Molecular Recombination in T4 Bacteriophage Deoxyribonucleic Acid

JOURNALOFVIROLOGY,Mar.1970,p.368-380

Copyright( 1970 AmericanSociety forMicrobiology

Vol.5,No. 3 Printed inU.S.A.

Molecular

Recombination

in

T4

Bacteriophage

Deoxyribonucleic

Acid

III.

Formation of Long Single

Strands

During

Recombinationl

ROBERT C. MILLER, JR.,2 ANDRZEJ W. KOZINSKI, AND SAMUEL LITWIN

Department of Medical Genetics, University of Pennsylvania, Philadelphia, Pennsylvania 19104

Received for publication3November 1969

Evidence was presented to support thehypothesis thatlong single strands

ap-pearingatlatetimes(15 min afterinfection) areproducedasaresult of

recombina-tionand not asa continuous elongation during thereplication process. The

pro-duction oflong strands does notdepend onthemultiplicity ofinfection, and the

first long strandsappearatthetime when20to50phage equivalent units of

deoxy-ribonucleic (DNA)aresynthesized,and notearlier.Theaddition ofchloramphenicol

at5 min, which preventsmolecular recombinationbutallowsreplicationofDNA,

prevents the formation oflong, single strands. Chloramphenicol added between 8

and10minafterinfection,atimeatwhichmolecularrecombinationisfully expressed

andcovalentrepair of recombinant molecules isallowed, doesnotpreventformation

of long single strands. Cutting of single-strand DNA with a limited amount of

endonuclease I allows confirmation that the fast-sedimenting characteristic of

intracellular denatured DNA is caused primarily bythelength of thestrands, and

not by the formation of aggregates.The computer simulation of two

recombina-tion models indicates the feasibility ofrandom breakage and rejoining of

mole-cules in generating long concatenates.

Thedeoxyribonucleic acid

(DNA)

moleculesof

T4 bacteriophage are

circularly

permuted and

terminally

redundant

(8,

12).

Circular

permuta-tion can be created by at least two different

mechanisms.Onewouldinvolveforminga

physi-cal circle of the DNA during the replicative

process. Thesecondprocesswouldrequire, prior

tomaturation,theformation of molecules much

longerthan onephageequivalentunit.This would beconcludedbyexcision of moleculesof aboutone

phageequivalent unitinlength

during

the

matu-rationprocess. The

long

molecules could be

cre-atedbysome mechanismof

replication

inwhich

the DNA iscontinuously elongated,orbya

mech-anism of recombination, which we favor. Since

neutral sucrose gradient analysis doesnotreliably

measure the molecularweight ofreplicative DNA

(6),

thepresence oflong single strandsinan

alka-line sucrose gradient would be a desirable

indica-tion that double-stranded DNAlongerthan one

phage equivalentunit does exist in the

intracellu-lar DNApool.

1Submitted byR. C. Miller,Jr., to the University of

Penn-sylvania inpartial fulfillment of the requirements for the Ph.D degree.

2Present address: InstituteforEnzymeResearch, University ofWisconsin, Madison, Wis.

When newly synthesized DNA is labeled with

3H-thymidine during later stages ofinfection of

Escherichia coli B bybacteriophageT4, some of

the label is found in material which sediments

faster than reference DNA in analkaline sucrose

gradient (1). It has been postulated that this

material representssingle-strandedDNAwhich is

longer than the single strands which can be

iso-lated from maturephage.Experiments testingthis

hypothesis are reported here. Net synthesis of

DNA was measured simultaneously with the

production of long single strands to determine

whether thelongsinglestrandswereformed

dur-ing the initial stages of replication or arose at

later times, concomitantly with the onset of

re-combination. Furthermore, it will be

demon-strated that one caninhibitlongsinglestrand

for-mationwithout inhibitingDNAsynthesis.

Studies tobe reported here support the

conten-tion that the fast-sedimenting DNA, in alkaline

sucrose gradients, is indeed composed of single

strands longer than those isolated from mature

phage. Estimatesof net DNAsynthesis

concomi-tant with long single strand production indicate

that approximately 20 phage equivalent units of

DNA are synthesized before a measurable

frac-tion of intracellular DNA enters single strands

368

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exceeding in size the reference mature DNA.

Addition of chloramphenicol (CM) to infected

cells at a time which leads to the inhibition of

re-combination inhibits the formation of longsingle

strands even though DNA synthesis is not

im-paired. Furthermore, an insignificant extent of

long singlestrand production canbe detected in

cells grown in 5-bromodeoxyuridine (5-BUdR) medium even though this condition allows

pro-ductionof viablephageof normalburst sizes. Two

modelsof recombinationwill be simulated in the

computer, and the results will be evaluated in

respect to thefeasibilityof the formation oflong

molecules asa result ofrecombination.

MATERIALS AND METHODS

Most of the procedures for density or isotopic

labelingandsucrose ordensity gradient analysishave

beendescribed elsewhere (6).Some sucrosegradients

wereunderlayeredwithapad ofsaturatedsucrose to

guarantee quantitative recovery ofthe input

radio-activity; these gradients were prepared by

under-layering3.5 mlof 5to20%sucrosegradientwith 1.0

ml of saturated sucrose with a canulating needle

attached to a syringe. The lysozyme-Triton X-100

lysis (LTL) method wasperformed in the following

manner. Cellswere suspendedinamixture of 0.05 M

tris(hydroxymethyl)aminomethane (Tris), 0.05 M

NaCl, 0.05 M ethylenediaminetetraacetate (EDTA),

andlysozyme (100,ug/ml), pH 8.0,andchilledat4C

for 10 min. Triton X-100 wasaddedtoa final

con-centration of1%,and themixtureswerechilledforat

least 10minat4 Cbeforethe addition ofalkali. The

programmingapproachesfor computersimulationwill

beoutlinedin thetext.

RESULTS

Net DNA synthesisversus long strand. Iflong

single strandsare

produced simply

as a function

of

replication,

theyshould be detected beforetwo

or three phage

equivalent

units of DNA per

bacterium are

synthesized.

The

following

experi-ment was conductedto determinetheamountof

DNA synthesized before

long

single

strands can bedetected.

E. coli B23 was grownto 3 X 108 cells/ml in

low phosphate TCG medium. The culture was

splitinto two

samples;

thefirst one

(part 1)

was

infected with T4

bacteriophage

[multiplicity

of infection

(MOI)

=

5.0]

labeled with

32p

at a

specific activity of 5.0 mc/mg of P. The second

sample was

(part

2)

infected with cold

phage

(MOI = 5.0).

P2p

at a

specific

activity

of 0.5

mc/mgofP was added tothe second

sample

at

2 min afterinfection. DNA

synthesis

was

meas-ured by uptake of 82P to trichloroacetic

acid-precipitable, alkali-resistant material

(9).

At

varioustimes after

infection, samples

from part 1 weretransferredtoKCNand

lysed

with

lysozyme

and sodium lauryl sarcosinate (1) or with lyso-zyme and Triton X-100. At the same time, sam-ples from part 2 were precipitated with

trichloro-acetic acid and the amount of DNA synthesized was determined after alkali digestion of the sam-ples by procedure of Schmidt et al. (9). The num-ber of phage equivalent units of DNA

synthe-sizedwas calculated on the basis that 1 ,ug of P =

5 X 10'° phage equivalent units. 8H reference

phage were added to the lysates from part 1; themixtureswere treated with alkali and layered

on 5to 20% sucrose gradients which were

under-layed with 1 ml of saturated sucrose. The satu-rated sucrose pad provided quantitative recovery

of the material from the gradients.

Alternatively, if progeny strands were labeled

with 32p incorporated after infection (part 2),3H

reference phage was added, similarly to part 1, butfractionswere collected into conical tubes and werealkali-digested (9) prior to being counted in a scintillation counter at various times after in-fection (Fig. 1). Figure 2 shows the results of

sucrose gradient analysis of lysates obtained at

corresponding timesafter infection. This result is

independent of the MOI up to at least 15 phages per bacterium. Figure 2 represents the fate of

parental 32p label. Progeny label was analyzed at

the same times, and the patterns obtained in

sucrose gradient analysis were virtually

indistin-guishablefrom thoseofparental DNA; therefore,

they are not documented inthis paper.

Inhibition of long strand formation. Kozinski

has shown that addition ofCM at about 5 min

after infection inhibits recombination without im-pairingDNAreplication (6). If long singlestrands

are formed as a result of recombination, the

addition ofCM to the infected bacteria at this

time shouldinhibit their assembly. Indeed, if CM

isaddedat 5 minafter infection andthecellsare

incubatedfor 45minutes, eventhough50 to 100

phageequivalent units of DNA weresynthesized

(a figureatleasttwiceaslargeasthat whenlong single strands arefirst formedduring normal

in-fection),nolongsingle strandsarefound(Fig. 3).

This indicates thatsynthesiscanproceed without

necessarily forming long single strands. In

con-trast, addition ofCM at 8 to 10min allows the

appearance of

fast-sedimenting

DNA.

When the infected bacteria were pregrown in

5-BUdR, an unmeasureable amount of long

singlestrands was formed in the absence of CM.

When CM was added to bacteria infected in

5-BUdR medium, the amount of long single

strands formed was much less than in

light

medium (Fig. 4). When one notesthat

5-BUdR-labeledDNA sediments faster thanan

unsubsti-tuted DNA molecule of the same size

(10),

the

actualamountoflong

single

strands which were

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MILLER, KOZINSKI, AND LITWIN

Z 200

LLC

0t

/

unZ/

z/ ow

z

Z

100->Sz/

X. 50

w

0 5 lo 15 20 25

MINUTES AFTER INFECTION

FIG. 1. Synthesis ofDNA in E. coil B23, by

in-fected bacteriophage T4B01r. E. coli B23 was grown

to 3 X 108 cells/ml in low-phosphate TCG medium. The culture was divided into two parts: (i) infected

with T4 bacteriophagelabeledwith 32p (see Fig.2for

sucrosegradient analysis ofthispart); (ii) infectedwith

coldphage. To the latter 32pwasaddedat 2 minafter

infection,and DNAsynthesiswasmeasuredby uptake

of 32ptotrichloroacetic acid-precipitable,

alkali-resis-tantmaterial.

synthesizedinthepresenceof CMdecreases

fur-ther. However, bacteria infected in 5-BUdR

medium liberates5-BUdR-labeled, viableprogeny

in normal quantities. It appears, therefore, that

the formation of long single strands is not an

obligatory intermediate of DNA replication in

T4-infected cells.

Inallcases,theprocedure yielding thegreatest amountoflong singlestrandswasto add CMat

10to 12 min afterinfection, a time whenall the

enzymes necessary forrecombination and repair

of recombinantmoleculeswerepresentandyeta

timewhen maturationwasinhibited.

Length of denatured DNAasafactorprimarily

responsible for fast sedimentation. Incubation of

the infected bacteriawith lysozymepriorto

alka-linelysis isimperative; withoutlysozyme present

in the lysing solution, material which sediments

much faster than reference DNA in alkaline

sucrose

gradients

canbe detectedeven if CM is

added at the moment of infection. When this material is incubated with lysozyme, the parental label is found to sediment

identically

with the

reference DNA. The

alkali-resistant,

lysozyme-sensitivecomponent

presumably

isanassociation oftheparentalDNAwithcell wallmaterial. This association takes placeevenifCMisaddedatthe

momentofinfection,eveniftheparental phage is

inactivated with ultraviolet light, and it takes place with ambermutants, deficient inDNA

syn-thesis.

Figure

5 illustrates the results of alkaline

sucrose

gradient

analysis

ofonelysate treatedin

threeways:

(i) lysed

in 0.25MKOH,

(ii)

lysed by the

sarkosyl method,

and

(iii) lysed

by the Triton method. The lysate was E. coli Binfected with

T4BO1r

inthepresenceof CMandwas

incubated

for30minbefore

sampling.

Oneexplanation ofthelysozyme-resistant, fast-sedimenting material could bethatthereplicating pool ofphage DNA is such a tangled mass of

nucleicacid that during alkali denaturation indi-vidual single strands become trapped in a

"chicken wire-like" mesh which would sediment

as an aggregate in alkaline sucrose gradient. A

secondexplanation might bethat theassociation with cellwallmaterial which is sensitiveto

lyso-zyme when thetrapped DNA is of conservative

nature might be resistant when the DNA is in

replicative form, since replicativeDNA may have

a structurewhich facilitates the association with

cell wall material. Recombinant DNA is known

to have

"puffs,"

"gaps," and a highly tangled

structure(3,

6);

thisstructuremight maintainan

association withcell wallmaterial which couldbe more intimate and resistant to lysozyme. This would

impart

a

fast-sedimenting

characteristicto the DNA. Two experiments argue against this hypothesis.

First,fast-sedimenting, denaturedDNAcan be

isolated from an

alkaline

sucrose gradient,

dialyzed, and heated for2min at95 Cin standard saline-citrate

containing

1%

formaldehyde, and

thematerialwillstill sedimentas a

fast-sediment-ingcomponent. A partially renatured, mesh-like

structure might be expected to decompose at

meltingtemperatures in thepresence of

formalde-hyde.

Second, if the fast-sedimenting, denatured

DNAisincubated with

endonuclease

I so that the

DNA receives, on the average, one to two single

strandbreaks perphage equivalent unitof length,

the fast-sedimenting DNA is cut to short

frag-ments as predicted for a

linear

molecule. The

molecular-weight distributionofthe fragments of

a moleculewhich has been randomly cut can be

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[image:3.497.56.249.52.373.2]

C]

LLI

H

H 0

C)

0

1-0 H

z

Lli

w-'.'V28.3%

:L

.1.

9 min.

-BOTTOM

FRACTION

OF THE

LENGTH

OF THE

GRADIENT

FIG. 2. Alkaline sucrosegradient analysis of intracellular parental 32P-labeled DNA. Samples from part iof

the experiment (describedinFig.1)weretransferredatvarious timesafter

infectiont

toKCN,chilled,andlysed with

lysozymeandTriton X-100.3Hreference phagewereadded to the lysates and the mixtures were treated with

alkali andwerelayeredonS to20% alkalinesucrosegradients whichwereuniderlayeredwith1.0ml of saturated

sucrose. 3H = reference, integralDNA.

predicted; furthermore,thesedimentationpattern

of thesefragmentsina sucrose

gradient

has been

simulated in a computer

(7).

Figure

6 illustrates

thepredicted distribution of

fragments produced

by cutting molecules of different

length (L

=

phageequivalentsof

length)

with various average

numbersofcuts(x = number of

cuts)

per

phage

equivalent unit of DNA. When one to two cuts

per phage equivalent unit have degraded the

target molecules, the products all have

approxi-mately the same distribution regardless of the

originallength of the molecule. Afteroneto two

cuts per phage equivalent unit of DNA, the

products of size 1 molecules will sediment in a

sucrosegradient similarlytothoseof sizes3, 5,or

15, for example. However, ifthetargetmolecules

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MILLER, KOZINSKI, AND LITWIN

I-C)

H

llJ

0

llJ

0

11

0

A

5'cm

0

.5

-BOTTOM

B

8'cm

C

lO'cm

FRACTION OF THE

LENGTH

OF THE

GRADIENT

FIG. 3. Effect ofCMonformation oflong single strands. Inanexperimentsimilartothat describedin Fig.

1, CMwas addedat the indicated timesafter infection,andthecellswere incubatedforanadditional 45 min.

3H= reference,integralDNA.

are notlinear single-stranded DNA, theproducts

ofcuttingmaynotbeexpected tosediment simi-larly to reference DNA treated in an identical

manner. Figure7 illustrates theprinciple of this

experiment. If the fast-sedimenting material

ac-quires its sedimentation characteristics from a

structureresemblinga "chicken wire-like" mesh,

it would notbeexpected tofallcompletely apart

afterone to two cutsperphageequivalent unitof

length. Also, ifthelabeledDNAwereattachedto somelysozyme-resistant, alkali-resistantmaterial, a portion ofthe label would still sediment fast

after cutting. With these ideas under

considera-tion, thefollowing experiment wasconducted to testthehypothesis that thefast-sedimenting

ma-terialwascomposedofsinglestrandslongerthan

those isolated from mature phage.

E. coli B23 was grown to 3 X 108cells/ml in

high phosphateTCG medium and infected with

T4BOir

(MOI = 5.0) labeled with 32p at a

specific activity of 3.0. Chloramphenicol (100

Ag/ml)

was added to the infected cells 10 min

after infection, and the cells were incubated for

45min at 37 C. Thecells werelysedbythe

lyso-zyme-Triton method;

38H

reference DNA was

added to the lysates, and themixture was incu-batedfor 20minwith 0.25 M KOH. Themixture

was analyzed in an alkaline sucrose gradient

which had been underlayered with a pad of

saturated sucrose. 32P-labeled DNA reachingthe

pad was isolated, dialyzed against 0.07 M Tris

(pH 7.6) and supplementedwith single-stranded

3I reference DNA. This 32P-labeled DNA was

considered to be apparently long single strands.

Partof themixturewasreanalyzedin an

alkaline

sucrosegradient (Fig. 8A);this servedasacontrol

which showed that fast sedimenting strands

re-tained their characteristics after the isolation procedure.Partof the mixturewasheated to 95 C

for 2 min in standardsaline-citratecontaining 1%

formaldehyde; this part was analyzed on a 5 to

20% sucrose gradient containing 1%

formalde-hyde(Fig. 8B). Onecanseethat suchheat

treat-mentdoesnotdrastically affect thesedimentation

characteristic of the 32P-labeled DNA. Another

part of the fast-sedimenting moiety was

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[image:5.497.60.448.63.362.2]

F-C)

LLI

llJ

0

Q

LLJ

bi

11

0

0 5

0-BOTTOM

9'CM

1.0

(

D

0

12'

CM

. .

::1 ::1 : :,

: :, . I

: :,

I,

.,_.,

::~

.5

1.0

FRACTION

OF THE

LENGTH OF THE GRADIENT

FIG. 4. EffectofCMonformationoflong single strands producedinbacteria infectedin5-BUdRmedium. Cells

weregrownfortwogenerationsin5-BUdRmedium andwereinfectedwith 32P-labeledT4BO1r. CMwasadded at

various timesafterinfection,andthe cultureswereincubatedfor45min.Thecellswerelysed,andalkalinesucrose

gradientanalysis oftheintracellular, 32P-labeledDNAwasperformedwith the 3H integral DNA asa

referenice

bated with a very low concentration of

endo-nuclease I. As an internal

control,

3H-labeled

maturephageDNAindenatured formwasadded

prior to

digestion.

After treatment with endo-nuclease I, the mixture was incubated with 0.25

M KOH-0.02 M EDTA for 20 min and then

analyzed by

alkalinesucrose

gradient

sedimenta-tion

(Fig.

8D).

Itisobviousfrom this

graph

that the

32P-

and 3H-labeled DNA sedimented in a

similar fashionaftertreatmentwithendonuclease

I. The

apparently long

single

strands werecut to

thesize

fragments

predicted

for linear molecules. To test the size of

resulting

fragments,

fresh 32p referenceDNAwasadded inexcesstothemixture

treated with endonuclease Iand themixturewas

analyzed in an alkaline sucrose

gradient (Fig.

8C).By

comparing

thedistance sedimented

by

the

32p reference DNA (D1) with the distance

sedi-mentedbythe3H DNA

(D2),

one candetermine

thenumber of

single-strand

scissionsreceived

by

the DNA per

phage

equivalent

unitof DNA.This

canbe done

by

comparing Fig.

8Cwith

Fig.

7or

by

using

the

graph

of number of scissions

(X)

versusD2/D1ofLitwin,Shahn,and Kozinski (7).

Thenumberofscissions whichthe DNA received

from the endonuclease I was between one and two perphage equivalent unitof DNA. This

ex-periment supports the contention that the

fast-sedimentingDNAis actually composed of single

strandslonger thanonephageequivalentunit and

argues against thepossibilityoftheartefacts

dis-cussedbefore.

Simultaneous maturationof progeny and paren-tal phage DNA. Some theories of the in vivo replicationof T4bacteriophageDNApredict that

labeled parental DNA should appear in mature

progeny phage at a rate different from that of

labeled progeny DNA. Forexample, if T4 phage

DNAreplicatedinacontinuouslyelongating

con-catinate (11), 50% of theoriginal parental label

would be extendedout at oneend. IfphageDNA

then is encapsulated at this end, 50% of the

parental label would beencapsulatedintheinitial

stagesof maturation. The

"rolling

circle" model

predicts, on the other hand, that some of the

parentalDNAresides ina sanctuary attached to

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[image:6.497.52.438.65.366.2]

MILLER, KOZINSKI, AND LITWIN

L-a

w

w

0

w

0L

0

0 .5

*-BOTTOM

0

"TRITON

X-1OO

LYSIS"

p

:..

N

.5

lb

[image:7.497.63.448.57.366.2]

FRACTION OF THE

LENGTH OF

THE

GRADIENT

FIG. 5. Effect oflysing procedureonsedimentationcharacteristicsofparentalDNAin alkalinesucrosegradients.

E. coli B23 cellswereinfectedwith32P-labeledT4BOi',andCMwasadded at themomentof infection. The

cul-tureswere incubatedfor30minandchilled in NaCI-EDTA. Thesuspensionsweredivided into threeparts;each

partwaslysedbyadifferentmethod:A,lysedin0.25mKOH;B, lysed bythesarkosylmethod(I);andC,lysed

by the Triton method. 3H referenceDNA was addedtothe lysates, andthe mixtures were treated withalkali. The alkali-treatedmaterialwas layered on S to 20% alkaline sucrose gradients underlayered with a pad of

saturatedsucroseandanalyzedasusual. 3H = reference, integralDNA.

thecell membrane or resides as the core of the

rolling circle (2). Parental label would, in this

case, beexpectedtomature atarateslower than

that ofprogenyphage.Incontrast,thepossibility

that recombination disperses all parental

ma-terialuniformly throughout the pool of

replica-tive DNA should guarantee that both parental

andprogenyDNAin thepoolshould haveequal

chance ofbecoming incorporated into maturing

phages. To test this hypothesis, parental phage

DNA was labeled with 82p and progeny phage

DNA was labeled with tritiated thymidine, and

then the percentage of each label which became

resistanttodeoxyribonucleasewasdetermined as

afunction of time afterinfection.Theexperiment

wasconducted in thefollowingmanner.Aculture

of E. coli B23 was grown at 37 C to 3 X 108

cells/mlinhigh phosphateTCGmedium

supple-mented with 5 Mg ofthymidine per ml, 5 ,ug of

5-fluorodeoxyuridineperml,and 25,gof uridine

perml. The culture was infected at an MOI of

5.0 with T4 bacteriophage labeled with 32p at a

specific activity of 0.3 mc/mg ofP. At 11 min

after infection, 3H-thymidine was added to the

cultureat a final specific activity of 2 mc/mg of

thymidine. At 12minafterinfection,the infected

cells were chilled, centrifuged, washed with and

suspended in high phosphate TCGmedium

sup-plemented with 100 Mgof cold thymidineperml.

Theincubation of the cellsat37 Cwascontinued,

andatintervalsthereafter samplesoftheinfected

bacteriawerechilled; oneportionof each sample

was tested for the presence of mature progeny

phage, andoneportionwaslysed by the

lysozyme-Triton method. The lysates were incubated with

pancreatic deoxyribonuclease; before and after

treatmentwith deoxyribonuclease, aportionwas

precipitated with trichloroacetic acid, transferred

toaglass-fiber filter, dried, and countedfor

radio-activity. (Aseparate testshowedthat inamixture

v

.

SARKOSYL

LYSISN

.:1

.,

_J

0

w

a

374

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D

A B

Reference DNA Reference

DNA

ENDO I +ENDO I

[image:8.497.50.462.61.658.2]

*

r_

FIG. 7. Hypothetical results of cutting long single

strandswith limited amount ofendonuclease I. A, true

Ll long strand; B, fast-sedimenting artefact.

of 3H-labeledDNA and 32P-labeled phage which

was treated in a reconstruction

experiment,

the

3H was rendered completely soluble in 0.3 M

trichloroacetic acid, whereas the 82p was

com-pletely

insoluble;

i.e.,

the

procedure truly

dis-criminated betweenDNA outside and insidethe

phage

head.)

In

Fig.

9, the percentage of each

label which became resistantto

deoxyribonuclease

is plotted, as well as the

production

of mature

progeny phage. It is apparent from this graph

that the SP and 3H become resistant to

deoxy-ribonuclease at almost identicalrates; this

indi-catesthat the

parental

andprogeny DNAformed

priortomaturation become enclosed inthephage

L-l headatthesamerate.

L.2

Computer

simulation

of

recombination

and the

L*3

Lz15 FIG. 6. Predicted size distributions of DNA

frag-mentsresultingfromrandom breaks. The graphs

pre-sentedin thisfigure were produced by the methodof

Litwin,Shahn,andKozinski (7).The graphsrepresent

computer-simulated sedimentation patternsoffragments

of long molecules. Thefragments were produced by

0 cuttingdifferent-length "molecules" (L= phage

equiv-alentunitsoflength) withvariousaverage nwnbersof

12/D TOP cutsperphageequivalentunitofDNA (X = cuts per

phage equivalentunitofDNA).

A

=

0.5

Ls2

Lw2

A=2.0

2

Botom

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MILLER, KOZINSKI, AND LITWIN

20-c

3ap

I-10

-0

.S 1.0 0 .5 1.0

LGBA D

20

LL_ 0

Z 10

0~

0 .5 0 .5

-i--BOTTOM

FRACTION OF THE LENGTH OF THE

[image:9.497.258.457.46.315.2]

GRADIENT

FIG. 8. Sedimentation of long single strands after

heatingat95 CoraftertreatmentwithendonucleaseI.

Long single strands labeled with parental 32p were

isolatedfrom thepad ofanalkalinesucrose gradient,

supplemented with 3H integral DNA, and dialyzed

against0.07MTrisbuffer (pH 7.6).This material was

treated in thefollowing manner. (A) Part was

reana-lyzedinanalkalinesucrosegradientunderlayeredwith

apad. (B)Partwasheatedto95Cfor2minin standard

saline citrate containing 1% formaldehyde and

ana-lyzedinasucrosegradientcontaining1%formaldehyde.

(CandD)Partwasincubated with endonuclease Iand

split into twoportions; one (D) was analyzed on an

alkaline sucrose gradient and one (C) was

supple-mented with a 10-foldincrease of "2P-labeled,

single-strandedreferenceDNA.

production oflong molecules. Evidencepresented

in this paper supports thehypothesis previously

expressed (6) that recombination between

frag-mentsof DNAmayleadtotheformation of long

molecules and ultimately to the production of

circularly permuted, mature DNA molecules. It

remainstobeproven,however, howfeasiblethis

mechanismis,froma stochasticpoint of view.

Thisquestioncanbeapproached by the method

cn

4-c

0 0

30

a

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0

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0

0 101

I..

C

0~

-10 aX

C

-8 0

80

16 t, a}

C

'4

12 CL

Minutes after Infection

FIG. 9. Maturation ofprogeny ('H) andparental

(32p) phageDNA. A cultureofE. coliB23wasgrown

to 3 X 108 cells/ml in high-phosphate TCG medium

and was infected with "2P-labeled T4BO,r

bacterio-phage. 3H-thymidine was pulsed from 11 min after

infectionito12min,and thenwasfollowedbya"chase"

as described in the text. At intervals thereafter,

in-fectedcellswerechilled andlysed bythe LTLprocedure.

The lysates were treated with pancreatic

deoxyribo-nuclease, andaportionwasassayed fortrichloroacetic

acid-precipitable material.

of computer simulation. The T4 DNA molecule

can be represented by a series of numbers

(2 X 105) arranged in a circularly permuted

manner and endowed with a 5% terminal

re-dundancy (7). Two basic hypothetical modes of

recombination can be considered: (model 1, cut

and strip model) one which would demand

double-strand cutting and enzymatic removal of

opposite strands, resulting, in effect, in the two

original neighbors being unabletorecombine with

each other [a model similartothat postulated by

Thomas(11)]; and (model2,diagonalcutmodel)

onewhichwillgeneraterecombining fragmentsby

a diagonal cut between two nicks located in the

trans position. Nicks or cuts can be introduced

intothesimulatedmolecules byarandom-number

generator by a procedure previously described

(7).Theprograminvolved in simulating

recombi-nation ina simulated cell is as follows. A

popu-lation ofsimulated bacteria are infected with a

poissonly distributed MOI of12 phages, 2 light

and 10 heavy. (The difference in the density in

376 J. VIROL.

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[image:9.497.58.252.58.393.2]

thecontext of this paperplaysno role, butwillbe

used foranalysis ofdensity classes of simulated

recombinants presented elsewhere.) Each of the

infecting phage DNA receives a randomly

dis-tributed number of nicks or cutsassigned bythe

random-number generator. After completion of

thisstep, theprogram calls forseparationof each

infecting molecule intosubunits eitherby (model

1) introduction ofa cut on the opposite strand, which will be followed by the removal of 400

"nucleotides" on twoopposite strands (let'ssay, fromsimulated 5' end ofstrands) or(model2) by

performing a diagonal cut between two closest

translocated nicks. The resulting fragments are

characterized

according

totheirsize, number,and

sequence of numbers within the open

comple-mentary area, a sequence, which being unique,

offers no ambiguity. The

listing

ofthe resulting subunitsand their terminalcomposition isstored

in thememory ofthemachine. Thenext stepisa

simulated recombination which proceeds as

follows. Thecomputerretrievesatrandomoneof thefragmentsand matchesit

against

arandomly retrieved additionalfragment out ofthepool of

fragments in the simulated bacterium. Ifthereis

no homology between the two fragments ofat

least 12bases, thesecond

fragment

istransferred

backtothepool.Themachineproceedsto

pick

at

random a third subunit, checking it for possible homology. If

homology

isindeed

found,

adimer

(recombinant)

is formed which is returnedtothe

pool in which further random

matching

opera-tions are performed until all such possibilities

have been exhausted. After

completion

of the

recombinational process, recombinant molecules

are characterized

according

to their

lengths,

densities, possible

unmatched areas, and sizes of gapsand"whiskers."The results ofthese

charac-terizations are stored in the memory of the

machine. After

completion

of this

matching

processwithinthe

bacterium,

thecomputer

keeps

tallyof the

resulting population

of

fragments

and

recombinants and proceeds to evaluate another

bacterium in a similar manner. The simulation usually involves

103

to 10 simulated bacteria. Upon

completion

ofthe

experiment,

the results

are retrieved from the memory of the machine

and canbe

represented

ina

variety

ofways. The

machinemaybe instructedtowithdrawthe

mole-cules fromonebacterium and drawthemolecular

configurations

of the recombinants

(Fig. 10).

Alternatively, the machine may be instructed to

subdivide the

resulting fragments

in the entire

experiment

according

to their molecular lengths

and draw the results as

simulated,

sucrose

gradient distribution

(7).

In this case, the

dispersion

of r = 0.0323 has

been allowed asthis

corresponds

to the best

ob-served dispersion of intact molecules in sucrose gradient in our laboratory. The simulation of both

models ofrecombination reveals that whereas the

model invoking cutting and denuding the ends, "cut and strip model" proposed by Thomas (11), is not compatible with the formation of long molecules and, for that matter, produces

recombi-nants of short size, the model ofdiagnonal cut

produces a sizeable proportion of recombinant

molecules which are larger than size one. Signifi-cantly, the ends of the participating molecules

were not allowed to be engaged in

recombina-tional events, i.e., to be partially denuded. Even

without this option, the distributionof observed

sizes of recombinants, for a limited number of

nicks and limitednumber ofinfectingmolecules,

resembles pretty well the distributions observed

in an in vivo experiment.

Wedo want toemphasizethatcomputer

simu-lation proves the stohastical feasibility of the

diagonal cut-typerecombination as a mechanism

generating long molecules. Scrutiny of the draw-ing represented in Fig. 10 shows that recombinant

molecules obtained by this method contain

nu-merous single-stranded gaps and "whiskers."

Those could quite feasibily be filled or excised,

respectively, by the proper enzymes. Indeed,

electron microscopy of recombinant molecules

isolated from CsCl reveals a sizeable amount of

"whiskers" and branches (3).

DISCUSSION

T4bacteriophage DNA iscircularly permuted

and terminally redundant (8, 12). Circular

per-mutation could be achieved by a number of

different mechanisms. One mechanism could

in-volve theformation of acircularDNA molecule

during thereplication process. A second

mecha-nismcouldinvolve alongmolecule of intracellular

phage DNA which would becut into units ap-proximately one phageequivalent unit in length

during the maturation process. Thelongmolecule

could be created either through replication of

progeny DNA or through recombination.

Ob-servation of

fast-sedimenting

DNAinanalkaline

sucrose gradient indicated that long molecules

mayindeed exist atlatertimes after infection.

Experiments were undertaken to demonstrate that this

fast-sedimenting

DNA was actually composedofsinglestrandslongerthanonephage equivalent unit. Because neutralsucrosegradient analysis had been shown to be an unreliable

indicator of molecularweightofreplicativeDNA

(6), it was feared that analysis atpH 12.5might

also be unreliable. For

example,

itwasconceivable

that DNAsedimentedfast in thesucrosegradient

atpH12.5notbecause of itslengthbut because of

its association with some

lysozyme-resistant,

on November 11, 2019 by guest

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MILLER, KOZINSKI; AND LITWIN

I

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FIG. 10. Computerdrawing of the contentsofa "single cell" infectedwith 10 heavy (thick line) and2light

(thin line) phage. After completion ofthe recombinationprocess, performed according to the "diagonal cut"

model, 16fragmentsresulted. Thosearedrawn bythe computeras twoparallel linesrepresentinga bistranded

structure. Note thediversity of length (some sizably largerthan onephage equivalent unit) andthe presence of

single-strandedgapsand"whiskers." Note also that thediagonalcutmodel allows reunionoftwooriginally

neigh-boringfragments,areunionwhich, exceptforanick (thinslashacrossthesingle strand),isperfect,notendowed

withagap or"whisker,"andshould berepairablein vivoby ligase without the involvementofa "filling"or

ex-cising enzyme. Fragment #2 illustrates thefinedetails ofthematchingprocedureperformed by thecomputer,

namelyby displaying thebase numbers initiating andending each ofthe constituents oftherecombinant. The

drawings wereperformed bythecomputerforarandomlychosen "bacterium."

alkali-resistant material, or because individual

single strands in the pool of replicative DNA

could not, for any number ofreasons, be

dis-associated. Two experiments presented here

argue in favor of the hypothesis that the

fast-sedimentingDNAwasactuallycomposedoflong

single strands. First, the DNA still sedimented

"fast" after reisolation and heating to 95 C.

Second,andmoreimportant,theapparentlylong

singlestrands werecut tothe sizefragments

pre-dictedbycomputer analysisfor linear molecules

exposedtoalow concentration of endonuclease I

(aconcentration which gaveonetotwo cutsper

phage equivalent unit oflength). Onthe basis of

any of the proposed artefacts, a residual,

fast-sedimenting core of this DNA moiety should

have remained after only one to two cuts per

phage equivalentunit ofsingle strandedDNA.

Having supportedthe contentionthat the

fast-sedimenting DNA, atpH 12.5, was indeed long

singlestrands, the nextquestioninvestigatedwas

whether the long single strands were produced

bya mechanism ofreplication orby

recombina-tion. A computer simulation had in fact shown

that long molecules could be produced by a

mechanism of random breakage and reunion.

Thisshould be followed by efficient repairof the

single-strand intersections in the polynucleotide

chain of the recombinant molecules.

If the long singlestrandswerebeing produced

asa result ofrecombination, one should expect,

first ofall, to seethe appearance oflong single

378

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on November 11, 2019 by guest

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DIAGONAL CUTMODEL

SI. t.00 6.00 0.60o .es LENGTH (PEU) a

DIAGeNAL CUT MODEL SIGMA = 0.0323

CUTANDSTRIP MODEL SIGMA= 0.0323 DIAGONALCUT MODEL SIGMA =0.0323

Ws S _8-NICKS

D2/0l '?oo I00D2/D o

I~~~~~~~~~~~~~~~~

FIG. 1 1. Simulatedsucrose gradient distributions ofthleproducts of recombination in bacteria infected with 12 phages (no replication allowed). The infecting phages were distributed poissonly and received 4, 6, or 8 nicks (also distributed poissonly). For 6-nicks class, two models ofrecombination were simulated, diagonal cut model and cut and strip model. ineach panel, a broken sigmoid curve represents the summed percentage of recovery (integral graph) ofunnicked, integral DNA. Notethiatfor any given number of nicks in the diagonal cut model, recombina-tion generates a sizeable fracrecombina-tion of molecules larger than size 1.This can be appreciated while comparing integral graph of per cent recovery of recombinants (solid, continuous sigmoid line) with that of reference DNA (broken line). The cut-and-strip model is notcompetent for production of long molecules, andlrecombinants actually tend to peak at approximately 0.4 phage equivalent unit length (PEU).

strands coincidentallywith theonsetof

recombi-nation, and, second, theaddition of CM at the

propertime would inhibittheformation oflong

single strands while not affecting replication of

DNA. Addition of CM around5min after

infec-tionwasknowntoinhibit recombination without

inhibiting replication (6).Infact,addition of CM

at 5 min inhibited theproduction oflong single

strands, as predicted, even though 45 phage

equivalent unitsof DNAweresynthesizedinthis

particular experiment. (Themaximumfigure,not

reported here,canbeashighas100phage

equiva-lentunits.)

Many of the experiments documented in this

paperhave beenperformedwith aparentallabel;

it should be emphasized here that most of the

results havebeenconfirmedalsoforprogeny DNA

labeledby uptake of 3H-thymidine or32p. The

in-hibition of long single strands by CM without

inhibiting DNA replication is important when

considering another unlikely but possible

ex-planationof theprevious results. Iftherewere20

hypothetical"sites" forreplicationinthecell(as

might be inferred from the fact that 20 phage

equivalent units ofDNA are synthesized before

anylong singlestrandsareformed), and ifallthe

sites hadto beoccupied bya phageDNA

mole-culebeforeanylong singlestrandswereproduced,

then it wouldnotbesurprisingthatnolong single

strandswereproduced duringthe first rounds of

replication. However,sincelong singlestrandsare

not produced when recombination is inhibited

even though 50to 100 phage equivalent units of

DNAaresynthesized, thishypothesis isvery

un-likely.

Theabsenceoflong singlestrandsproduced by

phage-infecting bacteria in 5-BUdR medium

argues against the hypothesis that long single

strands are an obligatory intermediate of DNA

replication.

I NEGRAL DNA

S 4-NICKS I;

A6s.60 1.20 1.00 0.00 0.60 0.6 0.20 -'?bo

D2/01

I

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[image:12.497.46.449.67.350.2]

MILLER, KOZINSKI, AND LITWIN

Several results presented here and elsewhere

bear directly on various published models of

DNA replication. In any model explaining the

replication of T4 bacteriophage DNA, the

fol-lowing experimental results should be taken into consideration. A pulse of 3H-thymidine

incor-porated in the initialstages ofreplication is not

covalently bonded to the parental DNA; i.e.,

parental 32p and progeny 3H labels separate in

an alkalinesucrose gradient and arerepresented

by both T4 bands (4). An interesting

permuta-tion of this resultisthatapulseof 5H-thymidine

from 3to6.25min after infection is incorporated

intoa piece ofprogenyDNA the samesizeasa

piece labeled from 6to 6.25 min;the size of this

piece is about that of the fragment Kozinski called FSBP (3). The size of the fragment labeled

from 3 to 3.25 min, however, is much smaller.

None of this pulse-labeled progeny DNA was

covalently attached to parental DNA. It should

be noted that these results were obtained by

infection with a 32P-labeled parental phage, so

the integrity of the parental DNA was always

assured in theseexperiments; i.e., the size of the

progeny pieces could not have been caused by

some random nicking by an enzyme such as

endonucleaseI,norcouldthe bulk oftheprogeny

DNA have been separated from parental

ma-terialbysome random-nicking process.

Any model explaining the replication of T4

bacteriophage DNA should be consistent with

theresult that there isnopreferential maturation

of parental DNA into progeny phage. This

proposal is basedonthe result that the increase

in percentage of labeled DNA which becomes

resistant to deoxyribonuclease as maturation

proceeds is the same whether the labeled DNA

is of parental or progeny origin; the progeny

DNA was labeled from 11 to 12 min after

in-fection with a pulse of 3H-thymidine. This

ex-periment discriminates againstamodelbasedon a

continuously elongating concatenate ofprogeny

DNA. This model predicts that when

matura-tionproceeds from the end, 50% of theparental

label should be encapsulated immediately at the

beginning of the maturation process. Even if

parental label were recombined away from the

end of theelongatingconcatenate, someresidual

label would mature preferentially. Predictions

of the rolling circle model (2) of DNA

replica-tion do not agree with this result either; no

sanctuaries such asthe core ofthe rolling circle

or the end attached at a membrane can be

oc-cupied by parental label. In addition, thereare,

apart from physical-chemical controversies,

genetic phenomena which are not included in

the model of the continuously elongating

con-catenate, such as unquestionable fact of

recom-binationand the phenomenon ofclonal

distribu-tion of mutants or recombinants. The best

hypothesisfitting the data is that recombination

distributes parental label randomly through the

replicative pool, so all replicative DNA has an

equal chance of being encapsulated. Thus the

mechanism generating long molecules is a

mo-lecular recombination, a phenomenon

ulti-mately determining circular permutation in T4

DNA.

ACKNOWLEDGMENTS

Thisinvestigationwassupported by NationalScience Founda-tion grant GB 13048 and by Public HealthService grantCA 10055 from the National Cancer Institute. Thecomputer evalua-tion of data was performedin the University of Pennsylvania Medical School Computer Center, supported by Public Health ServicegrantFR15-06.

Oneof the authors (R.C.M.)wassupported by Public Health ServicegrantGM-006-94-09 awardedtotheGraduate Groupon

Molecular Biology, University of Pennsylvania.

LITERATURE CITED

1. Frankel,F. F. 1968. Evidence forlongDNAstrands in the replicating poolafter T4infection. Proc. Nat. Acad. Sci. U.S.A. 59:131-138.

2. Gilbert, W., and D. Dressler, 1968. DNA replication: the rolling circle model. Cold Spring Harbor Symp. Quant. Biol. 33:473-482.

3. Kozinski,A. W. 1968. Molecularrecombinationinthe ligase-negative T4 amber mutant. Cold Spring Harbor Symp. Quant. Biol. 33:375-391.

4. Kozinski, A. W. 1969. Unbiasedparticipation ofT4phage DNA strands in replication. Biochem. Biophys. Res. Commun. 35:294-299.

5. Kozinski, A. W., and P. B. Kozinski, 1963. Fragmentary transfer of82P-labeled parentalDNA toprogenyphage. II. The average size of the transferred parental fragment. Two-cycle transfer. Repair of the polynucleotide chain after fragmentation. Virology 20:213-229.

6. Kozinski,A.W.,P.B.Kozinski,and R.James.1967. Molecu-lar recombination in T4 bacteriophage deoxyribonucleic acid:I.Tertiarystructureofearly replicativeand

recom-bining deoxyribonucleicacid.J. Virol. 1:758-770. 7.Litwin, S. E.,S.Shahn,andA.W.Kozinski, 1969.

Interpreta-tion ofsucrose gradient sedimentationpattern of DNA fragmentsresultingfrom randombreaks. J. Virol.4:24-30. 8. MacHattie, L.A.,D. A.Ritchie,and C. A.Thomas. 1967. Terminalrepetition inpermutedT2 bacteriophage DNA molecules. J. Mol. Biol. 23:355-363.

9. Schmidt, G., B. Hershman, and S. J. Tannhauser. 1945. The isolation oflambda-glyceryl-phosphorylcholine from incubated beefpancreas; its significance for the inter-mediary metabolismof lecithin. J. Biol.Chem. 161:523. 10. Shahn, E., and A.W. Kozinski. 1966.Fragmentarytransfer

of32P-labeled parental DNA toprogeny phage. III. In-sertion ofa single parental fragmrnt to the progeny

molecule.Virology30:455-470.

11.Thomas, C. A. 1966. Recombination of DNA molecules. Progr. Nucl. AcidRes.Mol. Biol. 5:315-335.

12. Thomas, C. A.,andI.Rubenstein. 1964. The arrangements

of nucleotidesequencesinT2 and T5bacteriophageDNA molecules. Biophys.J. 4:93-106.

380

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