Localization of single-chain interruptions in bacteriophage T5 DNA I. Electron microscopic studies.

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Localization of

Single-Chain

Interruptions

in

Bacteriophage

T5 DNA

I. Electron

Microscopic

Studies'

PATRICIA P. SCHEIBLE,2 ELLEN A. RHOADES, AND MARC RHOADES* DepartmentofBiology, The Johns Hopkins University, Baltimore, Maryland 21218

Received forpublication29 December 1976

BacteriophageT5 DNA wasexamined in anelectronmicroscope after limited

digestion withexonuclease IIIfrom Escherichiacoli. The effect of the exonucle-asetreatment was to converteachnaturally occurringsingle-chaininterruption

in T5 DNA into a shortsegment of single-stranded DNA. The locations of these segments were determined for T5st(+) DNA, T5st(0) DNA, and fragments of

T5st(0) DNAgenerated byEcoRI restrictionendonuclease. The results indicate

thatsingle-chain interruptions occur in avariable, butnonrandom, mannerin

T5 DNA. T5st(+) DNAhas four principal interruptions locatedat sites approxi-mately 7.9, 18.5, 32.6, and 64.8% from one end ofthe molecule. Interruptions

occur atthesesites in 80 to 90%of the population. Alargenumber of additional sites, located primarilyatthe ends of the DNA, containinterruptionsatlower

frequencies.The average number ofinterruptions per genome, asdetermined by

thismethod,is 8. Asimilar distributionofbreaksoccurs inT5st(0)DNA,except

that the 32.6% site is missing. At least one ofthe principal interruptions is

reproducibly located within an interval of 0.2% oftheentire DNA.

Mature particles of the virulent coliphage T5 contain a single molecule of double-stranded DNA, which has a nonpermuted, terminally repetitious nucleotide sequence (21, 29). T5

DNA is distinguished from most other viral

DNAmoleculesby the presence of a small

num-ber of single-chain interruptions (1). These

sites can be repaired by DNA

jigase

and are

located within one strand ofthe duplex (11).

Although the interruptions in T5 DNA had

been thoughtto occur atdefinedpositions,

sev-eral studies have suggested that theexact

dis-tribution of these sites is variable.

Denatura-tionof T5 DNA resultsintheformationof over

40discrete classes ofsingle chains, which can

be resolved by agarose gel electrophoresis (9).

The relative abundances of these fragments

varyoverawiderange.Evidencethat

interrup-tions occur atvariable positionsatoneendof T5

DNAhasbeenobtainedby electronmicroscopic

studies of circular molecules produced by

an-nealing exonuclease-treated DNA (21).

Thepurpose of theexperimentsreportedhere was to determine the locations of the

single-chain interruptions in T5 DNA. This was

ac-complished by examining T5 DNA in an

elec-IContribution912fromtheDepartmentofBiology,The JohnsHopkinsUniversity,Baltimore,Md.

2Presentaddress: DepartmentofSurgery, Duke

Univer-sity MedicalSchool, Durham, NC27710.

tronmicroscope after limited digestion with

ex-onuclease III ofEscherichia coli, an enzyme

thathydrolyzes both internal and external 3'-terminiinduplex DNA (17). Under the appro-priateconditions, the short single-stranded

seg-mentsproduced by the exonuclease can be

visu-alized and thepositions of the interruptions can be determined. By performing analogous

exper-iments onfragmentsgenerated by EcoRI endo-nuclease, we constructed an unambiguous physicalmapof T5 DNA.

MATERIALS AND METHODS

Purificationofexonuclease III. E. coli 1100, an

endonuclease I-deficient strain (6), was grown at

37°Cin4liters of M9 medium(2)supplemented(per

liter) with 2 g ofCasamino Acids, 1 g oftryptone

(Difco Laboratories, Detroit, Mich.), and 1 mg of thiamine hydrochloride. At a concentration of5 x

105/ml, the cellswere chilled to 0°C, harvested by centrifugation at 10,000 x g for 15 min, and

sus-pended in 30 ml of EB (0.02 M Tris-hydrochloride

[pH7.8], 10-3M2-mercaptoethanol, 10-4MEDTA). All subsequent steps were carried outat 0 to 4°C. The resuspended cellsweredisrupted bysonic treat-ment, andtheresultingextract wascentrifugedfor 90 min at 25,000 rpm in a Spinco 40 rotor. After

dialysisagainst 0.01 Mphosphate buffer(potassium

phosphate [pH 6.8] containing 10-3 M

2-mercapto-ethanol and 10-4 M EDTA), the supernatant was

applied toacolumnofDEAE-cellulose (100-mlbed

volume). Elution was carried out with a 350-ml lin-725

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726

SCHEIMLE,

RHOADES, AND RHOADES

eargradient formedwith 0.01and0.30 Mphosphate

buffer. Exonuclease III activitywasrecovered ina

single peak elutingbetween 0.075 and 0.140 M phos-phate buffer. The active fractions were dialyzed

against 0.01 M phosphate buffer and appliedto a

phosphocellulose column (35 ml). Elution was

car-riedoutwitha150-ml lineargradientformed with

0.01and 0.30 Mphosphatebuffer. Activitywas

re-coveredinasharp peakat0.15 Mphosphatebuffer.

This materialwasdialyzedagainstEDTA-free 0.01

Mphosphate buffer and appliedtoacolumn of

hy-droxyapatite (3.0 ml). Elution was carried out as

described above. ExonucleaseIII wasrecoveredin

fractions ranging from 0.075to 0.115 M phosphate buffer. Contaminating endonuclease activity was

foundtoeluteslightlyahead ofthe exonuclease. The fractions showing the highest ratio of exonuclease/ endonucleaseactivitywerepooled and concentrated

to 1mlbypressuredialysisagainstEBcontaining 2

X 10-3 M MgC12. This material was applied to a

Sephadex G-75 column (1.5 by 30 cm, superfine)

equilibrated with thesamebuffer. ExonucleaseIII

eluted from this column justafterthe void volume

andjust ahead of the endonuclease activity. The leading fractions containing exonuclease III were

free from detectable endonucleaseactivityandwere

used for theexperimentsreported here (seeFig. 1).

Assays for exonuclease III were routinely

per-formedby measuringthe formation of acid-soluble

radioactivity from 32P-labeledT5DNAasdescribed

below. At variousstagesthe identity of the

exonu-cleaseIIIactivitywasverifiedby the3'-phosphatase assayof Richardson andKornberg (22). Endonucle-aseactivitywasmeasuredbyalkalinesucrose

gra-dient sedimentation.

Digestion of T5DNA with exonuclease III. Reac-tion mixturescontained0.067 MTris-hydrochloride (pH8.0), 10-3MMgCl2, and10 ggof32P-labeled T5 DNA perml. The concentration of exonuclease III wasadjusted sothatapproximately4%of the DNA

washydrolyzed within 15minat37°C. The

forma-tion of acid-soluble radioactivity was measured by

thin-layer chromatography on

polyethyleneimine-cellulose(12).

Sequential treatmentof T5 DNA with

exonucle-ase III and EcoRI endonuclease. Exonuclease III

incubations were carried out as described above.

The reactions were terminatedby heatingto 650C

for 5min. The solutionswerethen made0.01 M in

MgCl2 and 0.10 M inTris-hydrochloride (pH 7.6), and sufficientEcoRIendonucleasewasaddedto

pro-duceacompletedigestwithin 30 minat37°C.These

incubations wereterminated by adding1/5 volume of 0.1 MEDTA. EcoRI endonucleasewaspurifiedas

describedby Rhoades (19).

Electron microscopy. DNAwasprepared for

mi-croscopybyaproteinfilm procedurepatterned after

that ofWestmoreland et al. (30). The hyperphase

contained0.15 MNaCl, 0.5,ugof DNAperml,0.01%

(wt/vol)cytochromec,and 30to50%(vol/vol) form-amide. (Lower formamide concentrations produce clearer junctions between single- and double-stranded DNA. Higher concentrations render the

single-stranded regions more visible.) The

hypo-phasewasglass-distilledordeionizedwater.

Speci-mens wererotary-shadowed with platinum and ex-aminedinaPhilips EM200or a JEM100Belectron microscope. Tracings of projected molecules were measured with a Numonicsgraphics calculator. The double-stranded, circular DNAs ofbacteriophages

4)X174 and PM2 and the single-stranded DNA of

4)174 were employed as internal molecular weight

standards.

Other methods. The proceduresemployedfor the preparation ofradioactively labeled T5 phageand DNA, for sucrose density gradient centrifugation, and for the preparation of

qSX174

DNA have been described previously (21). PM2DNA was agift of S. Rogers.

RESULTS

Molecular weight of T5 DNA. A wide range of values has been reported for the molecular

weight of T5 DNA. In the case of T5st(+) (wild type),the valuesvary from 66 x 106 to68 x 106 asmeasuredby hydrodynamic methods (5, 27) to83 x 106 asmeasured by 32p autoradiography (15). The heat-stable deletion mutant T5st(0)

has generallybeen reported to contain 6 to8%

less DNA thanthewildtype (9).

To obtain areliablevalue for thisstudy, the

length of T5st(+) DNA was determined, in an

electron microscope, relative to the circular,

double-stranded forms of 4X174 DNA (3.4 x

106;28) and PM2 DNA (6.4 x 106; 18). Allthree

DNAs were present on the same grid, and at

least 5 ofeach ofthe reference genomeswere

included in every field. A sample of28

mole-culesyieldedavalueforT5st(+)DNA of76.1 ±

1.6 x 106relative to 4X174 DNA and of 77.0 ±

1.5 x 106relative to PM2 DNA. The averageof

thesetwodeterminations,76.5 x 106,is ingood

agreement withthevalueof 77.4 x 106recently

determinedby electron microscopyforT5st(+)

DNA (14).This value also providesaclose fitto

the empiricalrelationship between

sedimenta-tion coefficient and molecular weight

con-structed by Freifelder (7) for other coliphage

DNAmolecules.

The molecular weight ofT5st(0) DNA was determined relative to T5st(+) DNA by com-paring thefragments derivedby cleavageof the two genomes with restriction endonucleases.

Aspreviously described(19), EcoRI

endonucle-asedigestsofT5st(0)DNAlackthreefragments

foundinwild-type digestsand containone new

fragment. The molecular weight of this

frag-ment was shown by electron microscopy to be 5.6 x 106less than the sum of thethreemissing

wild-type fragments. A similar analysis

per-formed withHpaI endonucleasegave a value of 5.4 x 106 for the deleted segment. The average ofthese values represents a 7.2% deletion if T5st(+) DNA has a molecular weight of 76.5 x

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INTERRUPTIONS IN T5 727

106. (The details of these experiments will ap-pearinasubsequent publication.)

Digestion of T5 DNA with exonuclease III. E. coli exonuclease III catalyzes the stepwise

removal of 5'-mononucleotides from internal

andexternal 3'-termini in duplex DNA(17,23).

Since the mode of attack of exonuclease III is

random or nonprocessive (16), uniform

degra-dation of all susceptible sites can be achieved

with subsaturating levels ofenzyme. Limited

digestion of T5 DNA with exonuclease III should, therefore, generate short segments of single-stranded DNA atthe site ofeach

inter-ruption, aswell asatall duplextermini.

The exonuclease III used in this study was

extensively purifiedtoremoveothernucleases.

The principal contaminant during the later stages of purification was an endonuclease,

which introduced a limited number of

ran-domly placed single-chain breaks in T5 DNA. This endonuclease canbe separated from

exo-nuclease III by gel filtration as described in

Materials and Methods. Figure 1 shows the alkaline sucrose gradient profiles ofT5 DNA

afterdigestion to 3.3% with purified and

par-tially purified exonuclease III preparations.

The sedimentation profile of the DNA treated with purified exonuclease III is virtually

un-changed from that of untreated DNA.

Treat-ment with the partially purified preparation, however, resulted inasignificant loss of

mate-rial from the fastest-sedimenting peak, which

representsthe intact strand of T5DNA. Thefully purified preparation of exonuclease IIIhas also been analyzed in experiments

em-ploying both alkaline sucrose gradient

sedi-mentation andagarosegelelectrophoresis (20).

Under the conditions described here, T5 DNA

canbedegradedto atleast 7.8% without

detect-able endonucleolytic cleavage.

Electron microscopy of exonuclease III-treated T5 DNA. Exonuclease III-treated T5 DNAwas prepared for electron microscopy by

theformamide modification of thebasic protein film procedure. As originally described by Westmorelandetal. (30), this technique main-tainssingle-stranded DNA inanextended

con-figuration. Regions of single- and double-stranded DNA canbe distinguished, since the

former has athinnerand kinkier appearance.

Digestion of T5 DNAto3 to 4%with

exonucle-aseIII creates single-stranded segmentsof 600 to 1,000 nucleotides, which can be recognized

without difficulty in an electron microscope.

Except where noted, this level ofhydrolysiswas

employed in all of the experiments reported below.

Analysis of EcoRI fragments. The positions 200

100

a-W)I

100

200~

100

5 10 15 20

Fraction No. 25

200

100

200 0

a-100

100

FIG. 1. Effectof partial digestion with

exonucle-aseIIIon the alkaline sedimentationprofile of T5

DNA. 32P-labeled T5st(O) DNA was denatured and

centrifuged in alkaline sucrose gradients (a) after

incubation in a control reaction mixture, (b) after

digestion to3.3%withpurifiedexonucleaseIII,and

(c) after digestion to3.0% with apartially purified

preparationofexonuclease III.Thelatterpreparation

wasobtainedfromthegel filtrationstepdescribed in the text. Untreated 3H-labeled Xb2 DNA was

in-cluded in eachgradientas asedimentationreference.

Centrifugationwascarriedoutfor90minat40,000

rpminaSpincoSW50.1 rotor.Thedirectionof sedi-mentation is from right to left. Symbols: *, 32P_

labeledT5st(0) DNA; 0, 3H-labeled xb2DNA.

of the single-stranded segments generated by exonuclease IIIweredetermined for intact

mol-ecules ofT5st(+)and T5st(0) DNAaswellasfor

EcoRIfragments of T5st(0) DNA. Itwas

antici-pated that the measurements on intact

mole-cules wouldprovideasatisfactorydescription of

the locations of the single-chain interruptions. Theinitial results, however, revealedadegree

of intermolecular heterogeneity that made it impossible toalign individual molecules in an

absolute manner. This problem was circum-vented by combining the results obtained from intactgenomeswith those obtained from EcoRI

fragments.

EcoRIdigests of T5st(+) DNA containseven

fragments, of which thetwolargestoccuratthe

ends of the DNA (19).Fragment 1,representing

37%of thegenome,defines therightend of the

molecule, and fragment 2,a25%fragment, oc-cursinthesame half of the moleculeasthest

deletions (26). In the present experiments,

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EcoRIdigests wereprepared aftertreatmentof

the DNA with exonuclease III. As a result,

fragments 1 and 2 each had one duplex end,

generatedby EcoRIendonuclease,andone

sin-gle-stranded end, generated by exonuclease III. The orientation ofbothfragmentscould thus be unambiguously determined.

The positions of the single-stranded

seg-mentsin29molecules of EcoRI fragment1and 26 molecules of EcoRI fragment 2 of T5st(0) DNAareshown in Fig. 2. The fragments have

been alignedasthey wouldoccurin wild-type DNA, with the position of each single-strand/

double-strand junction plotted to the nearest

0.1% of the total length ofT5st(+) DNA. It is

apparent that single-stranded segments occur

frequently at two sites within EcoRI fragment 2, located at approximately 8 and 18%, andat

onesiteinfragment 1, locatedat65%.

Interrup-tionsoccurlessfrequentlyatanumber of other

sites,especiallyateach end oftheDNA,within

the terminalrepetition.

The nine molecules ofEcoRI fragment1 plot-ted at the top of Fig. 2a possess two duplex termini. These molecules, however, allappear to containthe single-stranded segment at65% and were aligned on that basis. The probable cause ofthis will bediscussedbelow.

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To determine which end of each

single-stranded segment represents theoriginal

inter-ruption, itisnecessarytoknow thepolarity of

thestrands inT5 DNA. Several studies (3, 10,

11) have indicated that most, ifnotall, of the interruptions inT5 DNAarelocated withinone

strand of the duplex. The polarity of this

strand, which is designated the "interrupted"

strand, can be determined by analyzing the

effect ofX exonuclease digestionon the single-chain fragments contained in T5 DNA. As orig-inally reported by Rhoades and Rhoades (21), and confirmed in the accompanyingpaper(20),

the 3'-terminus of theinterruptedstrandoccurs

at the left end of the molecule. Consequently,

theleft side of each single-strandedsegmentin Fig. 2 marks the site ofan interruption.

Histogramsdescribing the density of single-chain interruptions along EcoRI fragments 1 and 2 are shown in Fig. 3. Each vertical bar

represents the frequency of interruptions within a segment equal to 0.2% of T5st(+)

DNA. Of particular interest is the siteat65%, which, because of its proximitytothe EcoRIcut at62.8%,canbemapped with considerable

pre-cision. Over 90% of the interruptions that de-fine this siteoccurbetween 64.7 and64.9%, an

interval ofapproximately 200 base pairs. A

his-65 70 75 80 85 90 95 100 0 5

% WildType Length

10 15

% Wild Type Length

20 25

FIG. 2. Positions of the single-strandedsegmentsinexonucleaseIII-treated EcoRI fragments1 (a)and2

(b) of T5st(O) DNA. T5st(O) DNA was treated with exonuclease III and EcoRI endonuclease, and the

unfractionateddigestwasexaminedinanelectron microscope. EcoRIfragments1and 2wereidentifiedonthe basis of size. The lengths of the molecules in the twopopulations were normalized to 37.2 and 25.0%, respectively, of the wild-type length.Thegapswithineachmoleculerepresenttheduplexlengthequivalentto eachsingle-stranded region.

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FIG. 3. Histograms of the frequency of single-chain interruptionsinEcoRIfragments1(a)and 2(b) ofT5

DNA. Theresults shown in Fig.2 were used to determine thefrequency of interruptionswithin segments equalto0.2%of intact T5st(+) DNA.

togram of the positions of the right end of the single-strandedsegments at65% (notshown) is significantly less sharp. This result would be expected, duetoheterogeneity inthe extentof exonuclease IIIdigestion, if the polarity of the interrupted strandwereas described above.

Electron micrographs of exonuclease III-treated EcoRI fragments are shown in Fig. 4

and 5. Figure 4 shows acomplete molecule of

fragment 1, in whichsingle-strandedsegments

defining interruptions at65 and 78% are

visi-ble. Figure 5 shows portions ofseveral mole-cules in which single- and double-stranded ends, as well as internal single-stranded

re-gions, arevisible.

Analysis of intact DNA. The positions ofthe

single-chain interruptions in 27 molecules of

T5st(+) DNA and23 molecules of T5st(0)DNA

are shown in Fig. 6. These molecules were

alignedon the basisofthe principal

interrup-tions at8, 18, and 65%observed in EcoRI

frag-ments 1 and 2. For every molecule ofT5st(0) DNAandmostof thoseofT5st(+) DNA, oneof

the twopossibleorientationsgaveabetter fit at

thesepositions. Thepattern ofinterruptions is

sufficiently complex, however,thatsomeof the

molecules maybeincorrectly aligned. Thegap inthe molecules of T5st(0) DNArepresents the

7.2%deletion thatoccursfrom 26.3 to33.5% in

wild-type DNA. Theseendpointsreflectminor

revisions ofthosereported previously (26).

Histograms of the positions of the single-chain interruptions in T5st(+) and T5st(0)DNA

are shown in Fig. 7. Each vertical line repre-sents0.5%ofT5st(+) DNA. Anelectron

micro-graph ofamolecule ofT5st(+)DNAcontaining

five single-strandedsegmentsis shown in Fig. 8.

The results in Fig. 3 and 7 reveal that T5st(+) DNA contains four sites, locatedat7.9, 18.5, 32.6,and 64.8%, where interruptionsoccur

inmost,butnotall,molecules. Except for these sites, the internalregionsof T5 DNAare

rela-tively free from interruptions. The only other internal sites where interruptions appear to

occur inmore than 10% of the population are

locatedat45 and 78%. In contrast, the endsof

the DNA contain a relatively high density of

interruptions. Theseterminal sites arelargely

clustered between 0 and 5% and 91 and 96%.

Since theterminalrepetitionin T5 DNAequals 8.3% ofwild-type DNA (20, 21), these clusters

occur at analogous positions within the

re-peated segments. A more detailed analysis of

the left end is presentedin the next section.

The distribution ofinterruptions in T5st(0) DNA issimilartothatinT5st(+) DNA, except that the interruption at 32.6% lies within the

deleted region. The histograms in Fig. 7 sug-gest that, compared with wild-type DNA, T5st(0) DNA contains fewer interruptions at

the right end of the molecule. However, a

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730 SCHEIBLE, RHOADES, AND RHOADES

FIG. 4. Complete EcoRI fragment 1 of T5st(0) DNA after 4%digestion with exonuclease III. The

arrowsindicate thetwoendsof thefragment,oneof

which is single-stranded. Two internal single-strandedsegments, specifying interruptions at64.8 and 77.8%, are also visible. The bar represents a

duplex length of 500 basepairs.

larger number of molecules of bothtypeswould

havetobeanalyzedtosubstantiate this

conclu-sion.

The total number of interruptionsper

mole-cule is highly variable. Thesample ofT5st(+)

DNA studied here contained an average of eight interruptions. Some molecules hadasfew

as four, whereas others had as many as 13 interruptions. ForT5st(0) DNA,the range was

from3to10, withanaverageof6interruptions

permolecule.

Analysis of the interval from 0 to 8%. As

describedintheprecedingsections,theinterval

between 0 and 8% atthe left end of T5 DNA

contains a high density ofsingle-chain inter-ruptions. Thisregion wasinvestigatedinmore

detail byexaminingT5st(0)DNA in anelectron

microscope after degradationto0.9%with

exo-nucleaseIII.The single-strandedsegments

cre-atedby this level of hydrolysis (250 nucleotides)

werebarely large enoughtobe identified. Only

unambiguous single-stranded segments were

included in this analysis. An electron micro-graph in which several short single-stranded segmentsarevisibleisshowninFig.9.A histo-gramof the frequency of single-chain interrup-tions attheleftend of T5DNA isshowninFig.

10. This histogram was derived from measure-ments on 14 intact molecules and 8 molecules

whose right ends were either too tangled to

measure orobscuredby agridbar.

The resultsinFig. 10indicate that, in

addi-tion totheprominent site at 8%, interruptions

are clusteredat 0.8, 2.4, and 3.4 to 4.0% from

theleftendof the molecule. Evidence for sites

at2.4and3.4 to 4.0% canbeseeninFig.3.The

siteat0.8%,however,isnotvisible in themore

highly degraded molecules. Interruptions

ap-pearto occur at these sites in 20to30% of the

population. The level of interruptions at the

right ends of the molecules sampled in this

experimentwas toolowtopermitanalysis.

DISCUSSION

The results presented above demonstrate thatT5 DNA possessesalarge number ofsites

where single-chain interruptions can

poten-tiallyoccur. The frequencieswithwhich

inter-ruptions actually occurredatthese sites inthe

mature form of T5 DNAwere highly variable.

Although amajority ofthe interruptions were

found at four sites and atthe two ends of the

genome, asignificant fraction of the

interrup-tions occurred at siteswhere the frequency of breakswasless than0.10. Individual molecules of T5 DNA thus differ with respecttoboth the number and location ofsingle-chain interrup-tions. Within thepopulations of intactT5st(+)

andT5st(0)DNAsampledinthisstudy,no two

moleculespossessed identical structures.

The validity of these conclusions depends

largelyontheproperties of E. coli exonuclease

III. For an accurate picture ofT5 DNA to be

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FIG. 5. Portions of several EcoRI fragments ofT5st(0) DNA after 4% digestion with exonuclease III. The arrowsindicate single-stranded regions, both internal and external. The molecule centered in (A) is the left half of EcoRI fragment 1. The bars represent duplex lengths of 500 base pairs.

obtained, it is essential that exonuclease III

initiate hydrolysis at all of the natural inter-ruptionswithoutintroducingadditional

single-chain breaks. The latterrequirement appears

to have been satisfied, since itwaspossible to

prepare exonuclease IIIthatwas free from de-tectable endonuclease activity. It would be

dif-ficulttodetectalow level of activity, however,

anditisconceivable thatasmall fractionof the

observedinterruptions werespurious.

Thefinding thatinterruptionsdo notalways

occur ateach ofthe principal sites might

indi-cate the occasional resistance of internal

3'-termini to hydrolysis. In fact, Hayward and Smith (9)suggested, on the basis of their elec-trophoreticanalysis of denaturedT5DNA, that

theprincipalinterruptionsarealwayspresent.

Theelectrophoretic studiespresentedinthe

ac-companying paper (20), however, demonstrate

that eventhe most frequent interruptions are

occasionally missing. Moreover, studies onthe

properties of exonuclease III have indicated

thatvirtuallyallligase-repairablesingle-chain

interruptions in apopulationaresusceptibleto

hydrolysis(17). It isthusunlikelythata

signif-icantfraction of the natural interruptionswas

overlookedbythe exonuclease.

A moreserious problem concerns the extent

of exonuclease III hydrolysis. In most of the

experiments reported here, single-stranded

segmentsof 600 to 1,000 nucleotides were cre-ated at the site of each interruption. These

conditions clearly preclude detection of

inter-ruptionslocatedwithin 1,000base pairsfroman

endorfrompairsofinterruptionsseparated by

less than this distance. This situation can be

remedied,inprinciple,bydecreasing theextent

of exonuclease III hydrolysis. One experiment inwhichsingle-strandedsegmentsof 200 to 300

nucleotidesweregenerated did allow the

iden-tification of an additional site,located0.8%(900

nucleotides)from the left end of the genome. In

practice, however, very short single-stranded

segments aredifficult toidentifywith certainty (Fig. 9). Furthermore, as demonstrated in the accompanying paper (20), interruptions occur

inT5 DNA at alocation that would be virtually impossible to detect regardless of theextentof

digestion. This site occurs 0.4% (450 basepairs)

from therightend of the molecule. Since the

3'-terminusofthe intactstrandalso occurs at the right end of the molecule, the 0.4% segment will bedegradedfromboth directionsby

exonu-clease III. Removal of225 nucleotides per

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* UL

II

I Il

*

,, _

.I

(a)|

I Il I Il I II I I I I

0 10 20 30 40 50 60 70

I I

80 90 100 % Wild Type Length

FIG. 6. Positions of single-chain interruptions in intact molecules of T5st(+) and T5st(0) DNA. After normalizationto 100%of the wild-type length, the molecules were aligned to give the best fit at the 7.9, 18.5, and 64.8% sites, where interruptionsoccurat ahigh frequency. The vertical bars represent the left end ofeach internal single-stranded segment. The deleted segment inT5st(0) DNA has been left blank so that the maps of thetwogenomescanbecompared directly. (a) T5st(+) DNA; (b)T5st(0)DNA.

II II

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40 50 60 70 80 90 100

%Wild Type Length

0 10 20 30

FIG. 7. Histogramsof the frequency ofsingle-chain interruptions in T5st(+) and T5st(0) DNA. The results shown inFig.6wereusedtodetermine the frequency ofinterruptionswithinsegmentsequalto0.5%of intact

T5st(+)DNA.(a) T5st(+)DNA; (b) T5st(0) DNA. 0.50

0.40 0.30 8 0.20

.2 CL 0.10

C, 0.50 0.40

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0.30 0.20 0.10

I I I I I

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INTERRUPTIONS IN T5 DNA 733

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FIG. 9. T-st(.)DNAafterdigestionto0.9%with exonucleaseTI Thearrows indicateshort, internal

segments ofsingle-stranded DNA. The bar

repre-sentsaduplex length of500 basepairs.

terminus will therefore result in loss of the right (5') end ofthe interrupted strand. This

effectisundoubtedlyresponsibleforthe appar-ent absence ofsingle-stranded materialat the

right ends ofsomemolecules (Fig. 2).

Many of the structural features of T5 DNA described in this study were first proposed by

Hayward and Smith (9, 10), who fractionated denaturedT5DNAbyagarosegel electrophore-sis into 4major andover40minorsingle-chain

fragments. The majorfragments werethought

todefineprimary interruptions thatoccurinall

molecules, whereas the minor fragments were

proposedtoresult fromsecondaryinterruptions

that occur less frequently within the major

fragments. The sizes of both the major and minorfragmentswerediscrete, and the

electro-phoreticpatternswereunaffected byvariations

in host bacteria and in the method of DNA

preparation.Theonlyqualitativedifference

be-tween the model proposed by Hayward and

Smith(10)and the conclusions obtainedinthe

present study is that, as mentioned earlier,

eventhe mostfrequent interruptions are

occa-sionally missing. However, because of the

as-sumptionthat theentiremoleculecanbe

recon-stituted from major fragments, these authors

failed

torecognize the segmentbetween 0

and

7.9%. The density of interruptions in this re-gion is too high to yield a major

fragment,

although a minor fragment that defines this

segment canbe identified(20).

Several other studies on T5 DNA have

mapped single-chaininterruptions at sitesthat

agreerelatively well with the positions

deter-mined for the principal interruptions in the

presentstudy. Inanelectron microscopic exam-ination of partially denatured T5st(+) DNA,

Bujard (3) identified interruptions at sites

lo-cated 7.9, 19.0, and 60% from one end ofthe

molecule. Bujard and Hendrickson (4)

deter-mined the positions of the major

single-stranded fragments of T5st(+) DNA,

isolated

by sucrose gradient sedimentation, by

anneal-ing eachfragment tothe intactstrand.

Analy-sisofthesestructures in anelectronmicroscope

gavepositionsfor single-chain interruptionsat

3.9, 7.8, 17.8, 30.6, and 65.0% of the wild-type

length. Hayward (8)demonstrated that

hydro-dynamic shearing breaks duplex T5 DNA at

single-chain

interruptions. The positions of

these shear-sensitive points were determined

by electrophoretic procedures to occur at 7.4,

16.9, 29.5, and 64.0% of the wild-type length.

Morerecently, Saigo (24, 25)haslocated

inter-ruptions at 8.2, 17.9, 32.6, and64.0% by exam-iningsheared tail-orghost-DNA complexes in

an electron microscope and at 18.0, 33.1, and

0.30

0.20

0.10

0 2 4 6 8 10

%WildTypeLength

FIG. 10. Histogram of the frequency of single-chain interruptionsattheleft endofT5DNA. The positionsof thesingle-strandedsegmentswere deter-minedin 22moleculesofT5st(0)DNAthathad been digestedto0.9%with exonucleaseIII.Themolecules

were aligned as described in the legend toFig. 6,

exceptthat thelengthswere notnormalized.Instead, the positionof each interruptionwasplottedrelative

totheleft end of the DNA. Each vertical line

repre-sents0.2%of theintactT5st(+) DNA.

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64.5% by denaturing mapping. Except for the

60% site reported by Bujard (3), all of these

values agreereasonably wellwith the positions

determinedinthe presentstudy.

The T5st(0) deletion mutant has been shown by heteroduplex mapping to lack the segment between 26.3 to 33.5% on the wild-type genome (26; M. Rhoades, unpublished data).

Conse-quently, the prominent interruption locatedat

32.6% shouldbe missing inT5st(0) DNA. The absence of this site had been previously

in-ferredfromthe electrophoretic studies of

Hay-ward and Smith (9, 10) and Hayward (8). A

different structure for T5st(0) DNA has been

proposed by Labedanetal. (13). These authors

analyzed the products formed by shearing

T5st(0) DNA after attaching one end of the

molecule, by phage infection, to the bacterial

membrane. Under the assumption that a uniqueendof the DNA was injected, amodel

for T5st(0) DNA was proposed, with

interrup-tions located 8, 19, 35, and 60% from the end

that initiated transfer. This result indicated

that the leftendof the genome, as describedin

the present study, initiated infection. T5st(0)

DNA, however, does not contain a detectable

levelof interruptionsinthe vicinityof35%from the leftend. The origin of this discrepancyhas

notbeendetermined.

Itwasinitiallyanticipatedthatknowledgeof

the positions of the single-chain interruptions in T5 DNA would provide some insight into their originand function. The major

contribu-tionof thisstudy,however, hasbeento

demon-stratethecomplex and heterogeneousnatureof

T5 DNA. A more detailed description of the locations of single-chain interruptions in T5

DNA ispresented in the accompanying paper

(20).

ACKNOWLEDGMENTS

Wearegrateful to Rolf Benzinger and the Biology De-partment, University of Virginia,Charlottesville, for pro-viding supportand facilities for portions of this study. P.S. was supported by Public Health Service training grant 5 T01-GM-5717 from the National Institute of General Medi-cal Sciences. This research was supported by grant GB-20460from theNational Science Foundation.

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