Multidimensional analysis of intracellular bacteriophage T7 DNA: effects of amber mutations in genes 3 and 19.

Copyrighti) 1987,American Society for Microbiology

Multidimensional Analysis of

Intracellular Bacteriophage

Effects of

Amber Mutations in Genes

3

and 19

T7 DNA:

PHILIP SERWER,* ROBERT H. WATSON,AND SHIRLEYJ. HAYES

Department of Biochemistry, The University of Texas Health Science Center, San Antonio, Texas 78284-7760

Received13 February 1987/Accepted 20 July 1987

By use of rate-zonal centrifugation,followed by either one- or two-dimensional agarose gel electrophoresis,

theformsof intracellular bacteriophage T7 DNA produced by replication, recombination, and packaginghave

beep analyzed. Previous studies had shown that at least some intracellular DNA with sedimentation coefficients between32S (the S value of mature T7 DNA) and 100S is concatemeric, i.e., linear and longer than mature T7 DNA. The analysis presented hereconfirmed thatmost ofthisDNA is linear, but also revealed a significant amount of circular DNA. The data suggest that these circles are produced during DNA packaging. It is proposed that circles are produced after a capsid has bound twosequential genomes in a concatemer. The size distribution of the linear,

concatemeric

DNA had peaks at the positions of dimeric and trimericconcatemers.

Restriction endonuclease analysis revealed that most of the mature T7 DNA subunits ofconcatemers were joinedleftendtoright end. However, these data also suggest that a comparatively small amount of left-end to left-end joining occurs, possibly by blunt-end ligation. A replicating form of T7 DNA that had.an S value greater than 100 (100S+ DNA) was also found to contain concatemers. However, some of the 100S' DNA, probably the most branched component, remained associatedwith the origin after agarose gel electrophoresis. It hasbeenfound that T7protein 19, known to be required for DNA packaging, was also required to prevent loss, probably by nucleolytic degradation, of the right end of all forms of intracellular T7 DNA. T7 gene 3 endonuclease, whoseactivityisrequired for both recombination ofT7 DNAanddegradationof host DNA, was required for the formation of the 32S to 100S molecules that behavedasconcatemersduring gel electrophoresis. In theabsence of gene 3 endonuclease, the primary accumulation productwasorigin-associated 100S+ DNA withproperties that suggest the accumulation of branches, primarily at theleft end of mature DNA subunits within the 100S+ DNA.

Tounderstand the processesofDNAreplication,

recombi-nation, and condensation, the DNA inbacteriophage-infected

cells has been fractionated and characterized (reviewed in

references8, 13, and54). In thecaseofbacteriophageT7, the

mature DNA is linear

and

terminally repetitious. Mature T7

DNA has a unique permutation of its sequence (9, 33). The

sedimentation

coefficient,

S,ofmature-length

(monomeric)

T7

DNAis32(51). Afterfractionation of T7-infectedEscherichia

coliby rate-zonalcentrifugation, most ofthe DNAnot

pack-aged in

bacteriophages

is also not monomeric. DNA that

sediments between32S and 100S is observed. LinearDNAs

longer thanmatureT7 DNA(concatemers)are atleastsome

of the 32Sto100SDNA(16, 21, 23,37, 50). Inadditiontothe

32S to lOOS DNA, a replicating (and therefore branched)

DNA that appears collapsed when viewed by electron mi-croscopyandthatsedimentsmore

rapidly

than100S

(100S+

DNA)hasbeenobserved (23, 29, 38). The 100S+ DNAhas

also beenreferredto asflowers (23, 29). SomeT7

concatem-ers are partof

100S+

DNA(43, 44). In the case ofseveral

double-strandedDNA

bacteriophages,

including X,

P22, T2,

T3, and T4as well as T7, concatemers are

packaged by

a

preformed, DNA-free

capsid (procapsid);

a concatemer is

cutto maturesize either

during

orafter

packaging

(reviewed

in references 10, 11, 12, 46, and59).

To understand theformation andpackagingof

concatem-ers, their characteristics have been determined. Profiles

suggestingthe presenceof

discrete-length

concatemers

(dim-ers,etc.)aresometimesbutnotalways seen

a.fter rate-zonal

*Corresponding author.

centrifugation in sucrose gradients, followed by

fractiona-tion ofthegradient and assay for DNA (16, 21, 23, 37,50).

However,theaccuracyofthe assay andtheresolutionpf the

sucrosegradientarenotsufficienttoreliablydetect

discrete-lengthconcatemers in thepresence of othertypes ofDNA

(variable-lengthconcatemers,forexample).A morereliable

procedureis to couplerate-zonalcentrifugation with

proce-dures of agarose gel electrophoresis developed (40) for

optimal resolution of concatemers. In

addition,

two-dimensional agarose gel electrophoresis procedures have

beendeveloped for

determining

the conformation

(circular,

linear, branched) of DNA (1, 41, 45).

Application

ofthe

latterprocedurestoT732Sto100SDNAwould

improve

the

characterization of this DNA,

particularly

the detection of

comparatively small amounts of a conformer

(circles,

for

instance)present amongmoreabundant linear DNA. In the

present study, these procedures ofagarose gel

electropho-resis and

analysis

of

fragments

released

by

restriction

endonucleases were applied to 32S to 100S T7 DNA with

somesurprising results.

Apparently, before

becoming

partof

either

100S'

DNAor

a pure concatemer, T7 DNA

replicates

as a linear

(as

opposed

to

circular)

molecule with the

primary origin

of

replication 17% of the length of the mature DNA from the

geneticleft end(17, 60, 61).

Therefore,

itis

presumed

that T7

concatemers are formedby end-to-end

joining of

the

prod-uctsofreplication (24, 58). Inadditiontoend-to-end

joining

during

concatemerization, joining

of genomes to form

branchesoccurs

during

DNArecombinationand

replication.

Genetically

removing

the

product

of T7 gene

3,

p3

(the

protein productsof T7 genes will bereferredto

by

pfollowed

3499

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by the gene number [53]), an endonuclease that assists in degradationof the host DNA (4, 36) and selectively cleaves branched DNA(7)causes accumulationofrapidly sediment-ing(>32S) DNA(16, 24, 27). Although it has been assumed that the high S value of this DNA was caused by concatemerization (16, 24, 27), the participation of p3 in genetic recombination (22, 26, 27, 31, 49) and the branch-cleaving activity of p3 suggest that absence of p3 would

causeaccumulationof branches. Accumulation of branches is anotherpossible cause of thehigh S value in the absence ofp3. However, the rapidly sedimenting DNAobserved in the absence of p3 has never been characterized beyond determination of its S value. Thus, inthe present study the above analysis applied to DNA from wild-type T7-infected cells has also been applied to DNA from a nonpermissive host infected by T7 carrying an amber mutation in gene 3

[referred to as T73; other amber mutants will also be indi-cated byaddingto T7 the number ofthe mutant gene(s)].

In the absence of p19, a protein necessary for DNA packaging and no other known process (34), rapidly sedi-menting DNA, presumed but not proven concatemeric,

accumulates (24). Thus, to complete the above analysis of intracellularDNA, the rapidly sedimentingDNAthat

accu-mulatedwheneitherT719orT73,19infected anonpermissive host hasbeenanalyzedasdescribed above. The implications

formechanisms of T7 concatemerformation andutilization are discussed forthe results obtained with all of the above bacteriophages.

MATERIALSAND METHODS

Strainsofbacteriaand bacteriophages. Bacteriophages T73 (mutant29[52])andT719 (mutant10[52])werereceivedfrom

F. W. Studier. T7319 was constructed by a genetic cross,

performed as previously described (52). Lysates and the

contents of lysates obtained by infecting a nonpermissive

host withT7 ambermutantswillbe referredto by thename

of the mutantbacteriophage.Forexample,alysate obtained

with T73 will be referred to as a T73 lysate. To avoid confusion, wild-typeT7 will bereferredtoasT7WT. The host

forT7WTand thenonpermissive host for ambermutantswas

Escherichia coli BB/1. The permissive host for amber

mu-tantswasE. coli 0-11'.

Buffersandreagents. Standard/G buffer contained0.15 M NaCl, 0.05 M Tris chloride, pH 7.4, 0.005 M EDTA, and gelatin (100,ug/ml).Cultures forradiolabeling of intracellular

T7 DNA were grown in M9 medium (20). Electrophoresis buffer A(0.05Msodiumphosphate, pH 7.4, 0.001M EDTA) was used for unidirectional agarose gel electrophoresis.

Electrophoresisbuffer B (0.01Msodium phosphate, pH7.4, 0.001 M EDTA) was used fortwo-dimensional agarose gel

electrophoresis. Sample buffer contained 0.005 M sodium phosphate, pH 7.4, 0.001 M EDTA, and bromphenol blue (400 ,ug/ml) with the indicated concentration of sucrose.

SeakemLEagarose(Marine Colloids Division,FMC Corp., Rockland, Maine), wasusedfor allagarosegels. 3H-labeled

thymidine (40to70Ci/mmol)waspurchased from

Schwartz-Mann (Orangeburg, N.Y.). Nycodenz was purchased from

Accurate Chemical and Scientific Corp. (Westbury, N.Y.). All restriction endonucleases were purchased from New

England Biolabs (Beverly, Mass.).

Infection, radiolabeling, and lysis ofcells. To detect and characterize T7DNA in E. coli infected with eitherT7WTor

aT7mutant, cellsweregrowninlog phaseto4.0 x 108/ml in

M9 medium at 30°C with aeration. The cells were infected

(multiplicity of 15), and incubation was continued at 30°C

with aeration. Intracellular T7 DNA was then labeled with

3H by adding

[3Hlthymidine

tothe infectedcellsat thetime

indicated.T7 stopsthesynthesis ofDNAbyits

host,

and the

DNAlabeledbythisprocedure isprimarilyT7 DNA

(21,

23).

To quench the process ofinfection, either part or all ofan

infected culture was mixed with an equal volume of an

ice-cold solution that contained 0.3 M NaCl, 0.1 M Tris

chloride, pH 7.4, 0.01 M EDTA, 0.008 M

KCN,

and 50%

sucrose. After quenching, infected cells were pelleted and lysed by a procedure described previously (38), except that the detergent used was Sarkosyl NL97 instead ofBrij (58).

Sarkosyl NL97 was used because in preliminary

experi-ments it wasfound to inactivate DNasesthat were presentin infected cells and that were not inactivated by Brij (58).

These DNases interferedwithanalysis ofDNAby

digestion

with restriction endonucleases. The above lysis procedure did not disrupt mature bacteriophage T7 and also avoided

partitioning the lysate before centrifugation. Partitioning can result in selective lossof either one or more typesof DNA in thelysate. However, the capsids of the previouslydescribed

T7 capsid-DNA complexes are released from DNA

by

Sarkosyl (39).

Fractionation of DNA by centrifugation.Afterradiolabeling

ofintracellular T7DNAandlysis of infected cells, 100 ,ulof

lysate was diluted into 500 ,ul of standard G buffer, mixed gently, and then centrifuged through abiphasic gradientof

sucrose and Nycodenz (sucrose-Nycodenz gradient). To avoid breakage from hydrodynamic shear (6, 38), thelysates were pipetted slowly (<0.2 ml/s) through a comparatively wide-bore (1 mm diameter) micropipette. The gradient con-sisted of a 9.4-ml linear 5 to 25% sucrose gradient poured over a2.4-ml linear Nycodenz gradient (1.13 to 1.26g/ml); all partsof the gradientwere in standard/G buffer. The density of solutions of Nycodenz was determined from the refactive index (32). After layering the entire diluted lysate on the sucrose-Nycodenz gradient, centrifugation was conducted for 120 min at 35,000 rpm and 18°C. During this centrifuga-tion, intact, mature bacteriophages sedimented through the entire sucrose gradient and 50 to 70% of the Nycodenz gradient without detectable loss of DNA. All 100S+ DNA sedimented to the sucrose-Nycodenz interface and was buoyed there because double-stranded DNA has a density of 1.126g/ml in a gradient of Nycodenz (14). Thus, the100S+

DNAdid not obscure the peak of bacteriophage-associated 3H after sedimentation in a sucrose-Nycodenz gradient. In the sucrose portion of the biphasic gradient, DNAs below 100S were fractionated by S value. The position of 32S DNA (monomeric T7 DNA) was assumed to be the position of the peak intensity of the monomeric DNA band observed after unidirectional agarose gel electrophoresis-fluorography (see below) of DNAs in fractions of the gradient (see Fig. 2). The positions in the gradient expected of dimeric, trimeric, and tetrameric concatemers (no. 2, 3, and 4 on Fig. 2) were calculated from the position of monomeric DNA (no. 1 on Fig. 2) by using a relationship described in reference 15. Gradients were fractioned by tube puncture; all fractions had the same number of drops. DNA-associated 3H in fractions of sucrose-Nycodenz gradients was assayed by acid-induced precipitation of the DNA, collection of precipitated DNAon a filter, and liquid scintillation spectrometry, performed as described previously (38). Recovery of 3H-labeled DNA from gradients was greater than 90%.

The concentration of cells in the lysates (before dilution) was 5 x

1010/ml,

25% less than themaximum concentration that can be used without observing cell concentration-dependent changes in sedimentation profile (data not

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shown). Anear-maximum concentration of cells was used to

maximize

the amountof

3H

incorporated

intoDNA.Raising

the ratio of

[3H]thymidine

concentration to cell

concentra-tion did not raise the amount of

3H

incorporated (data not

shown).

The profile of acid-precipitable

3H

in the fractions of a

sucrose-Nycodenz gradient is presented as a plot of the

percentage of the total

3H

versus fraction number. To

calculate this percentage, acorrection was made for

differ-ences in the volume of fractions as follows. Fractions in the sucrose portion of the gradient were found to have a volume greater than the volume of fractions in the following regions of the gradient, by the factor indicated: Nycodenz portion, 1.38; interface between Nycodenz and sucrose, 1.16; Sarko-syl-containing four fractions at the origin, 1.24. Fractions are referred to by the number that they were given in subsequent analyses of their contents by gel electrophoresis.

Agarose gel electrophoresis,

unidirectional.

Agarose gels

were poured and samples were subjected to electrophoresis through the gels as described previously (42). For 0.4% and more concentrated gels, a 30- or 31-finger comb was used to fractionate the bottom 29 or 30 fractions of a sucrose-Nycodenz gradient. These gradients had a total of 32 to 35

fractions. To

avoid

breakage of the septa between sample

wells for 0.2% agarose gels, a 13-finger comb with wider septa was used (details are given in reference 42). In addi-tion, to avoid breakage of 0.2% gels during fluorography (see below), they were poured above a 1- to 2-mm layer of 1.8% agarose in the same electrophoresis buffer used for the 0.2% gel.

Lowering

the salt concentration during dilution of bacte-riophages for electrophoresis releases sufficient monomeric DNA from bacteriophages to form a band during agarose gel electrophoresis. Before digestion with restriction enzymes

(see below), monomericDNA was completely released from

bacteriophages by heating to

75°C

for 10 min.

To determine the molecular weight of a linear DNA from its distance of migration during unidirectional agarose gel electrophoresis, the following procedure introduced by Southern (48) was used. (i) The molecular weight of markers

was plotted

as

afunction of the reciprocal of their distance of

migration. (ii) With this plot, the molecular weight of a DNA was determined from its distance migrated in the same gel

used for the markers. For linear DNAs with

molecular

weights between 0.25 and 1.0 times the molecular weight of monomeric T7 DNA, the plot for calibration was linear (r > 0.999) for the conditions of electrophoresis used below to analyze restriction endonuclease-produced fragments. Be-cause of curvature in this plot as molecular weight decreased

to 0.14 times that of monomeric DNA, a separate plotwas

made for DNAs shorter than 0.25 times the length of

monomeric DNA.

In the case of DNAs longer than monomeric T7 DNA,the

sample analyzed was

3H

labeled and detected by

fluorog-raphy (see below). The markers, which included themature

DNAs of bacteriophages T4, T5, and T7 (40), were unlabeled

and detected by staining with ethidium bromide (seebelow).

The distances migrated by unlabeled DNAs were scaled to

the distances migrated by labeled DNAs by use of the

mature T7 DNA present in the3H-labeled samples and also

in the collection of unlabeled markers.

Agarose gel electrophoresis, two-dimensional.

Two-dimen-sional agarose gel electrophoresis was performed byinitially

layering a sample at the origin of a 0.2% agarose gel

(first-dimension gel) embedded within a more concentrated

1.5% agarose gel (second-dimension gel). The sample was

subjectedto electrophoresisthrough the first-dimension

gel

at 0.34V/cmfor 16 h with

buffer

circulation

(42)

at20ml/min

started2 h afterthe start of

electrophoresis.

After this first

electrophoresis, a second

orthogonally

directed

electropho-resiswasperformedat thevoltageandforthetime

indicated,

causing the molecules of DNA to migrate into the

second-dimension gel. Embedding of gels and two-dimensional

electrophoresis were performed by procedures described

previously (41). Ethidium bromide

staining

of

gels

to be

analyzedbyfluorography(see

below)

revealed

that

noband

wasformed bymore than 10 ng ofDNA. Tennanogramsis

belowtheupper limitfornodistortion ofaband

position

(40).

Detection of DNA after agarose gel

electrophoresis.

To

detect

3H-labeled

DNA after agarose gel

electrophoresis,

gels were subjected to

autoradiography

after

permeation

with a compound (fluor) that fluoresces when struck

by

,B

particlesproduced by decayof

3H

(fluorography

[3,

25]).

For

fluorographywithpolyacrylamidegels,it has been shown

(5)

that salicylic acid can replace the fluors used

originally (3,

25). The advantages of fluorography with

salicylate

were

comparatively short time of

penetration

and

comparatively

lowcost(5). Therefore, attempts to use

salicylate

as afluor

withagarose gels were made.

In the presence of 0.72 M sodium

salicylate,

drying

of

agarose gels eitherin a vacuum or in air resulted in brittle

gels that

exuded

crystals of sodium

salicylate.

The use of

glycerolto dryagarose gelsfor silver

staining (30)

suggested

the use of glycerol

together

with sodium

salicylate

for

fluorography of agarose

gels.

The inclusion of

10%

glycerol

during drying for

fluorography

prevented

the above

prob-lems. The following procedurewas used.

Agel(volume, 95 to 120ml;5 to 6mm

thick)

was soaked

at room temperature for 2.5 h in 500 ml ofa solution that

contained10% glyceroland0.72Msodium

salicylate,

pH

7.0

to7.4. Forsupport,the agarosegelwas

subsequently

placed

on water-saturated filter paper, and a second

piece

of

filter

paper was placed beneath the

supporting

piece.

The

gel

on

filterpaperwas thenplaced inanovenat

56°C.

Foreither1%

ormore concentrated gels, a

1/8-in.

(ca.

0.3-cm) thick

glass

plate over plastic wrap was

placed

on the

gel

to prevent

curling. Lessconcentratedgelsweredried without eitherthe

glass plate or the plastic wrap. Gels were allowed to

dry

overnightat atmospheric pressure.

Drying

required

noless

than 5 and no more than 15 h. The dried

gel

was even less

brittle than agarose gels vacuum dried without either

glyc-erol orsodiumsalicylate. Thedried

gels

were

slightly

sticky

butusableforfluorography.

Increasing

the concentration of

sodium salicylate increased the stickiness of the

gel

after

dryingwithoutchanging detection

efficiency.

After

drying,

a

gel was covered with plastic wrap and

clamped

against

preflashed (25) X-ray film

(Kodak

SB-5).

Exposure

was

madeat -70°C. Inaccordance with the recommended

cau-tions (5), gloves should be worn while

handling

salicylate.

Salicylic acid forms an

extremely

light

powder

whose

dis-persion in the laboratory is difficult to prevent unless

work-ing under a hood with

negative

air flow. Gels more

con-centrated than 0.3% agarose and used for unidirectional

electrophoresis usually do not

undergo

distortion in

shape

duringthe drying stepin the above

fluorography

procedure.

However, less concentrated

gels

do

usually

distort

slightly

(see Fig. 3). Because of the presence of the

comparatively

weak first-dimensiongel embeddedin themoreconcentrated

second-dimension gel used fortwo-dimensional agarose

gel

electrophoresis,these

gels also

usually

distort

during drying.

However, the distortion is

usually

small

enough

so that the

pattern

of DNA inthe

gel

is not

significantly

disturbed.

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Theintensityofbandsproducedbythe aboveprocedureis not a linear function of the time that the X-ray film is

exposed. Conclusions drawn from band intensities are

semiquantitative.

To observe nonradioactively labeled DNA, gels were

stainedwithethidium bromide as describedpreviously (40).

The gel was photographed through a Tiffen 23A (orange) filter on Kodak Tri-Xfilm.

Digestion

with restriction endonucleases. The

following

restriction endonucleases were used to cleave intracellular

T7 DNAaftercentrifugationin asucrose-Nycodenz gradient

(fragments in order of left to rightplacement in mature T7

DNA are indicated, asis thepercentage of total monomeric DNA mass inthefragment; dataarefromRosenberg et al. [35]). XbaI: A (32.10), C (25.25), B(28.55), D(14.10); AvaI: A(26.20), D (13.60), B (25.58), E(9.92), C (24.70);BgII: B

(28.76), A(71.24). In

addition,

enzymeswhichdo not cut T7

DNA (35) but do cut E. coli DNA (not shown) were used

(EcoRI, BamHI, andPstI).

Todigest with the above restriction

endonucleases,

frac-tions fromasucrose-Nycodenzgradientwereheatedto75°C

for 10 min to eject DNA from mature

bacteriophages.

A

10- I amount from a fraction was diluted with 20 ,ul ofa

bufferthatcontained0.01 MTrischloride, pH 8.1,0.015 M

MgCl2, and 150 ,ug of bovine serum albumin per ml, with

enzymes addedin 1to3 ,ulportionsto afinal concentration

of2to 4U/ml. This mixturewasincubatedat37°C for2h. To

stopdigestion andprepare the DNAfor

electrophoresis,

10

pul ofthefollowing solutionwasadded: 15%sucrose, 400 pLg

ofbromphenol blue per ml, and 0.08 M EDTA, pH 7.4.

EcoRI, BamHI, and PstI were tested for activity with mature DNAfrombacteriophageA andcompletelydigested

1 ,ugofDNAunderthe conditions usedhere.

Fragments of T7 DNA produced by digestion with a

restriction endonuclease are labeled either by the letters

described above and below or, when appropriate, by a

numberwhich indicates thefragment's fraction ofthe mass

ofmature T7 DNA.

Digestion with nuclease Si. Tocleave single-stranded

re-gions of DNA without cleaving double-stranded regions,

digestions with nuclease Siwereperformed (57). To 10,ul of

afractionfrom a

sucrose-Nycodenz

gradient

wasadded 20

,ul ofa mixture that contained 1 part standard G buffer, 14

partsofabuffer that contained0.39 MNaCl,0.015 MZnCl2, and0.15 Msodiumacetate(plI4.6) andnuclease S1to afinal

concentration of600 U/ml. Digestion was continued for 15

min at30'C andterminatedby additionof 10

,u1

ofasolution

containing

10.5% sucrose,bromphenolblue(400

p.g/ml),

and 0.05 M EDTA, pH 7.4. The concentration of nuclease Si

useddegrades

OOS'

DNAbut notmonomericT7 DNA (43).

RESULTS

Sedimentation and gel electrophoresis ofDNA from cells infected byT7WT. To fractionate and detectintracellular T7

DNA, T7WT-infected cellswere labeled with

[3Hlthymidine

from 14 to 22

mnin

after infection. After concentration and

lysis of the infected cells, the

3H-labeled

DNA was

centri-fuged throughasucrose-Nycodenz gradient. Intheprofileof

acid-precipitable

3H

from this gradient, a peak formed by

100S+

DNA was observed at the interface between the sucroseandNycodenz (fractions23to 25, Fig. la). Particles

of mature

bacteriophage

penetrated this interface and

formedapeakin fractions28and 29 (arrow marked by

4)

in

Fig. la). In the sucrose-containing portion ofthe

sucrose-Nycodenz gradient, DNA sedimenting at 0 to 100S was

;_

30Q-

I5201-5 23

3

3-89.5

0

1

1 5 10

I5

20 25 30

Fraction

No.

FIG. 1. Rate-zonal centrifugation of DNA in lysates of cells infected withT7WT, T719, and T73. A 15-ml cultureof nonpermissive host was infected with either (a) T7WT, (b) T719, or (c) T73 by procedures described in Materials and Methods. The infectedcells werelabeled from14 to22 min fterinfection with [3H]thymidine (6.7 ,uCi/ml, final concentration). After labeling, the cells were quenched, pelleted, and lysed by procedures describedin Materials and Methods. The lysates were centrifuged through sucrose-Nycodenzgradients, andportions of the fractionswereassayed for acid-precipitable 3H by procedures described in Materials and Methods. Thepercentage of the total 3H in each fractionisplotted as afunction of fraction number.Sedimentation is fromlefttoright. found. The percentages ofacid-precipitable 3H in mature

bacteriophage, 100S+ DNA, 32S to 100S DNA, and DNA

sedimentinglessrapidlythan32S (<32SDNA) are shown in

Table1.

In the 32S to 100S region, a peak was observed at the position of monomeric DNA(32S) (vertical arrow 1 in Fig. la). To further test for both monomeric DNA and other discrete-sized DNAs in

the

gradient,

portions offractions 1 to 30 were subjected to agarose gel

electrophoresis-fluorography,with 0.4%agarose and 0.34 V/cm, conditions

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[image:4.612.328.564.66.490.2]

[image:5.612.316.553.65.287.2]

TABLE 1. Acid-precipitable

3H

inintracellular particles after the labeling and fractionation shown in Fig. 1

%of total3H

P bae

Mrophage

100S+ DNA

32S-100S

DNA <32SDNA

T7WT 40.7 20.9 35.4 2.7

T719 0.0 46.6 47.1 5.8

T73 0.55 77.8 17.4 4.14

T73,19 0.37 84.7 10.8 4.0

found previously (40)to resolve monomeric T7 DNA from T7 concatemer-sized DNAs (Fig. 2a). To avoid

overexpo-sure ofbands from fractions that contained comparatively large amounts of 3H-labeled DNA, selected fractions from the mature bacteriophage-containing and 1OOS+ DNA-containing regions of the gradientweredilutedasdescribed

in thelegendtoFig. 2 beforeuse.MonomericDNA thathad

emptied from mature bacteriophages was observed at the

2---0.7t--

-1 5 10 15 20 25 30

I ~ 0 I1

t 3 t

2 34

0.71-I _ *.4_-w

0.71

-2- 3 4

FIG. 3. Agarose gel electrophoresis ofconcatemer-sedimenting DNAathigherresolution. To 30,ul ofsamplebuffer that contained 3% sucrose was added 15 ptl of the indicated fraction from the sucrose-Nycodenz gradient ofFig. la(T7WT). A 40-pul amount of thismixturewassubjectedtoelectrophoresis ina0.2%agarosegel at0.34 V/cm for 50 h, andthe gelwas subsequently subjectedto fluorography as described in Materials and Methods. The

arrow-headsindicate theorigins of electrophoresis; theopen arrow indi-catesthedirection ofelectrophoresis.

'4

_-0.71-'- a

!

0.z29-_

_

[image:5.612.51.296.87.167.2]

I--.

FIG. 2. Agarose gel electrophoresis of DNA fractionated by centrifugationinasucrose-Nycodenz gradient.To 20,ulofsample buffer with 3% sucrose and 1% Sarkosyl was added 10 pulofthe indicatedfraction from thesucrose-Nycodenz gradientsused inFig. 1tofractionate DNA from E.coliBB/1infected with(a) T7wT, (b) T719,or(c) T73.Some of these fractionswerediluted beforeusein standard G buffer as described below. A 25-pul amount of this mixture was layered in a sample well of a 0.4% agarose gel in

electrophoresisbuffer A. Thesamplesweresubjectedto electropho-resisat0.34 V/cm for 40h,and thegelwassubsequentlysubjected

tofluorographyasdescribedinMaterials and Methods. The

arrow-heads indicate theorigins ofelectrophoresis; theopen arrow indi-catesthedirection ofelectrophoresis.Thefollowingdilutionswere

made beforeadding samplebuffer:fraction29,1/5;fraction28, 1/6; fraction24,1/4.

position of bacteriophage T7 (Fig. 2a, fractions 27 through 29). The peak of monomeric DNA previously observed in the sucrose portion of this gradient (Fig. la) was also

observed in theprofile of monomeric DNA in the gel of Fig. 2a(verticalarrow 1). However, some monomeric DNAwas

alsofoundthroughout the 32Sto 100Sregion of thegradient andattheposition of

10OS'

DNA.

Inadditiontotheband formed by monomeric DNA in Fig. 2a, twobandswereobserved closertotheorigin of electro-phoresis. The band closestto the bandof monomeric DNA (indicated by horizontalarrow2attheleftof Fig. 2a)wasat

theposition ofadimericconcatemer +20% in theagarosegel (see Materials and Methods). The DNA that formed this band alsoformedaskewedpeak in thesucroseportion of the

sucrose-metrizamidegradient. Thispeakwasapproximately

at the position expected of a dimeric concatemer (vertical arrow 2 in Fig. 2a). The band above the band of dimeric concatemer in Fig. 2awas formed by DNA that could be

either a trimeric or a tetrameric concatemer. A separation with resolution higher than the resolution in Fig. 2 was

needed to resolve trimeric from tetrameric concatemers.

Thus, assuming that the bands of Fig. 2a were formed by

linearDNA (this assumptionwastested andconfirmed; see

below),thepresence of dimericconcatemerswas confirmed witharesolution by length of ±+20%, and the presenceofat

leastone longer, discrete-lengthconcatemerissuggested. To improve the resolution by length of T7

concatemer-sizedDNAs,agarosegel electrophoresisof DNA in fractions 8to19(Fig. la and2a)wasperformed ina0.2%agarose gel

at 0.34 V/cm (Fig. 3). The two bands in the

concatemer-containing region of the gel were found at the positions of

dimeric concatemers +10% and trimeric concatemers ±20%. In other experiments, a less intense band at the

8 13 18

4x..

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a b

24 24

2.- X i

t-

-FIG. 4. Digestion ofT7WT100S+ DNA with nuclease Si. Frac-tions 23, 24, and25of thegradient of Fig.la wereseparately diluted into standardG buffer by1/3, 1/5, and 1/1, respectively. To 10

RI

of eachof these dilutionswasadded 20 ,ul of either(a)Sl bufferor(b) Si buffer with nucleaseSi(600U/ml). Digestionwascontinuedfor 15minat30°C and wasstopped by addition of10pu1 ofasolution containing bromphenol blue (400,ug/ml), 10.5% sucrose, and 0.05M EDTA, pH7.4. A

20-RIl

amountofaquencheddigestwassubjected toelectrophoresis in a0.4% agarose gel for40hat 0.34V/cm, and thegelwassubsequentlysubjectedtofluorographyasdescribed in Materials and Methods. The arrowheads indicate the origins of electrophoresis; the open arrow indicates the direction of electrophoresis.

position of tetrameric concatemers wasalso observed(data

notshown).

Inadditiontothe monomeric DNA and multimeric DNAs discussed above, DNAatthe origin of electrophoresis was observedattheposition ofT710OS+DNA(Fig. 2a,fraction 24). DNA, most heterogeneous in distance migrated, has previously been released from 100S+ DNA by digestion with nuclease Si, specific for single-stranded DNA (43). Diges-tion with nuclease Si (400 U/ml) of10OS+DNA reduced the amount of theorigin-associated DNA and released heterog-eneousDNA(digested: Fig. 4b, fraction 24; control: Fig. 4a, fraction 24). The nuclease Si did not digest monomeric T7 DNA (Fig. 4, fractions 23 and 25). An interpretation of this result is that branches connected by single-stranded DNA are necessary for the comparatively slow migration ofthe origin-associated 10OS+ DNA.

Two-dimensionalagarosegelelectrophoresis: circularDNA in lysates. Although it has been shown by electron micros-copy that most32S to 100S T7 DNA is linear (21, 23), it is possible thatsomeof this DNA iscircular, inanamount too

small for reliable detection by electron microscopy. The procedure of two-dimensional agarose gel electrophoresis described in Materials and Methods separates the linear DNA from the open circular DNA of bacteriophage X

(molecular weight, 32,100,000) and should do the same for

anyDNAofcomparable orgreatersize (P. Serwer and S. J. Hayes, Electrophoresis, in press). During the second elec-trophoresis at 6 V/cm, such circles are immobilized in the

first-dimension gel; linear molecules migrate out of the first-dimension gel and forman arcin the second-dimension

gel. According to the data of Bell and Byers (1), branched molecules should fall between thearcof linearDNA and the

immobilized circles. Circles too small to be immobilized shouldalso befoundbetween thearcof linearDNAs and the

first-dimension gel (45).

Thus, to determine whether circles were present among

themolecules of 32Sto100S DNA, this DNAwas subjected

totwo-dimensionalagarosegel electrophoresisasdescribed

in Materials and Methods. Initially unradiolabeled

prepara-tions ofDNA wereused,and the DNAprofilewasobserved

by staining with ethidium bromide. By DNA-DNA hybrid-izations performed in a previous study (23), no more than 20% of the unlabeled DNA was likely to be host DNA. The profileofall detected DNAsedimenting at35S to 45S (Fig. 5a)and 45S to 52S (Fig. Sb) was a single arc. This arc was coincident with an arc formed by the collection of linear DNAspreviously (45) used as markers (thearcofmarkersis not shown). This observation confirms the previous finding (21) that most 32S to 100S T7 DNA is linear.

Although some distortion ofgels used for two-dimensional agarose gel electrophoresis occurs during drying for fluorography, fluorography is more sensitive than staining for detecting comparatively small amounts of nonlinear DNA.Fluorograms of gels after two-dimensional agarose gel electrophoresis confirmed that most of the 3H-labeled T7 DNA, including DNA that formed a band, was linear for DNAs from thefollowing regions of the sucrose-Nycodenz gradient inFig. 1: 59S to 75S (Fig. 5c), 41S to 59S (Fig. Sd), 30S to 41S (Fig. Se), and 13S to 30S (Fig. Sf). However, a comparatively weak band bent during drying (filled arrow, Fig. Sd) was observed in the first-dimension gel. This band was not detected in Fig. 5c, e, or f. It was formed byDNA thatmigrated 14% more slowly than linear monomericDNA

I.t-/> b

I

ew f

FIG. 5. Two-dimensional agarose gel electrophoresis of 32S to 100ST7 DNA. UnlabeledDNAfrom30 mlofT7wT-infectedE.coli BB/1 was sedimented through a sucrose-Nycodenz gradient as described inMaterials and Methods. DNAs(40,ul) from the 35Sto 45S region (a) of the gradient and 45S to 52S region (b) of the gradient were separately subjectedtotwo-dimensional agarose gel electrophoresis (without dilution)at6V/cm for1.8hfor the second electrophoresis. Electrophoresis was followed by staining with ethidium bromide as described in Materials and Methods. In a separateexperiment, fractions from the gradientin Fig. la(T7wT) werepooled and diluted, as indicated below. To 30 ,ul of each of these dilutions was added 10

pul

of sample buffer with no sucrose, and 35 p,loftheselatterdilutions wassubjectedto two-dimensional agarosegel electrophoresis for2.0 hfor the second electrophoresis. Thegelwassubjectedtofluorography. Other panels: (c) fractions 15 to 19(59S to 75S), no dilution; (d) fractions 9 to 14(41S to 59S),1/1.5 dilution; (e) fractions Sto 8(30S to 41S), 1/2 dilution; (f) fractions 0 to 4(13Sto30S),nodilution.Thearrowheads indicate the origins of electrophoresis; theopenarrowsindicate thedirections ofthefirst (I)and second(II)electrophoresis.

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during

the first electrophoresis (0.34 V/cm) but did not

migrate during

the second electrophoresis (6V/cm). When

the

voltage gradient

for the second electrophoresis was

lowered from6 to 1

V/cm,

the arrestedDNAmigrated (not

shown).

This behavior of the DNA that formed the band

indicated

by

thearrowinFig. Sd is thesame as the behavior

ofopencircles of

bacteriophage

A DNA(Serwerand Hayes,

in

press).

Itistherefore concludedthat the DNA that formed

this bandwas circularDNA. Analysis of single fractionsby

the

procedure

in

Fig.

5 revealed that this circular DNA

formeda

peak

atthe42± 2S

position.

The S valueof theT7

circles was

significantly higher

than the 36S expected for

opencircularT7DNA

(2,

19). The measured S value forT7

circles was,

however,

consistent with a DNA that was

closed circularatthetime of

centrifugation

(2, 56). Thus, the

data suggest that the bandindicatedby an arrow inFig. Sd

wasformed

by

either circles thatwere closedatthe time of

centrifugation

and

subsequently

convertedtoopencirclesor

circles thatwereopenatthe time ofcentrifugationbut whose

S value wasaltered

by

DNA-DNA interactions during

cen-trifugation.

The correctalternative is notknown. The

inte-grated intensity

of the band ofcircles was always less than

3% of the

integrated intensity

ofthe arcof linearDNA.

To

help

determine the

pathway

forthe production ofthe

comparatively

small amount of circular DNA

produced

during

a

T7WT

infection,

thekineticsofappearanceof circles

was determined

by

a kinetic

labeling

experiment similarto

those

previously performed (38),

but with assay for circles

by

the

procedure

of two-dimensionalagarose

gel

electropho-resis used here. After

labeling

T7wT-infected

cells for 1.5

min

(13.0

to 14.5 min after

infection)

at

30°C,

no

circle-associated 3H was detected

by

the

procedures

used above.

Similarly,

nocirclesweredetected after

stopping

incorpora-tion with an excess of unlabeled

thymidine

and further

incubation for 1.5 min.

However, by

3.5 minafter

stopping

incorporation

of

[3H]thymidine,

circle-associated 3H was

detected and itsamountincreased

monotonically, roughly

in

proportion

totheincreasein

bacteriophage-associated

3Hat 5.5 and 7.5 min after termination of

labeling (data

not

shown).

This result indicates that thecirclesarethe

product

ofaprocess thatoccursafter DNA

replication.

The

mecha-nism for the

production

ofcircles is discussed further below.

Alterations caused

by

amber mutations ingenes 3 and 19.

To determine the effects on intracellular DNA of

removing

either

p3,

p19,

or

both,

the

experiment

of

Fig.

lawasalso

performed

with

T73, T719,

and

T73,19.

A

peak

atthe

position

ofmature

bacteriophage

was notpresent in the sedimenta-tion

profile

for either

T719

(Fig. lb), T73 (Fig. lc),

or

T73,19

(not

shown),

as

expected.

The

T719

lysate

had both 32Sto lOOS DNA and

10OS+

DNA

(percentages

for

T7WT,

T719,

T73,

and

T73,19

areall

given

inTable

1).

The

T73

and

T73,19

lysates

had a percentage of

10OS+

DNA that was

signifi-cantly higher

than that in both

T7WT

and

T719

lysates.

The percentageof 32StolOOSDNAinthe

T73

and

T73,19 lysates

was

comparably

reduced

(Table 1).

These observationsare in agreement with those made

previously

for

T73

and

T719

lysates

(24).

To furthercharacterize the

T719

and

T73

DNAs

fraction-ated in

gradients

suchasthosein

Fig.

lband c, these DNAs were

subjected

tobothtwo-andone-dimensional agarose

gel

electrophoresis.

By

two-dimensionalagarose

gel

electropho-resis,

thecircularDNAdetected in

T7WT

32Sto lOOS DNA was absent from

T719

32S to lOOS DNA

(not

shown).

The

T719

32S tolOOS DNAwaseitherarrestedatthe

origin

ofthe

first

electrophoresis

or

migrated

to a

position

on the arcof

linear DNAs.

During

unidirectional

electrophoresis,

most

32S to 100S DNA in T719 lysates migrated as concatemers, but did not form thebandof dimericconcatemersobserved

forT7WT in a 0.4% agarose gel (Fig. 2b). Analysis of T719 concatemersin the0.2%gel of Fig. 3 did reveal weak bands of dimeric andtrimeric concatemers, but these bands were reduced in intensity to levels that were at the limit of detection (data not shown). In addition, only a trace of monomeric DNA was observed in the T719 lysate (Fig. 2b). Thus, the presence of p19 was necessary for the appearance ofmost DNA that migrated as either monomeric DNA or a multimeric concatemer. The origin-associated DNA ob-served in the fraction that contained T7WT 100S+ DNA in Fig. 2a was also observed in the case of T719 100S+ DNA

(fraction24, Fig.2b). Some origin-associated DNA was also

found tocosediment with32S to 100S DNA in Fig. 2b. Inthe case of T73, almost all of the 100S+ DNA detected was origin associated (fraction 24 in Fig. 2c). In addition, origin-associated DNA was found in the 32S to 100S region of thegradient. These observations have also been madefor

T73,19

DNA(notshown). No evidence for the accumulation of32Sto100Sconcatemersin either T73 or

T73,19

lysates was observed. Onthecontrary, thedata in Table 1 indicate that

32Sto100S concatemers were decreased by at least a factor of 3 in theT73 lysate. The data of Fig. 2c suggest that the

decrease was even higherthanthis. The abovedata do not

reveal whether concatemers arepartof theorigin-associated

T73100S+ DNA.

Digestion with restriction endonucleases. (i)T7WT. If either

concatemeric or circular DNA is a constituent of either

100S+or32Sto100SDNA, then digestion with a restriction

endonuclease will produce a fragment (termini-joined [TJ]

fragment) that consists of joined terminal fragments of

monomeric DNA(24, 44)ifno region of theTJfragmentis

branched. Thus,tofurther characterizethe DNAsobserved

inFig. 1, asampleof each fraction wasdigestedwith either

one or more restriction endonuclease and the fragments

produced were analyzed by agarose gel

electrophoresis-fluorography. The fragments released from T7 DNAs in

fractions of the gradient in Fig. la

(T7WT)

were observed

after digestion by the following mixture of restriction

endonucleases: XbaI (four fragments released from

mono-meric T7DNA) and EcoRI, BamHI, and PstI (none ofthe

latter three cut T7DNA, but theydo cut E.coliDNA). Such

analysis of DNA released from mature T7 bacteriophage

(fractions

26 to 29 in Fig. 6a) revealed the A, B, C, and D

fragments ofXbaIdescribedpreviously (35). At theposition

of monomeric DNA

(vertical

arrow 1 in

Fig.

6a), the same

profile wasobserved. However, as the S valueofthe DNA

increased from 32S, an additional band, closer than the A

band to the origin of electrophoresis, was observed to

increasein

intensity.

Also,theintensity ofthebandsformed

by

fragments

from theleft(Aband)andright (D band)ends ofmatureT7 DNA decreased in relation to theintensityof the B and C bands. Neitherthe

migration

distance northe

intensity of the additional band was altered when EcoRI,

BamHI, and PstI were omittedfrom the

digestion

mixture

(notshown),

indicating

that this bandwasformed

by

T7, not

host,DNA. These observationssuggest that this additional

band was formed by a TJ

fragment.

From its position in

agarosegels (seealso

Fig.

7),this TJ

fragment

hadalength

0.48±0.03timesthe

length

ofmatureT7

DNA,

equal

within

experimental error to the

length

of a

fragment (TJ[L-R])

consisting ofthe A

(left end)

and D

(right end) fragments.

This length is

significantly

different from the

length

of a

fragment(TJ[L-L])

consisting

oftwoleft endsora

fragment

(TJ[R-R]) consisting oftwo

right

ends. The data of

Fig.

5

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20 2 30

! I!

79

w

Iz

- TJ(L-L)

-TV{L-R) 4~*% --B

. -F 'I

D

rnu.;;; * --dl

4

,-*-, -*.. aU d.a..b 6II&..

---TJ(L-LI

-TJlL-

LRI1

-TJ(L-R)

-j-A

-8 C

-49

-Host

TJ(L-L)

--TJ(L-R)

_-A

-B

LC

-D

FIG. 6. Digestion of fractionated intracellular T7 DNA with restriction endonuclease XbaI. A portion of each fraction of a sucrose-Nycodenz gradientwasdigestedtocompletionwith restric-tion endonucleaseXbaI asdescribed in Materials and Methods. A

25-,ul amountof eachdigestwasthen subjectedtoelectrophoresis through a0.7%agarosegelat0.5 V/cm for24 hinelectrophoresis buffer A. After electrophoresis, the gel was subjected to fluoro-graphyasdescribed in Materials and Methods. Thelysatesanalyzed were(a)T7wT, (b)T7WT(the same gel butexposed three timesas long),and(c) T719. Thearrowheadsindicate theorigins of electro-phoresis;theopenarrowindicates the direction ofelectrophoresis. indicate that most of the TJ fragments were from linear

concatemers, not circles.

When thefluorogram ofFig. 6a was exposed three times as long as it was exposed for Fig. 6a, two additional,

comparatively weak bands appeared at the position in the

gradient of concatemer-sedimenting, but not monomeric,

DNA(Fig. 6b). Thesebandswere also present when EcoRI,

BamHI, and PstI were omitted from an XbaI digest (not

shown). Thus,itis concluded that thefragments that formed

these latter twobands were released fromT7 concatemers

by XbaI. The larger fragment (TJ[L-L] in Fig. 6b) had a

molecular weight 0.62 + 0.03 times that ofmonomeric T7

DNA, indistinguishable from twice the molecular weight of

the A (left end) fragment. It is therefore assumed that the TJ(L-L) fragment consists of two joined A fragments and was cut from a left-end to left-end joined region in a concatemer. The smaller fragment, marked with an asterisk

inFig. 6b,had a length 0.19 + 0.01 times that ofmonomeric

DNA,significantly differentfrom twice the molecular weight

of the A (right end) fragment. There are, thus far, no data

that indicate the nucleotide sequences in the fragment

marked withan asterisk in Fig. 6b.

(ii) T719. When the experiment of Fig. 6a was repeated

with T719, but omitting the EcoRI, BamHI, and PstI, the TJ(L-R) fragmentwasfoundin32Sto 100Sand100S+ DNA. Inrelationto the B and C fragments, theTJ(L-R) fragment was more abundant and the A and D fragments were less abundant at all S values in theT719 DNA of Fig. 6c than they werein the T7WT DNA of Fig. 6a. The D fragment and the fragment indicated withanasteriskwere notdetectedatall in the T719 32S to 100S DNA. Onlya comparatively small amount of D fragment was released from the T719 100S+ DNA(Fig. 6c). In relation to the A fragment,the Dfragment appeared depressed by a factor of more than 10 by the absence ofp19. The preferential loss ofthefragmentat the right end was alsoobserved when restriction endonuclease XbaI (left-end fragment larger than right-end fragment)was

replaced byBglII (left-end fragment smaller than right-end

fragment) and AvaI (left-end fragment approximately the same size as right-end fragment; see below) (data not shown). These observations indicate that the selective loss of the right-end XbaI fragmentin T719intracellular DNA is caused by the position of this fragment, notby its relative size. The DNAforming the band marked hostinFig. 6cwas digested by the mixture of EcoRI, BamHI, and PstI (not shown) and was thereforeE. coli DNA. This3H-labeledhost DNA has beenpresentin all lysates thus far examined and was the onlybandof host DNA observed.

(iii)T73.Digestion of T73 100S+ DNA tocompletionwith

restriction endonucleaseXbaI released some of the

origin-associated DNA as fragments A, B, C, and D. However, most of the T73 100S+ DNA detected remained origin

associated after digestion withXbaI (Fig. 7a, lane 1). The

intensityof the bands(Fig. 7a,lane1)decreased in the order

DtoA, the reverse of the order that was observed formature T7 DNA(Fig. 6a,fractions26 to29;Fig. 7a, lane 4)and that was expected if fragments were released in equimolar

amounts. In contrast to the results obtained with T7WT

100S+ DNA(Fig. 7a, lane2)andT719100S+ DNA (Fig. 7a,

lane 3), the TJ(L-R) fragment was not released from T73

100S+ DNA.

a 1L 34

*w --Host

_.

-TJ

( -R)

--,A

-. -B

40 -c

-GD

b

234

-. -TJ(L-R)

Lc

-D

FIG. 7. Digestion of 100S+ DNA from T7wT (lanes 2), T719 (lanes 3), and T73 (lanes 1) lysateswith restriction endonucleases XbaIandAvaI.Asample of100S+DNA fromT7wT,T719, and T73 lysateswasdigestedtocompletionwitheither (a)Xbal or (b)AvaI

as described in MaterialsandMethods. Monomeric T7 DNA was alsodigested (lanes 4). The digests were then subjected to electro-phoresis in a0.7% agarose gelat 0.5 V/cm for 21 h; the gel was subjected to fluorography. The left-hand lane of panel b contains mature,undigestedT7 DNA.Panels a and b were different sections ofonegel. The arrowheadsindicate the origins of electrophoresis; theopenarrowindicates the direction of electrophoresis.

t

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The alteration in relative band intensities observed in the T73 100S+ DNA can be a function of either the relative size

of the fragments involved or their position on T7 DNA or

both.Toobserve the effects of position independent of size,

the 100S+ DNAs weredigested with restriction

endonucle-ase AvaI, an enzyrme whose fragments from the left end,

middle, and right end of monomeric T7 DNA vary by no

more than 6% in size. Two smaller fragments that separate

the middle fragment from the two end fragments are also

produced (35). TheAvaI digest of T73 100S+DNA (Fig. 7b,

lane 1) revealed band intensitiesthat decreased in the order

C (right end) > B (middle) > A (left end); no TJ fragment

was present. In contrast, the A and C bands had depressed

intensities relative to the B bandforT7WT

100S'

DNA (Fig.

7b, lane 2) and T719 100S+ DNA (Fig. 7b, lane 3); the

intensities of the A, B, and C bands were indistinguishable

for the monomeric DNA (Fig. 7b, lane 4). The results for

T7WT and T719 100S+ DNA areinagreement with theabove

results obtained with restriction endonuclease XbaI in Fig.

6. The relative band intensities for the AvaI digest of T73

100S + DNAindicateaneffect of position. Thatis, fragments

werelost with increasing probability as distance from the left

end decreased. However, that an increased probability of

losing a fragment also occurs with increasing size of a

fragment was suggested by the observation that the C

fragment oftheXbaIdigestof T73100S+ DNA(Fig. 7a, lane

1) was moreintense than the largerBfragment,eventhough

the C fragment was at the left side ofthe B fragment(35).

The absence of the TJ fragment of XbaI and AvaI in T73

100S+DNAcouldhavebeencausedby eithertheabsenceof

left-to-right joined termini or the decreasing intensity of

bands with increasing size. A more extensive study of

left-to-right joining of termini in T73 100S+ DNA is in

progress.

As discussed above and below, the best explanation

fQr

the selective absence of fragments fromthe digests ofT73

100S+ DNA in Fig. 7 istheaccumulation of branchesinthis

DNA. To test this hypothesis, attempts were made to use

electron microscopy to compare the branching in T73 and

T7WT

100S+

DNAs. In

agreement

with

previously reported

observations(24), both ofthese DNAs looked

indistinguish-able from thepreviously described (24, 29, 38) complicated

tangles ofDNA (datanot shown). Amore advanced

proce-dure for

analysis

of

100S+

DNA structure is needed to

describe the branching of this DNA.

DISCUSSION

In the past, analysis of the DNA in lysates of

bacterio-phage T7-infected cells has been

performed by

rate-zonal

centrifugation

in a

density gradient, usually

followed

by

fractionation ofthegradientandassayforDNA(21, 23, 29,

37, 38, 50) (Fig. 1). In part because the DNA

analyzed

is

variable in both molecular weight and conformation and becausefractionation ofthe

gradient

limits

resolution,

mul-timeric concatemers could not be

reliably

detected in these

previous studies. The

asymmetric spreading

of both the

monomericDNAandthemultimericconcatemers

observed

hereisanadditional cause.

Although

attemptswere madeto

avoid

overloading during

centrifugation,

overloading

is a

possible explanation forsomeof this

spreading.

A

possible

additional

explanation

is

degradation

of more

rapidly

sedi-menting

DNA to fprm the

discrete-length

DNAs detected

here. This

degradation

could occur

either

during

or after

centrifugation.

Byuseofan

optical

procedure

forthedetection of DNA

aftereither agarose

gel

electrophoresis

orrate-zonal

centrif-ugation, bandshavepreviouslybeenfoundattheposition of

monomeric DNA and dimeric concatemers after

fractiona-tionof nuclease Si digests of 100S+T7DNA (43, 44). Inone

study, a band for tetrameric but not trimeric concatemers

wasalso observed(44). Byanalysisof all of theDNAinaT7

lysate with a combination ofrate-zonal centrifugation and

agarose gel

electrophoresis-fluorography,

in the present

study bands were observed at the positions of dimeric,

trimeric,and sometimestetramericconcatemers not

associ-ated with 100S+ DNA. The reason for variations in the

relative intensity of bands at the positions of trimeric and

tetramericconcatemersis not known. The DNA that forms

these bands was found to be linear by two-dimensional

agarose

gel electrophoresis.

The 32S to 100S linear DNA

also contained TJ restriction endonuclease

fragments,

pre-dominantly

theTJ(L-R)

fragment

expected ofa concatemer

that consists of monomeric DNAs

joined

left end to

right

end. Itis concluded, therefore, that thefirst twobands are

formed

by

concatemersthataredimeric

(±10%)

andtrimeric

(±20%), respectively. Locating

more

precisely

the ends of

these concatemers and testing for nicks and gaps in their

polynucleotide chains require analysis of the concatemers

afterseparation byagarosegel electrophoresis;thisstudyis

being performed.

Formation ofconcatemerswith left-endto

right-end

join-ing

ofmonomeric DNA can occur

by complementary

base

pairing

at the

repeated

ends made

single-stranded (21, 24,

58).

Because the

terminally repetitious

sequenceofT7 DNA

is not an inverted repeat (9), left-end to left-endjoiningof

monomersina concatemer cannot occur

by complementary

base

pairing. Thus,

the

TJ(L-L) fragment

observed here

would be produced by joining of blunt ends, presumably

catalyzed by

a

ligase

suchasthe

bacteriophage

T4

ligase

that

joins

DNAatblunt ends

(47).

Thepresenceof the

TJ(L-L)

band in

digests

of T719 concatemers indicates that DNA

packaging

isnot

required

forleft-end toleft-end

joining.

Thecircular T7 DNAfoundin

T7WT lysates

was notfound

in T719 lysates,

indicating

that DNA

packaging

was

neces-saryfortheformation of thecircularDNA. This conclusion is consistent with the kinetics of the formation

of

circles

during

infection with T7WT. A

possible

mechanism for the

formationof circles

during

the initiation of

packaging

(i.e.,

before DNA enters the

capsid)

is

juxtaposition

ofthe two

ends ofamonomericDNAwithinaconcatemer,followed

by

recombinationbetween these ends. The

juxtaposition

would

becaused

by

capsid-DNA

binding.

Evidence for

binding

ofa

capsid

ofT7concatemers at a

point

onthe DNA7%fromthe

right

end has been

presented (39).

Some monomeric and

concatemericDNA

observed

herewasreleasedfrom

capsid-DNA

complexes by Sarkosyl

used for

lysis (39).

The

pack-aging

specificity

ofT7-T3

hybrids

confirms the presenceofa

specific binding

sitenear

(but

not

at)

the

right

end

(62).

If the

right

endoftwo

sequential

monomersinaconcatemerwere

bound

by

a

capsid,

juxtaposition

of the two ends of the

monomerclosesttothe

right

endoftheconcatemerwould be

achieved

(Fig. 8).

Attheinitiationof

packaging,

the

binding

oftwo

sequential

monomers

by

the

capsid

in aconcatemer

also

explains

the

following

observations.

(i)

During

"in

vitro"

DNA

packaging

withaT3 extract,the

selectivity

for T3 DNA in

preference

to T7 DNA is greater for

concate-meric DNA than it

is

formonomericDNA

(18);

the

binding

specificity

would be increased for a concatemer

by

there

being

two

binding

sitesinstead ofone.

(ii)

The DNA

pack-aging

of

bacteriophages

Kand P22is

processive (reviewed

in

references 11 and

59);

the

processivity

would be a conse-quenceof increased

probability

for

binding

a

capsid

ifasite

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Capsid

I

FIG. 8. Mechanism for the formation of circular DNAduringT7 DNApackaging.Theprocapsidofbacteriophage T7,referredtoas

capsid I (46), simultaneously binds two adjacent genomes in a

concatemer;thebindingsiteonbothgenomes isnearthegenome's rightend. A result is thejuxtapositionof the left(L)andright (R)

ends of the genome bracketed by the two binding sites. It is

proposedthat thisjuxtapositionincreases theprobabilityof

recom-bination between thetwoterminally repeatednucleotidesequences

of thebracketed genome,producingthe circular DNAobserved.

on the DNAeither has previously oris presentlybound to another capsid. (iii) In vitro packaging of concatemeric X

DNA requires an amount of DNA-capsid linking proteins (reviewedin reference 11)that istwicetheamount required

for in vitro packaging of monomeric DNA (reviewed in reference 59).

The mechanism of the selective loss of therightendofall intracellular forms ofT719DNAis notknown. One explana-tion is that initiation ofDNA packaging requires p19 and

protects the right end from intracellular nuclease-induced damage. In agreement with the hypothesis that initiation of DNApackaging by the procapsid prevents loss of theright

endofconcatemers,this end is also lost in the absence ofp9 (p9 is a protein essential for the formation ofa procapsid

[34]) (P. Serwer and R. H. Watson, unpublished data). Possibly, to initiate packaging a procapsid with the assis-tanceofp19binds the DNA to bepackagedattherightend of the DNA, as shown in Fig. 8, butwith the DNA's right

terminusalso bound tothecapsid.

Thedeficiency of bands formedby discrete-length DNAs in T719 lysatescan be explained, at least in part, by

degra-dationoftherightend ofconcatemers. It is alsopossiblethat thisdeficiencyis caused inpartbyeitheradeficiencyin the production ofdiscrete-length T719 concatemers or

concate-merizationmorerapidandextensivethan the concatemeriza-tion of T7WT. For example, packaging of concatemers in T7WTwould limit thegrowthofconcatemers. As therateand extentofconcatemerizationincrease,the fraction of DNA in those concatemers toolongtoisolatewithoutshear-induced breakage (6, 38) increases.

Theaccumulationofaform of10OS+DNA inT73 lysates

(24, 50) has been previously interpretedas an accumulation

of concatemers(16, 24, 27, 50). However, itwasfound here that mostof the DNA in T73 lysates is 1O0S+DNA that is origin associated after agarose gel electrophoresis. The

remaining DNA is 32S to 100S DNA, almost all origin associated during agarose gel electrophoresis. This 32S to lOOSDNA is thereforenotlinear, concatemeric DNA. The comparatively slow migration ofT73 DNA during agarose gel electrophoresis (see Results) and the evidence for branch-cleaving activity of p3 (7, 55) suggest that the

intracellular DNA that accumulates in T73 lysates is

branched. Thebranchingisextensiveenoughsothatmostof

the T73 100S+DNA remains origin associated even after

digestionwithrestrictionendonucleases XbaI andAvaI.The

data also indicate that thereare more branches on the left than on the right end of T7 DNA in T73

1OOS'

DNA complexes. A possible explanation is the previously demon-strated (17, 61)initiation of DNAreplicationonthe left end of T7 DNA. Inapparentanalogy withT7, bacteriophageT4 accumulates aDNA with theproperties of T73 10OS+DNA in the absence of T4 p49,aprotein that iscomplementedby

T7p3 (7). It has also been concluded thatT449DNA also has accumulated branches (28).

ACKNOWLEDGMENTS

We thank Elena T. Moreno and Helen C. Hoffer for technical assistance, Henry R.Hollyday III for constructingT73,19,and Anna M. Uriegas for secretarial assistance.

Supportwasreceivedfrom Public Health ServicegrantGM-24365 from the National Institutes of Health and the Robert A. Welch Foundation (grant AQ-764).

LITERATURE CITED

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8. Dressler, D., and H. Potter. 1982. Molecular mechanisms in geneticrecombination.Annu. Rev. Biochem.51:727-761. 9. Dunn, J. J., and F. W. Studier. 1983. Complete nucleotide

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10. Earnshaw,W.C., andS. R.Casjens. 1980.DNApackaging by thedouble-strandedDNAbacteriophages. Cell21:319-331. 11. Feiss, M. 1986. Terminase and the recognition, cutting and

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