingtothe LTL lysisprocedure (19),followed bydigestion and concomitant denaturation of DNA with 0.1 MNaOH

at 37°C for 10 min. For the isolation ofintracellular

phage DNA associated with proteins, agentle lysis

procedure using lysozyme and Triton detergent was

performedaspreviouslydescribed(19).

CsCl density gradients were prepared and run as

previouslydescribed(9).ThedenaturationoftheDNA forCsCI gradient analysiswasaccomplished byadding KOH to afinalconcentration of 0.1 M, followed by neutralization withKH2PO4. This stepof

neutraliza-tion is important, sincecentrifugation of T4 DNAin CsClgradientscontainingKOH(orNaOH) ata

con-centration of 0.1 M results in the introduction of

numerous single-stranded breaks (E. Shahn, Ph.D.

thesis, University of Pennsylvania, Philadelphia, 1965).Theobserved distribution of theparentallabel inaCsCI gradientwasusedtocalculate thepercentage ofparental DNA which hadreplicated (R). The

effi-ciency of replication(E)wascalculatedaspreviously

described(21),acceptingas100%efficiencythe extent of DNAreplicationobservedininfectionswith

unirra-diated phage.The E value for UV-irradiatedphageis expressed as a percentage of the replication of the

unirradiatedphage:

E= (RUV/Rnon-UV) x 100%

Alkaline sucrose gradients were prepared as 5 to

20%osucrose in1 MNaCl and0.2 MNaOHandwere

centrifugedat30,000rpmfor 3hinanSW50.1 rotor.

Unit-lengthT4or,where appropriate,T7phageDNA

wasaddedas asizereference.Thedistance

sediment-ed in alkaline sucrosegradients by the experimental

DNA relative to that sedimented by the unit-length reference DNA (D2/D1)serves as a measureof strand integrity. The interpretation of D21D1 asameasureof

the number of single-stranded breaks incurred by

genomic-length T4 DNA, as a result of processes

acting at random locations along the molecule, was

performed accordingtoLitwinetal.(16). Itshould be noted, however, that this method of analysis cannot

distinguish between true single-stranded breaks and alkali-labileareas.

Thefast-sedimenting DNA-protein complexeswere

isolated in neutralsucrosegradients underlaid with1

ml(apad)of saturatedsucrosesolution.(Inthispaper,

the term "padded neutral sucrose gradient" will be used whenreferringtosuchgradients.) The gradients

were runat15,000rpmfor90min inanSW50.1rotor.

UV irradiation (260 nm) of the bacteriophage was

performed withagermicidallamp (General Electric)

withoutafilter. Duringthe UV irradiation, the phage weresuspendedin Tris-saltbuffer (0.15 M NaCL,0.01 M Tris-hydrochloride, pH 7.4). The number of UV irradiationhits receivedwas calculated fromthe

sur-vival curves ofthe irradiated phage. One hit is the

amount of UV irradiation causing, on average, one

lethaleventperphage, where the probability

distribu-tion is Poissonian. In conformity with the data of

Carlsonand Kozinski (1), thetimerequiredtodeliver 1 hit toheavyphageisequal tothetime required to

deliver 0.5hitstolightphage.

Thesonication of DNA wasperformed in a Raythe-onsonicator. The molecularweights of the resulting DNAfragments ranged between 5 x

10'

and 1 x 106.

RESULTS

Effect of UV irradiation on the integrity of BUdR-labeled T4 DNA. 32P-labeledheavy phage

wereexposedto0, 5, or 10lethal hits of UV (260 nm)irradiation. The DNA was released from the phage by alkaline lysis, and 3H-labeled, unit-length heavy T4 DNAwasaddedas areference. The DNAwas sedimented through alkaline su-crose gradients. As expected, the DNA from unirradiated phage sedimented coincidentally

with the reference DNA (Fig. 1A). However, the

DNAfromUV-irradiatedphage was

significant-ly displaced from the reference DNA. This dis-placement was dose dependent. In particular,

D21D1 equaled 0.56 for the DNA ofheavy phage

exposed to 5 lethal hits (Fig. 1B). This ratio corresponds to seven randomly distributed

sin-gle-strandedbreaks (16). Forthe DNA of heavy

phage exposed to 10 lethal hits, D21D1 equaled 0.45 (Fig. IC), corresponding to 15 single-strandedbreaks.

Wewishedalso toassay for theproduction of

double-strandedbreaks in theDNA ofirradiated

heavy phage. Of some concern was the

possibili-tyofartifactuallyintroducing or promoting

dou-ble-strandedbreaksduring the phenol extraction

of native DNA from UV-damaged phage. To avoid this potential problem, the 32P-labeled

heavy DNAwas firstextracted fromphage and then exposed to 5 or 10 lethal hits of UV irradiation. The irradiated DNA was examined in native form in neutral sucrosegradientsalong

with 3H-labeled, unit-length heavy T4 DNA. In

the case of both 5 and 10 lethal hits, the UV-irradiated heavy DNA cosedimented with the reference DNA (Fig. 2A). Thus, in this dose range, UV irradiation of heavy DNA does not

causedouble-stranded breaks.

On the otherhand, alkaline sucrose gradient analysis revealeddose-dependent single-strand-ed breakage of UV-irradiated heavy DNA (Fig.

2B). For DNAreceiving5 lethalhits,theD21D1

ratio equaled 0.56, correspondingto seven

ran-domly distributed single-stranded breaks. For DNA receiving 10 lethal hits, D21D1 equaled 0.48, indicatinganaverage of 13 single-stranded

breaks. Therefore, UV irradiation ofheavy T4 DNA causes 1.3 to 1.5 single-stranded breaks per lethalhit, regardlessof whethertheradiation is delivered to the phage or to the extracted DNA.

Additionalbreakdownof UV-irradiated BUdR-substituted DNAafter infection. Theintegrity of theintracellularparentalDNAof UV-irradiated

heavyphage5min afterinfectionwasexamined

by alkaline sucrose gradient analysis. The

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154 RESTIFOET AL.

0

<

B

5 HITS Di

W10-

(?cu:s)

DI~~~D

LL5

o

0-C

10OHITS

D

10 (iScits)

--

0-C.)5

FRACTIONOF THE LENGTH OF THE GRADIENT

FIG. 1. Alkaline sucrose gradient analysis of

32p-labeled DNA extracted from heavy T4 phage which had received 0 (A), 5 (B), or 10 (C) hits of UV

sults documented here show thatUV-irradiated heavy DNAacquires additional single-stranded breaks afterinjection intothe hostcell.

Asshownabove (Fig. 1C), theDNAof heavy phageirradiated with10 lethalhitshad

approxi-mately 15 single-stranded breaks before

infec-tion,resulting inanaveragesizeof

approximate-ly one-eighth that of unit-length T4 DNA.

However, alkaline sucrose gradient analysis of

the intracellular phage DNA extracted 5 min

after infection (multiplicity of infection

[MOI]

= 6) revealed a bimodal distribution of the

parental label(Fig. 3A). The faster sedimenting

portion (which consisted of 25% ofthe

recov-ered parental label) correspondedto a size

ap-proximatelyone-eighththatof unit-length heavy

T4DNA. In otherwords, it appeared unchanged

in size fromthat of the UV-irradiated parental

DNAbeforeinfection.Theremaining75% ofthe

parental DNA was very small and located near

thetopofthegradient,whereresolutionwas too

poor toallowaprecise sizedetermination. Itis

possibletostate,however, thatthestrandswere

less than1/100the sizeof unit-lengthT4 DNA.

Clearly, this bimodal size distribution could

not be due to ageneralized degradation of the

total parental DNA. Rather, the data suggest that, although most DNA of highly irradiated

heavy phage isadditionallyandextensively

bro-ken after injection into the host cell, a small

portion is somehowprotected from thisprocess.

Indeed, experiments describedbelow implicate

the DNA-protein replicative complex in the

mechanismof thisprotection.

To determine whether host- or phage-coded

enzymes mediate the observed intracellular

breakdown, the protein synthesis inhibitor

chloramphenicol was added to aportion ofthe

bacterial suspension at the time of infection to

prevent theproduction of phage-coded proteins.

Figure 3B shows the alkaline sucrose gradient

sedimentation pattern ofDNA extracted 5 min

after infection with UV-irradiated (10 hits)

heavy phage. The pattern was very similar to

irradiation. 3H-labeled unit-size heavy T4 DNA was

added as reference. Note the distance sedimented

through the gradientby the UV-irradiated DNA

com-paredtothatby thereference DNA. TheD2/D1 ratio

hasbeen shownby computer simulationand in vitro

experimentstobeauseful index for approximatingthe average number of random breaks inflicted on an

[image:4.491.63.221.47.640.2]

experimental population ofDNAmolecules(16). This

figure demonstratesalinear,dose-dependent

relation-ship between the number of lethalhitsofUV

irradia-tion and the resulting number of single-stranded

breaks inthe DNAoftheirradiatedphage. Symbols:

0,3H;o,32p.

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20-

E10-a

ul

0 0

w

x

25-

U-0

20-0

15-t

10-A

NSG

.O

0,

!I

6 .,

10

..

!i

.

; O

B

ASG

5 HITS

__

10 HITS

1

7Ci A

C-AC.TNOF,F TH ENGTHCF THE GCRACE_NT

FIG. 2. Sucrosegradient analyses of32P-labeledheavy T4 DNA extracted fromphage and then exposedto0, 5,or10hits of UV irradiation. 3H-labeled unit-size heavyT4DNAwasaddedasreference. Uponanalysis in neutralsucrosegradients (NSG) (A), the irradiated heavy phage DNA bands coincidentally with genomic length reference DNA. Thus, UV irradiation in this dose range doesnot causeanydouble-stranded breaks in heavyT4

DNA. Note that thesedimentationpatternsin alkaline sucrose gradients (ASG) (B) look very similar to those shown in Fig. 1. Again, 1.3 to 1.4 single-stranded breaks per lethal hit areintroduced by UV irradiation of genomic length heavyT4DNA.Symbols: 0,3H; , 32p.

that seenwithoutchloramphenicol,with73% of

the parental label sedimenting as very small

fragments and 27% as larger (one-eighth ofT4

unit length) pieces. This strongly suggests that

the breakdown of UV-irradiated phage DNA after infection is performed by host-coded en-zymes.

Extent and nature ofDNA replication of UV-irradiated heavy phage as examined by CsCI density gradient analysis. The experiments de-scribed above demonstrate (i) the immediate

effect ofUVirradiationonthe structural

integri-ty ofBUdR-substituted phage DNAand(ii) the

furtherbreakdownoftheirradiatedDNA

subse-quentto infection. Todetermine the replicative

competencyof thedamaged heavy phageDNA, we examined its ability to undergo replication

(as evidenced by a change in density) under a

numberof infection conditionsinlightbacteria: low andhigh multiplicityand in the presenceof unirradiated, rescuing phage.

First, we observedphage DNA replication in

E. coli infected at a low multiplicity with

UV-irradiated heavy phage. Separate portions of

bacteriawereinfectedat anMOIof 0.2 with

32p_

labeled heavy phage irradiated with 0, 5, or10

MR-I} k

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156 RESTIFO ETAL.

- L

ASG

10 Hits HH Phage Without CM 0

11

1I-T7 ref. I

I I

19

_I)

bis

80TTOM

FRACTION OF THE lENGTH OF THE GRADIENT

FIG. 3. Alkalinesucrosegradientanalysisof

intra-cellular 32P-labeled heavy parental DNA isolated 5

minutes after infection with phage irradiatedwith 10

lethal hits (A). (B) The same experiment but inthe presenceofchloramphenicol (CM)added at thetime

of infection. 3H-labeled unit-lengthheavyT7DNAis

the reference.Thetwopanelsshowsimilar size distri-butions ofparental DNA: in bothcasesthe majority (75%)isverysmall,about1/100 the size ofunit-length

T4DNA, and the minority(25%)bandsatalocation

correspondingto one-halfT7unit-length or

approxi-matelyone-eighth unit-lengthT4 DNA. Thesimilarity ofpatterns indicates the intracellular breakdown of

UV-irradiated T4 DNA is not dependent on phage proteinsand suggestsitis host mediated.Symbols:0,

3H;o,32P.

lethal hits. Samples were taken at 20min after

infection, at which time, even at low MOI, the

DNAsynthesis ofunirradiated phage was well

A

underway.

The DNA was

extracted,

supple-mented with3H-labeledheavy and light T4 DNA

as density references, and subjected to CsCl

CSCL

5 HITS HH Phage A

MOI=0.2

15--HH ref.

E=58%

LL ref-,:C

I

-)

B

>0

0 WIJ

15 lo

HITS

HH

Phage

1l M01 0.2

l10 E=70%

CA

W Ad'.~LL

O-80TTOM

[image:6.491.62.232.66.519.2]

FRACTION OFTHELENGTH OF THE GRADIENT

FIG. 4. CsClgradient analysis of 32P-labeledheavy

parentalDNA 20 min afterlow-multiplicity (MOI =

0.2)infections with phagethat hadreceived 5 (A)or10

(B)hitsof UVirradiation. 3H-labeledheavy and light T4DNA wereaddedas references. Note theshift in

density of the parental label away from the heavy reference location to analmost-lightlocation. The E

valueisconsiderableandsignificantlyhigher for phage

exposed tohigher dosesof irradiation. Symbols: 0,

3H;O,32P.

30-_

20-0

<

10-a

w

~0

w

t

30-0

z

'

20-cc

w

0-u

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CSCL

5 HITS HH Phage

MOI=5

A

B

Sonicated Q

I'

R~~~~~~~~~~~~

it

.)

II ,_1,

a,

0

.

C

_SOTTOM 5 1

FRACTIONOF THE LENGTH OF THE GkADIENT

gradient analysis. In addition, portions of the

extracted DNA wereprecipitated with

trichloro-acetic acid. Despite the significant damage done to heavy DNA exposed to high doses of UV

irradiation, the parental label was never more

than 20% decomposed to trichloroacetic

acid-soluble fragments (datanot shown). Treatment

of separate portions of extracted DNA with

nucleaseSi (datanotshown)revealed no

detect-able digestion by this single strand-specific

en-donuclease. Thus, therewasnolarge proportion

ofparentalDNA in single-stranded form.

Twentyminutes after infection with

unirradi-ated heavyphageat anMOI of 0.2, 73%of the

parentallabelmoved fromtheheavytoward the

light location (data not shown). This density

shift was accepted as a normal (or 100%)

effi-ciency ofDNA replication (21).

Figure 4A documents CsCl analysis of the

DNAextracted 20min after thelow-multiplicity

infection with heavy phage whichhad received

five hits ofUV irradiation. Forty-three percent

of the 32p label movedfrom the heavy location

towardthelight referencelocation,indicatingan

efficiency of replication of58%. In the case of

thelow-multiplicity infection with heavy phage

irradiated with10hits, 58% oftheparental label

shifted toward the light location, reflecting an

efficiency of replication of 70%(Fig. 4B).

Note that the efficiency of replication was

greaterfor the phagereceivingthehigher dose of

UVirradiation. This is in seemingconflictwith

thefactthatapproximately 30% ofT4genes are

involved in DNA replication (24), and,

there-fore, 10 hits delivered to aphage will result in

damage, on average, to three of these genes.

Although not all these genes are absolutely

required, one should expect that, without the

fullcomplement ofgenes necessaryfor

replica-FIG. 5. CsCI gradientanalysis of32P-labeledheavy

parental DNA 20 min afterahigh-multiplicity (MOI =

5)infection with phage thathad received 5 hits of UV

irradiation. 3H-labeledheavy andlight T4 DNA were

added as references. Panel (A) shows a significant

density shift of parental label,representing an E value

of70o.Panel(B) shows theCsCl gradient distribution

of the same DNAafter sonication. About half of the

almost-light parental label moved to the hybrid

loca-tion. Thisrepresents relatively long stretches of

paren-tal sequences, whereas the parental label remaining

almost-light after sonicationmust be disposed as very short stretchesamong much longer light ones. Panel

(C)showstheextracted phage DNA after alkali

dena-turation. This treatment fails to shift the replicated

parental label back towardthe heavylocation,

indicat-ingthattheseparentalregionsarecovalently joined to

newly synthesizedprogeny DNA.Symbols:0,3H;*,

32p.

1. H

0)

U

a

w

CD

w

0

0 w

0 z

w

0

w

CL

11

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CSCL J. VIROL. 5 Hits HH Phage plus

15 Rescuing Phage tion, one would observe a lower efficiency of

1

RescuingPhage

A

DNA replication in an infection with heavy

0A phage irradiated with higher doses. However,

*A-HHref. these data are consistent with host-mediated A E=75% repair replication ofthe UV-induced damages, L

LLref. andabody of evidence insupportof this

hypoth-1 esis will be

presented

in latersections.

10

.In

a separate experiment, E. coli were

infect-ed at

high multiplicity

(MOI

=

5)

with

heavy

phage which had received 0 or 5 hits of UV

Il',1irradiation. As shownin

Fig. SA,

DNA

replica-tion ofthe irradiated phage DNA was

demon-S- ,. P 5 strated by the shiftofthe heavy parental label

v r > toward the

light

location.

Accepting

as 100%

it ; 1, Q efficiencythe extent of density shiftundergone

by unirradiated heavy parental label, this corre-______

i,,sponds to anefficiency ofreplicationof 70%.

C In a separate branch of the experiment, we

coinfected with cold (non-radiolabeled),

unirra-50 diated rescuingphage to ensure that the

infec-Sonicated B tivecycle ineachbacterium would be

complet-ed. E. coli were coinfected with UV-irradiated

32P-labeled heavy phage (MOI=0.2) and

unirra-diatedcoldheavyphage (MOI= 5). Figures6A

10J

6> and 7A document the

CsCl

gradient analysis of

CK

65o3

' Q the DNA extracted 20min aftercoinfection. In

wll

,, the rescue

experiment

with

phage

irradiated

>

6C) ¢, with 5 lethal hits, 64% of the 32p label moved

o fromthe

heavy

toward the

light

reference

loca-5- tion, reflecting an efficiency of replication of

w 1 > r, 8

75%.

Note that this

efficiency

of

replication

is

a:

F 45w

§9not

significantly

different from that seen in the

II-

_LL_.6 & _:° in _edhigh-MOI experiment_{with 5 lethal} _hits.with_Inheavy_the _casephage_ofirradiat-_DNA

0

,>9 s 5 t^, extracted after coinfection with heavy phage

irradiated with 10 lethal hits and the rescuing

Z phage, 60% ofthe parental label underwent a

W shift toward the light location, reflecting an

0 Denatured efficiency of replication of

70%.

We have demonstrated that UV-irradiated

W 15 heavyphage undergoaconsiderable amountof

;L

DNA replication under the conditions of low

FIG. 6. CsCl gradient analysis of 32P-labeled

pa-10V

rentalDNA 20minaftercoinfection with heavy phage

which had received 5hits of UV irradiation (MOI = 0.2) and unirradiated cold heavy phage (MOI=5).

3H-,'<,) 4 > labeled heavy and light T4 DNA were added as

references. (A)Theefficiency of replication is slightly

higher than in cells infected withoutrescuing phage.

5 - t The replicated parentalnowbands very closeto the

light reference, indicating it has acquired a larger progeny contribution. (B) Sonication reveals (similar

toFig. 5)thathalfof theparental contribution in the

*

,^;'* replicated parentalexists as short stretches and half

.

a exists2 aslong stretches, relativetosurrounding light

0 progeny sequences. (C) Denaturation demonstrates

-o0T Om 5 1 covalent attachment ofreplicated heavyparental and

FRACTION OF THE ILENGTH OF THE GRADIENT lightprogeny DNA.Symbols: 0,3H;o,32p.

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(9)

CSCL

10 Hits HH Phage plus MOI,high MOI, andinthe presence of

unirradi-atedrescuing phage. These dataare incontrast

with previous experiments with irradiated light phage, which showed that during low MOI infec-tions no detectable DNA replication (assayed by

density shift of parental label) occurred for

phagewhichhadreceived 10 lethal hits (21). The high efficiency of replication documentedabove,

aswellasthe increasedefficiency seenin infec-tions withheavy phageexposed to higherdoses,

make ithighly unlikelythatincompleteinjection

ofphage DNA is a secondary consequence of UV irradiation, since, if this were the case,

efficiency of replication would decrease with

increasing doses of UV.

Close scrutiny ofthe CsCl gradient data

re-veals anotable difference inthe density ofthe

replicatedmoieties when one comparesthe

low-and high-MOI experiments with the rescue ex-periments. In the rescue experiments, aportion

of the parental labelshifted to a location almost identical to the light reference DNA (Fig. 6A and 7A). In contrast, in the experiments without rescuing phage, a portion of the parental label

bandedat adiscerniblyheavierlocation thanthe

light reference DNA. This indicates that in the low- andhigh-MOI experiments, the proportion-alcontribution of 32P-labeledheavyDNA to the progeny DNA was greaterthanthat seen in the presence of rescuing phage. In the high-MOI experiment, the proportionalcontributionof pa-rental to progeny DNA was calculated by a

methodpreviouslypublished (8) and foundtobe

approximately 15% of the mass of the replicated

segment. In the two rescue experiments, the closeoverlapof thelightreferenceDNAandthe parental labelprecludedaprecisecalculationof

the smallfractionalamountof the parental

con-tributiontothereplicated segments.

To further analyze the form of the parental contribution tothereplicatedmolecule, samples

-BOTTOM v)

[image:9.491.54.213.48.672.2]

FRACTIONOF THE LENGIH OF THE GRADIENT

FIG. 7. CsCl gradient analysis of DNA extracted

aftercoinfection withheavy phagewhich had received

10 hitsof UVirradiation(MOI=0.2)andunirradiated

cold heavy phage (MOI = 5). 3H-labeled heavy and

light T4 DNA were added as references. (A) The

magnitudeandposition ofthedensityshift ofparental labelshowthat(i) muchof the irradiatedheavy phage

DNA has replicated (E = 70o) and (ii) that the

replicated parentalDNAisvirtually indistinguishable

fromlightdensity; therefore, lightprogenysequences

constitute thevastmajorityof thereplicated parental. (B) Sonication does notchange thebandingpattern, therefore virtuallyallparentalsequencesinthe

repli-cated DNA exist as very short segments dispersed

among light progeny. (C) Heavy parental and light

progeny sequencesarecovalently joined. Symbols:0,

3H;o,32P.

C0.

0 a

w

0 z

w

C.)

0L

VOL.47,1983

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(10)

160 RESTIFOET AL.

ofextracted DNA from the above experiments

were sonicatedtoasizerangeof 5 x 105to1 X

106 daltons and banded in CsCl equilibrium gradients. The degree of density shift of the parental label after sonication, relative to the position of the unsheared DNA in the original CsCl gradient, is related to the length of the parentalsegmentin the replicated moiety. If the lengths of the parental segments were larger

thanthose of the fragments resulting from soni-cation, the labelwould bandatthehybrid loca-tion. On the other hand, verylittle shift ofany

kind would indicate dispersion of the parental labelas verysmallsubunits, significantly shorter

thanthe fragmentsgenerated by sonication. (For mathematical reasoning and equations, see the appendix in reference 5.)

Figures SB, 6B, and 7B show the results of CsClgradient analysis of the sonicated DNA.In

thecaseof moderately irradiated heavy phage (5

hits), DNA was isolated after the high-MOI experiment (Fig. 5) and the rescue experiment

(Fig. 6). In eachcase,approximately half of the

replicated parental DNA underwent a shift to the hybrid location after sonication. This

sug-geststhere isabimodal distribution oflengths of heavyparental DNA within thereplicated

frag-ments. Itispossible that relatively long

stretch-es of DNA in the moderately UV-irradiated

heavy genomeremain intactafterinfection and replicate semiconservatively. These stretchesof

parental DNA were still small in proportion to the amount of light density DNA which was

synthesized; thus, the replicated moleculeswere

ofalmost-light density. Upon sonication,

how-ever,thesestretches ofreplicated parentalDNA

shiftedtothehybrid location.Theremainder of

the parental label, which after sonication still

banded at the light or almost-light location,

represented heavy parental DNA which was

dispersed in very small pieces within the repli-cated molecule.

The DNA extracted from cells infected with

heavy phage irradiated with 10lethal hits banded atthe same position in the CsCl gradient both

before and after sonication(Fig. 7).In thiscase,

theparentalDNA is present within the replicat-ed moiety in minute stretches which are much

shorterthan the lengths offragments produced by sonication.

Furtheranalysiswasperformedto test

wheth-ertheheavyparentalDNA and thelightprogeny DNA were covalently linked or merelyjoined

via hydrogen bonding between complementary

areas. The extracted DNA from the high-MOI

and rescue experimentwas denatured,

neutral-ized, and analyzed inCsClgradients. A shiftof

the parental label backto theheavy location in

the gradient would reflect a lack of covalent

joiningofprogenytoparentalDNA.Figures5C,

6C, and7C represent CsCl gradient analysis of

the denatured DNA. In all cases, the parental

label remained at the light or slightly heavier

than light location. Therefore, we conclude

there iscovalent attachmentbetweenthe

paren-tal andnewlysynthesizedprogeny DNA.

Effectofchloramphenicol on the replication of

UV-irradiated heavy DNA. Upon a

low-multi-plicity infection with light phage exposed to 10

hitsofUVirradiation, smallgaps arerepairedby

hostpolymerase (2),but no phage DNA

replica-tion sufficient to produce a density shift of

parental label in a CsCl gradient is observed

(21). However, we showed above (Fig. 4B) a

significant amount of DNA replication (E =

70%) upon a low-multiplicity infection with

heavy phageirradiated with 10lethalhits.

When unirradiated heavy or light phage is

usedtoinfect bacteria in thepresence of

chlor-amphenicol, there is neither phageDNA

synthe-sis norreplication of parental labelasmeasured

byadensityshift(9, 10). Since10lethalhitsare

likely to render three phage genes for DNA

replication nonfunctional, we considered the

possibility thatthe extensive phageDNA

repli-cation observed upon infection with heavy

phage is performed by host enzymes. To test

this hypothesis, we measured DNA replication

when chloramphenicol wasadded at thetimeof

infection (MOI = 8) with

32P-labeled

heavy

phage irradiated with 3, 5, or 10lethal hits. If

hostenzymes areresponsible for the DNA

repli-cation observedafter infection of UV-irradiated

heavy phage, then chloramphenicol should not

prevent the replication. Moreover, if

radiation-induced damage is indeed repaired by host

en-zymes, theninthepresence ofchloramphenicol

one would expect more DNA replication for

phagereceivinghigher doses of UV irradiation.

Figure8depicts theCsCl gradientanalysis of

the DNAextracted 45 minafterinfection. Panel

A shows that 15% of the heavy parental DNA

exposed to 3 lethal hitsshifted toward the light

location, banding justto the heavy side of the

light reference DNA. This indicates that15%of

theparentallabel has becomeincorporated into

fragments composed of mostly progeny DNA.

After infection withheavyphageexposedto5 or 10 lethal hits, 24 or 32%, respectively, of the DNA shifted to a location slightly heavierthan

thelight reference.

In summary, UV-irradiated heavy phage

un-dergo a significant amount of DNA replication

despitethepresenceofaproteinsynthesis

inhib-itor. Inagreement with ourprediction, the mag-nitudeofthisreplication increases withthedose

ofirradiationtowhich thephagewereexposed.

Thisstrongly suggestshostenzymesare

respon-siblefortheobservedDNAreplication andthat

thereplication is in response to UVdamage.

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UVIRRADIATION OF BUdR-LABELEDT4DNA 161

CSCL

LL ref-9

,3 Hits HH II

Vi 5% Repa

le)!

Repli

HH*ef.:

LLmf.

-5 Hits Hl 24% Rep

I I Rep

LLref.-1

10Hits HF I32%Reps

Repli

QL.HH r,L1

l

.5

A

Formation of

parental DNA-protein complexes

after infection with UV-irradiated heavy phage.

Soon after infection, light replicative T4 parental iPhage DNA can be found in a DNA-protein complex

which sediments rapidly to the bottom of a

catlion neutral sucrose gradient (11, 18, 19). To examine

the effect of UV irradiation of heavy phage on theformation of thefast-sedimentingreplicative complex, E. coli were infected(MOI = 6) with heavy phage exposed to 0 or 10lethal hitsof UV

irradiation. Five minutes after infection, the cells were gently lysed by theTriton-lysozyme method, and thelysates were analyzed in neutral sucrosegradients underlaid with apad of

satu-rated sucrose solution. (Part of the lysate was

supplementedwith 3H DNA[asareference]and

Mg and digested with pancreatic DNase, at 10

,ug/ml. The extent of solubilization [95%] was

identical for bothisotopes. Thus, parentalDNA

B

ofUV-irradiated

phages

was

injected

complete-ly.)

After infectionwithunirradiatedheavy phage

(Fig. 9A), approximately 90% of the parental

HPhage DNA sedimented tothebottom of the

gradient,

)I

Page

resting on the pad. In contrast, examination of

olication the

lysates

from cells infected with heavy phage

which received 10 lethal hits revealed that

only

26% of the parental DNA reached the pad (Fig.

9B). Theremainder of the DNA bandednearthe

topofthegradient. Thus,atSminafter infection

the percentage of irradiated heavy parental

DNAassociated with thefast-sedimenting

com-plex is significantly reduced when compared

with thatof unirradiated DNA.

Thesedimentationpattern in aneutralsucrose

gradientisdependentuponthe

three-dimension-al structure of the DNA-protein complexes.

Hence, the relative position of the moieties in

C

thegradient doesnotyield information

concern-ing the size of theDNA. Todetermine the sizes

of the DNAcontainingaparental contributionin

air the two moietiesof the neutral sucrosegradient,

iir

_{icat flo}

the_{mM EDTA, denatured with alkali, and then}appropriate fractionsweredialyzed against1

sedimented through alkaline sucrose gradients.

Note the striking difference between the size

-1

FRACTION OF THE LENGTH OF THE GRADIENT

FIG. 8. CsCl gradient analyses of32P-labeled pa-rental DNA isolated 45 min after infection, in the

presenceofchloramphenicol (CM; added atthe time ofinfection), withheavy phage (MOI = 8) which had received3(A),5(B),or 10(C) hits of UV irradiation.

3H-labeled heavy and light T4 DNA were added as

references. In all cases therewas adensity shift of

someof theparental labeltoanalmost-lightlocation,

despite the presence ofaprotein synthesis inhibitor during the infection. Theextentofdensityshift,which reflects repair replication, increases with increasing

dose of UV irradiation. Symbols: 0, 3H;0, 32p.

0 w

w

0 w

a:

0 1--H

w

0

,

w

a.

15-

10-

5-BOTTOM VOL.47, 1983

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162 RESTIFO ET AL.

NSG with Pad

0 Hits A

T4 ref.

X

5:40

F

~~~~~~~~~~~~~~~,,

,20

w W

0

B

LLJ

40-

26s

10

Hits

W

30 ,

O3

cr~~~~~~~~~~~~~~~~~~~~~~~~~~~I

Il

t20

10~~~~~~~~~~

FRACTIONOF TPE LENGTHOF THE GRADIENT

FIG. 9. Padded neutral sucrose gradient (NSG)

analysisoflysatesfrom cells 5minafter infectionwith

heavy phagewhich received 0(A)or10(B)hits of UV irradiation. Thegradientswereunderlaidwithapadof

saturatedsucrose, and 3H-labeledunit-sizeheavyT4 DNA was added as reference. Ninety percent of unirradiated heavy parental sediments to the pad, indicatingits associationwith theDNA-protein

repli-cative complex. Only 26% of the irradiated heavy

parental label becomes associated with the

fast-sedi-menting complex. The rest sediments very slightly, remaining near thetop of the gradient. Symbols: 0,

3H; o, 32P.

profiles oftheparentalDNAassociated with the

fast-sedimenting complex (Fig. 10A) and the

parental DNAisolated from the top of the neu-tral sucrose gradient (Fig. 10B). Themajority of

theDNAisolatedfrom thepadwasof

consider-able size, approximately one-eighth the size of

unit-length T4 DNA, whereas that from the top of the gradient was very small, less than 1/100 the size ofunit-length T4 DNA.

We also wished to measure the extent of DNA replication undergone by the parental DNA

re-siding in these two moieties. Heavy phagewhich had received 15 lethal hits were used to infect E. coli (MOI = 8). At 20 min after infection, the bacteria were gentlylysed,and the samplewas sedimented through a neutral sucrose gradient underlaid with a pad of saturated sucrose solu-tion. The overall sedimentation pattern (data

now shown) of the parental label in the neutral

sucrose gradient was similar to that seen at 20

min after infection with heavy phage which

received 10lethal hits. Fractions fromthe topof

the gradient and from the pad were recovered and dialyzed, and the DNA was extracted with phenol and analyzed in CsCl density gradients.

As seen in Fig. 11, the DNA from the fast-sedimenting complex replicated to a greater

ex-tent than did the DNA from the top of the

neutral sucrosegradient. The majority(64%) of

the heavy parental label isolated from the pad

underwent a shift toward the light location,

whereas only 28% of the DNA from the top

underwentsuch ashift. Insummary,after

infec-tion with UV-irradiated heavy phage,the

paren-tal DNA exists in two classes. One resides in

the fast-sedimenting DNA-protein complex, is

of considerable size, is apparently protected

against host-mediated degradative processes,

and has ahigh percentage ofreplicativeDNA.

The other classis notassociated with the

fast-sedimentingcomplex and consists ofvery small

fragments with a much smaller proportion of

replicative DNA.

Association of progeny DNA with the

fast-sedimenting complex. In an earlier paper, we

documented the sizes of theprogeny DNA syn-thesized at various times after infection with UV-irradiated heavy phage (15). The size of progeny DNA early after infection,

approxi-mately one-eighth of unit-length T4 DNA, is

similar to that of the UV-irradiated parental

DNA residing in thefast-sedimenting complex.

We therefore proposed that progeny DNA is

generated from those parental DNA fragments

in the complex which contain origins of DNA

replication.Inthissection,wewill show that the

majority of newly synthesizedprogeny is indeed

associated withthe fast-sedimentingcomplex.

E. coli were infected at an MOI of 8 with

heavy phage which had received 15 hits of UV

J. VIROL.

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[image:12.491.65.225.51.545.2]

(13)

5:10

cr

w

0

030

W Q

T7ref.-1

wI20

O-~~~~~~~~I

10r

_eo7om

.5 1

-BOTTOM

FRACTIONOF THELENGTHOF THL GRADIENT

FIG. 10. Alkaline sucrosegradient (ASG) analysis

ofDNA isolated fromthepadand top ofapreparative

padded neutral sucrose gradient (see Fig. 9). 32P-labeledparentalDNAwasextracted5min after

infec-tionwithheavy phage which had received10hits of

UV irradiation. 3H-labeled unit-size heavy T7 DNA

wasaddedas areference.The DNAfrom the pad (A)

is ofvariable size, but the predominant peak

corre-sponds to one-half T7 unit-length or approximately

one-eighthT4unit-length. TheDNAfrom the topof

theneutralsucrosegradient (B) isverysmall,lessthan

1/100unit-size T4 DNA. Symbols: 0,3H; o, 32p.

H

10 o

w

w5

U.)0

0 B

15 28%

Replication

0.

_,Q

_~~III

10

-BOTTOM .5 1

FRACTION OF THE LENGTHOFTHE GRADIENT

FIG. 11. CsClgradient analysis of32P-labeled pa-rental DNA isolated from the top andpad of a prepara-tive padded neutral sucrosegradient. DNA was

ex-tracted20min after infection with heavyphage which had received 15 hits of UVirradiation. 3H-labeled unit-sizeheavy T7 DNA was addedas a reference. The DNA fromthepad (A) ismoreextensivelyreplicated

thanis DNAfrom the top (B) of the neutral sucrose gradient as indicated by the proportion ofparental

label undergoing density shift. Symbols: 0, 3H; 0,

32P.

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164 RESTIFOET AL.

50

40

30

20

ic

r

60x

NSG with

Pad

11

II!l

II

ii

lII

:l

l-o

coccG

e-BOTTOM

FRACTION OF THE LENGTH OF THE GRADIENT

FIG. 12. Padded neutral sucrose gradient (NSG) analysisofprogeny(3H-labeled)DNAfrom thelysate

of cells infected with heavy T4 phage which had

received 15 hits of UV irradiation. A

[3H]thymidine-containing package wasaddedat 3.5minafter infec-tionand thesamplewastakenat20minafter infection. Note that 60% of the incorporated progeny label, in contrast toonly26%of theparentallabel(seeFig. 9),

isassociated with thefast-sedimenting complexatthe

pad.

irradiation. A[3H]thymidine-containingpackage (specific activity, 1 mCi/mg) was added at 3.5

min after infection, and samples were analyzed

at 20 and 40 min after infection. The infected

cellsweregently lysedand the lysates

sediment-edthroughpaddedneutralsucrosegradients.At

20min afterinfection,the majority (60%)of the

[3H]thymidine-labeled progeny DNA was

asso-ciated with the fast-sedimenting complex (Fig. 12), whereas, as documented in the previous section (Fig. 10B), only 26% of the parental DNA resided there. Importantly, even 40 min

after infection (data not shown), approximately

50% of theprogenyDNA still resided in the

fast-sedimenting complex, possibly replicating

au-tonomously.

Comparison ofmultiplicityreactivation of light and heavy T4 bacteriophage. To compare the extent of multiplicity reactivation ofheavy and light bacteriophage, suspensions of each were exposed to oneofarange(0to 27lethal hits) of

doses ofUVirradiation. The phage were used to

infectseparateportionsof E. coli at an MOI of 6.

The levelofmultiplicity reactivationwas calcu-latedbydividingthenumberof infectivecenters

produced by the UV-irradiated phage by the

numberofinfectivecenters produced by

unirra-diated (0 hits) control phage.

Fig.13showsthat, intherangeoflow doses (2

to 5 lethal hits), multiplicity reactivation was nearly identical in light and heavy phage. How-ever, heavy phage irradiated withmore than 5 lethalhits clearlyshowed a markeddeficiencyin

multiplicity reactivation. With increasing doses

ofUVirradiation, there was aprogressive

diver-gence betweenthetwolines. The relationship of

thisdeficiency in multiplicity reactivationtothe

intracellular fate of the UV-irradiated heavy

DNAwill be discussed below.

DISCUSSION

Inthis report, we have attempted to

charac-terize the moleculareventsafter the damageof

BUdR-labeled T4phageby UV irradiation. The

12 16 HITS

FIG. 13. Multiplicity reactivationin E. coli infect-ed(MOI= 6)withheavyorlightT4phagewhich had receivedoneofarange(0to27hits)ofdosesof UV irradiation. Atdoses of 5 hitsorlower, the levelsof

multiplicityreactivationareverysimilarinheavyand

light phage. Athigherdoses, however, heavy phage undergo progressively less multiplicity reactivation than do their light counterparts. Symbols: 0, light

phage;*,heavy phage.

H

0-C)

LLi

w

LLJ

>

01

0 w

0 z

w

0 w

0L

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UVIRRADIATION OF BUdR-LABELED T4 DNA 165

direct effect of irradiating heavy phage can be

distinguished from those further insults which

ensueafterinfection, resulting from the

interac-tion of the damaged phage DNA with the host

cell.Inconcert, these sequelae of irradiation not

only determinethe degree of phagelethality but

also biasthe genetic representation among

new-ly synthesized progeny phage DNA in favor of

areascontainingorigins of replication(15).

UV irradiation ofheavy phage or of heavy

phage DNA results in the introductionof

single-stranded breaks in the phageDNA. As wehave

shown here by analyzingthesedimentation

pro-file of the phageDNAinalkaline sucrose

gradi-ents, the number of single-stranded breaks

in-creases linearly with the number of lethal hits.

Specifically, overthe dose range tested, 1.3 to

1.5 single-stranded breaks per genomic strand

result from each lethal hit. When analyzed in

neutral sucrose gradients, UV-irradiated heavy

phage DNA showsnoevidence, for dosesup to

10lethal hits,of double-stranded breaks.

Exposure of heavy DNA to UV irradiation

causesdebromination of the BUdR,resulting in

uracil (23; H. H. Vogelbacker and S.-K. Ling,

Fed. Proc. 40:1764, 1981). Therelationship

be-tween the debromination and the induction of

single-stranded breaks hasnotyetbeen clarified

(22; H. H.Vogelbacker, Ph.D. thesis,

Universi-tyofPennsylvania, Philadelphia, 1982).

After infection ofE. coli with UV-damaged

heavy phage, the DNA is subjected tofurther

breakdown by host-mediatedprocesses. Athigh

doses of irradiation (10 lethal hits), the

intracel-lularparental phage DNAclearly exists astwo

size classes,oneapparently unchanged from its

preinjection size(representing 25% ofthe

paren-tal label) and the other class (75%) smaller by

more than 10-fold. (Note, however, that the

breakdown isnotaccompanied bya substantial

amountof solubilization to trichloroacetic

acid-nonprecipitable material.) Since a similar

amount and patternof breakdown occur in the presence ofa protein synthesis inhibitor added

at the time of infection, we believe the

post-infection breakdown is host mediated. (Of

course, we cannotruleoutthepossibility that,in

the presence ofchloramphenicol, some

phage-coded proteins are synthesized in such small

quantitiesthattheycannotbe detectedby

pulse-labeling with radioactive amino acids.)

Thebimodality ofthe distributionof

intracel-lular heavy parental label in alkaline sucrose

gradients is clearlynotconsistent with the

oper-ation of a single random intracellular cutting

event applied equally to all phage DNA. We

interpretthese twodistinct size classes of

intra-cellularphageDNAasrepresentingtheproducts

oftwocompetingprocesseswithinthe hostcell.

On one hand there exists a process which

de-grades UV-damaged DNAto an extentdirectly

related to the amount of damage. A series of

experiments recently undertaken inthis

labora-tory have specifically implicated the removal of uracil by the host-coded enzyme uracil glycosy-lase asinstrumental in this degradative process (H. H.Vogelbacker, Ph.D. thesis, Universityof

Pennsylvania,Philadelphia, 1982). Events after

uracil removal, e.g., counternicking, may

pro-duce double-stranded cuts which subsequently

interfere with replicationby preventing

elonga-tion from the 3' end of the fragment. On the

other hand, other simultaneous events protect someofthedamaged DNAfromfurther

break-down or, alternatively, ensureprompt and

effi-cient repair. Amoretrivialinterpretation would

suggestthatthedegradativeprocessis saturated

when 75% ofthe UV-damaged DNA is broken

down. However, the data presented above

sug-gest the existence ofan active protective proc-ess.

First, the percentage of heavy irradiated

pa-rental DNA not subjected to the intracellular

breakdown is thesame asthat percentagefound

associated withafast-sedimenting DNA-protein

complex. Second,the sizeof thisminority class

parentalDNAisthe same as thatfoundresiding

inthecomplex.Third,theparental labelresiding

in larger fragments associated with the complex

undergoes moreextensivereplication thandoes

the parental label in the very small parental

pieces not associated with the complex. (Of

course,phage which didnot inject, if

sediment-ed to the pad, should contain nonreplicated

DNA.)

The protection afforded damaged parental

DNA associated with the replicative complex

probably does not entail the prevention of all

corrective excisions inthat DNA. If those

exci-sions, by uracil glycosylase, forinstance, were

not introduced to DNA in the complex, then

upon sonication of the replicative DNA, one

should be able toresolve theparental

contribu-tion in the form ofa hybrid moiety. However,

forhighdosesofirradiation,whenamajorityof

parental DNA in the complex is replicated,

sonication does not result in the shift of the

parental label to hybrid density. Hence,

"pro-tection" refers to the providing ofa molecular

milieu in which large stretches ofDNA canbe

maintained despite the infliction of transient

breaks. This could be accomplishedby holding

damagedDNAstrandsin close linearproximity,

or by facilitating recombination between

frag-ments, thusresultinginrelativelylongsubunits. Previous workfromthislaboratoryshowedthat even double-stranded cuts introduced by 32P

decay in vitro do notdissociate DNA from the

replicativecomplex (19). Therefore, itwas

pos-tulated that theproteincomponentsofthe

http://jvi.asm.org/

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166 RESTIFO ET AL.

plex are firmly associated with the DNA at multiple sites. This is consistent with the protec-tive role webelieve thecomplex plays.

To explain our data, we propose that after

infection withUV-irradiatedheavy phage, some areas becomepreferentially associated withthe

replicative complex and are able to serve as

templatesforDNAsynthesis (inamanner tobe

explained below). On the other hand, those

areas notassociated with thecomplex are more likely to be exposed tohostdegradative

process-es and are further extensively broken. This

proposition isconsistent withourearlier

hybrid-ization data, which demonstrated gene

amplifi-cation in the progeny DNA synthesized after

infection with UV-irradiated heavy phage (15).

The biased representation of areas containing

origins among the progeny sequences suggests

that origins are also over-represented in the

parentalDNAassociated withthecomplex.Not

surprisingly, we showed in this report a large

proportion (60%) of progeny DNA associated

with the complex, in contrast with the

associa-tion of only26% oftheirradiatedheavy parental

DNA with the complex. This report also

pre-sents evidence that UV irradiation greatly

de-creases the association ofheavy parental DNA

withthereplicative complex, which is in keeping

with the concept of competition between the

degradative process directed at

radiation-dam-agedDNAand theprotectiveprocess related to

theDNA-protein replicative complex.

In ourview, themostinteresting implications ofourdata concern the nature ofDNA

replica-tion after infections with irradiated heavy T4

phage.In anearlierreport, wepresented

experi-ments in which we labeled the newly

synthe-sized progeny DNA. Aconsiderableamount of

light progeny DNA (36 phage equivalent units

perinfective center)wassynthesized after

infec-tion with heavy phage irradiated with 10 lethal

hits. The progeny label was not covalently

at-tachedto theparental molecules, and its

repre-sentation of the T4 genome wasstrongly biased

in favor ofareas containing knownorigins (15).

In the present series of experiments, we fol-lowed theprogress ofradiolabeledheavy paren-tal DNA through the course of an infection.

Despite moderate or high doses of UV

irradia-tion, theheavy parental DNA underwent

exten-sive replication, asevidencedbythemagnitude

of density shifts in CsCl equilibrium gradients.

The efficiency ofreplication in low MOI, high

MOI, and rescue experiments was between 58 and75%of that afterinfectionwithunirradiated

heavyphage. Incontrast, irradiatedlightphage

doesnotshowany DNAsynthesis detectable by

densityshiftanalysis. Itdoes,however, undergo

repair synthesis to fill in the very short gaps

generated by excision ofdamaged areas(21; E.

Shahn, Ph.D. thesis, University of

Pennsylva-nia, Philadelphia, 1965).

Our results illustrate four features of the

phage DNA replication during infections with

irradiated heavy phage. First, we observed a

higherefficiencyofreplication ininfections with

phage receiving more irradiation. Second, the

replication took place (and with comparable

efficiency) in the presence ofchloramphenicol.

Together, these dataindicate that the replication

is in responseto UV damage, i.e., isa form of

repair replication,andthat thisrepair replication

is host mediated.

The third notable featureconcernstheamount

ofdistribution of theheavy parental contribution

in repair-replicated molecules. Conventional

re-pair consists of damage recognitionandexcision

followedbygaprecognition, filling in,and

liga-tion. Inthis mode ofrepair, molecular integrity

is restored by theaddition ofafewnucleotides

and therefore withoutanyappreciablechange in

density of the parental subunit. However, in the

case of irradiated heavy phage, the parental

labelundergoesadramaticdensityshift, moving

to a location very close to the light reference

DNA (Fig. 4through 7). This indicatesthatthe

parental contribution constitutes a very small

percentageofthe massofthereplicated subunit,

withtheremaindercontributed by newly

synthe-sizedlightDNA. Inhigh-multiplicity infections,

the estimated proportional contribution of

pa-rental label to the repaired molecule was only

15%. Aneven smaller parentalcontribution was

seen when bacteria were coinfected with cold

unirradiated and radiolabeled UV-irradiated

phage. (The almost coincidental banding of the

parental label with the light reference may

re-flect recombination between UV-damaged

heavyDNA and the undamaged progeny DNA

oftherescuingphage.)

By conventional repairreplication, the

mini-malparental contribution to therepaired

mole-culeis50%, i.e.,hybrid, unless there is

homolo-gous recombination between the repaired

parental and whole progeny. However, since

progeny synthesis in infections with irradiated

heavyphage issite-specific (15), manyareas of

the parental genome would be unable to

recom-bineinthisway. Theexceptionallysmall paren-tal contribution suggests that therepair

replica-tion seen after infection with UV-irradiated

heavy phage isqualitatively verydifferent from

conventional gap-filling repairsynthesis.

Theparentalcontribution in therepaired

tem-plate isnotonly small,but alsowidelydispersed

among stretches oflight parental sequences as revealed by the sonication data. At moderate doses (5 lethal hits) the parental nucleotides

exist

partly

asrelativelylong(significantly

long-erthan106daltons) stretchesandpartlyasvery

J. VIROL.

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(17)

small(significantlyshorter than 5 x

105

daltons),

highly dispersed units. At higher doses, the

parentalcontributionin thealmost-lightrepaired

molecule is virtually entirely disposed as very small segments scattered among long stretches

oflight sequences.

These heavyparental segments arecovalently

linkedtonewly synthesizedlightprogeny DNA

as evidenced by the failure of denaturation to

change theCsClbandingpatternoftheparental

label (Fig. 5 through 7). This covalent

attach-ment between parental and progeny sequences

clearlydistinguishesthistypeofreplicationfrom

that detected when one follows the progeny

label. The majority of newly synthesized

3H-labeled light progeny DNA is not covalently

attached totheparental molecule (15).

Hence, the phage DNA synthesis occurring

upon infectionwith UV-irradiated heavyphage

consists oftwotypes ofreplication which take

place simultaneously. The firsttype is the usual

replication aimedatgeneratingnumerous

proge-ny phage segments with which tocomplete the

infective cycle. Since initiation ofDNA

synthe-sisoccurspreferentiallyatorigins of replication,

andsince theparentalDNAisfragmented,

elon-gationof theprogeny strands is limitedandgene

amplificationresults (15).Thisamplified, biased

progeny DNA is not covalently linked to the

parental template. The second type is repair

replication of UV-induced damagetothe

paren-tal molecule. We believe this occurs

preferen-tiallyin areasprotected from host-mediated

dis-integration by virtue of their associationwith a

replicative complex. The products of this

repli-cationareshort, heavy parental fragments

scat-tered amonglong stretches of newlysynthesized

light progeny to which they are covalently

at-tached. Note thatthisrepair replication

contrib-utes asmall fraction to netDNA synthesis and

can only be observed byfollowing the parental

label.

Before considering two models which might

explain this unusualrepair replication, a fourth

importantaspect mustbeemphasized.As

previ-ously mentioned, the production ofmostly-light

repaired molecules doesnotinvolve a

consider-able loss ofheavy parental nucleotides, as

evi-dencedby lack of extensive solubilization ofthe

parental label throughout the course of an infec-tion.Therefore,weconclude thereis no consid-erable exonuclease digestion extending from

ar-easofUVdamageorfrom gapsproduced by the

excision of uracil (H. H. Vogelbacker, Ph.D.

thesis, University of Pennsylvania,

Philadel-phia, 1982).

Inattemptng toexplain thefeatures of repair

replication of UV-damaged heavy templates

mentioned above, we considered two models

(Fig. 14). Inboth models, areas of UV-induced

damageareexcised fromtheheavyDNA

mole-cule. Accordingtomodel1 (Fig. 14, leftpanel),

the gapscreatedbytheexcisionaresimply filled

in with newly synthesized light progeny DNA.

Since simultaneous excision of all damages

would result inthesegment's disintegrating into

small fragments, the excisions would have to

occurin apartlystep-wisefashion. To be consis-tent with theCsCldatashowingreplicated

mole-cules ofalmost-light density, model 1 has as a

requirement theexcision ofvery long stretches

of heavyparentalDNA (surroundingorstarting

from eachareaofdamage), which would result

inahigh degree ofsolubilization of the parental

label. Since weobservedrelatively little

solubili-zation, model1 is not atenableinterpretationof

ourdata.

To explain therepair of UV-irradiated heavy

parental DNA such that the resulting subunits

areofalmost-lightdensity,yet withoutan

appre-ciable amount ofsolubilization of the parental

DNA, we postulate model 2 (Fig. 14, right

panel). Inthismodel, excision alsoproceeds ina

sequentialmannerbutthe areaofparental

nucle-otides removed at each damage is very small.

This would result in relatively little

solubiliza-tion ofthe parental label and is therefore more

consistent with ourfindings. The model further

proposes that, as the progeny DNA elongates

and the gap is essentially filled in, the leading

progeny strand displaces, instead of ligating to, the 5' end of theparental strand and continues

elongating. The displaced strand could itself

then act as a template for progeny synthesis.

The synthesis on this lagging strand is

presum-ably discontinuous. UV damages (i.e., uracil)

stillremaining in eitherparental strand will not

impede theiruse astemplates; the uracil canbe

removed in alatercycle oftheprocess. As the

schematic diagramindicates,thegradual

remov-al ofUVdamages followed by repair synthesis

ultimately results in a large proportion of

sub-units in whichthe parental contribution is very

small an flanked by much longer stretches of

covalently attached progeny DNA. For any

giv-en fragment of damaged parental DNA, the

fractional length of stretches of parental label

foundinrepair-replicated fragmentswilldepend

on the number and distribution of uracil

exci-sions. At high doses, a larger proportion of

repaired fragments will have shorter parental

contributions,asisdemonstratedbyour

sonica-tiondata.

The proposedscheme ofstranddisplacement

during repair synthesis of UV-irradiated heavy

phage is reminiscent of DNAreplication atlate

times after infection with unirradiated phage,

when reinitiation occurs at random locations

alongtheparentalmolecule(4).This late

reiniti-ation contrasts with that occurring early after

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