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0022-538X/84/050293-08$02.00/0

Copyright C) 1984, American Society for Microbiology

UV Irradiation

Impairs

In

Vivo

Encapsidation

of

Bacteriophage

T4

DNA

ARTHURZACHARY* AND LINDSAYW. BLACK

Department of Biological Chemistry, University of MarylandMedicalSchool, Baltimore, Maryland 21201

Received5 August 1983/Accepted10January 1984

T4 DNA structuralrequirements forencapsidation in vivowereinvestigated, usingthin-sectionelectron

microscopy toquantitate the kinetics and yields of head intermediates aftersynchronous DNA packaging into accumulated processed proheads. UV irradiation (254 nm) of T4-infected bacteria justbeforeinitiation

ofencapsidation resulted inareductioninthe rateof DNApackaged measured by electron microscopy and inthe yield of viable phage progeny. InUV-irradiatedinfections with excision-deficient mutants (denV-), theextentofpackaging declinewasproportionaltothe UV dose andphage yieldswerelower thanexpected

based on thepackaging levels observed by microscopy. Rescue analysis ofprogenyfrom such infections revealed elevatedlevels of nonviable virions. Pyrimidinedimerswereencapsidatedin denV-infections,but

in excision-competent infections (denV+) dimers were not packaged. A UV-independent, 15 to 20% packagingarrestwasalso observedwhen denV endonucleasewasinactiveduring encapsidation, indicating

a denV requirement to achieve normal T4 packaging levels. Pyrimidine dimers apparently represent or induce transientblockage ofDNAencapsidationorboth, causingadecline in therate.Thisis in contrast to

other DNA structural blocks to packaging induced by mutations in T4genes 30 and 49, which appearto

arrestthe process.

DNA encapsidation in bacteriophage T4 development

involveslinkage of newlyreplicated concatemericDNA toa

functionalcleaved proheadbyphage-specified

linkage/pack-agingproteins andsubsequenttranslocationof the DNA into

the prohead (for review, see reference 5). Many mutations

have beencharacterized which prevent DNApackagingas a consequenceof alterations inthe structureoftheproheador

inthelinkage/packaging proteins. However, onlytwo

exam-plesofmutations inducing changes inDNA structurewhich

interfere withencapsidation have been demonstrated.

Mutations in T4 gene 49

(endonuclease

VII) result in the

intracellularaccumulation of expanded,partially DNA-filled

head structures attached to concatemeric, very fast

sedi-mentingDNA (6, 9, 10, 16, 18-22, 43). In vitro the gene 49

nuclease cleaves veryfastsedimenting DNAtoform slower

sedimenting linearconcatemeric molecules (17, 30)and can

resolve Holliday structures in DNA (27). In vivo gene 49 nuclease apparently cleaves recombination-derived

branched strands in concatemericDNAwhich would

other-wise prevent DNA encapsidation (16,25-27).

Mutations in T4 gene 30(DNA ligase)and in Escherichia

coli DNAligase,like gene 49mutations,reversiblyarrest T4

DNA packaging (7, 13, 45). The ligase function may be

necessary to seal nicks in the DNA concatemer which

presumably preclude encapsidation, and ligase could also

functionas apartofthepackagingmechanism and therefore

be required duringthe translocation process itself (6).

In this study we found that after UV irradiation of

replicated concatemeric T4 DNA in vivo there is an

impair-mentof its subsequent encapsidation compared with

unirra-diated DNA. This packaging impairment was most evident as adecline in the rate of packaging and was shown to be due largely to the formation and repair of pyrimidine dimers in T4DNA. We have also found a UV-independent packaging

impairment associated with mutations in the T4 gene denV.

These resultsprovidesupport for the hypothesis that the rate

* Correspondingauthor.

ofT4 DNA encapsidation in vivo is influenced

by

DNA

structure and by processes, such as repair, which modify

DNA structure.

MATERIALS ANDMETHODS

Bacteria andbacteriophage. E. coli B40(SU1+),an

amber-suppressing strain, wasused in experiments involving

elec-tronmicroscopy and marker rescue; E. coli BE,an

amber-nonsuppressing strain, was used in experiments involving

radioactive labeling and marker rescue; and E. coli AS19

(kindly provided by L. Gold), a

rifampin-permeable

strain,

wasusedfor rifampinexperiments. ThefollowingT4strains

were used in this study: T4D+; cs2O(N33) ts2l(N12)

amt(A3); cs20(N33)

ts2l(N12) amt(A3) denV,; am23(H11)

am23(B17); am55(BL292); cs2O(N33) tsdenV(431)

[tsdenV(431) mutant kindly provided by M. Sekiguchi].

H-broth was used forgrowthand phage dilution medium, and M9A wasused forlabeling experiments (8).

Temperature shift experiments for electron microscopy. E. coli B40 was growninH-broth at37°C to aconcentration of

2 x 108cells per ml. The culture was thentransferredto 20°C

and infected with the desired phage at a multiplicity of

infection of5.After 22 min, thecells were superinfected at a

multiplicity of infection of 5. After 85 min at 20'C, the

infected cells maintained at 4°C were centrifuged and

sus-pended in phosphate buffer (8), exposed to UV irradiation,

and then centrifuged and suspended in H-broth. Samples were removed for assay of infective centers or for fixation

for electronmicroscopy, and the culture was transferred to

42°C. Samples forphage yield orfor fixationwere removed

at various times after transfer to 42°C. When the inhibitor rifampin (Sigma Chemical Co.) was used, it was added (final concentration, 200 ,ug/ml) 5 min before UV exposure and shift to 42°C. Each experiment was repeated two to five

times, and results presented are mean values for the

repli-cates.

Temperature shift experiments for radiolabeling. E.coliBE wasgrown in M9A at 37°C to a concentration of 3 x

108

cells per ml. Deoxyadenosine (250 ,ug/ml) and

[methVl-3H]thy-293

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FIG. 1. Thin sections through UV-irradiated (51J/m2)excisionrepair-competent (V+)and -deficient (V-) T4-infected E. coli B40cells, fromarepresentative temperature shift experiment. (A) V+ infection at 10 mps showing EH, PFH, and FH, indicating that packaging has occurred. Prohead I structures (PHI) formed after shift to 42°C cannot mature because of the 21 ts mutation. (B) V+ infection at 20 mps

showingthatEH and PFH have been converted to FH. (C) V- infection at 10 mps; predominance of EH (some are expanded) indicates reduced packaging. Arrow indicates a tailed EH. (D)V-infection at 30 mps.Presence of several DNA containing heads indicates that some

packaginghasoccurred; however, many EH remain. Magnification is the same in all micrographs; bar in (C)= 100nm.

mine (5

XCi!ml)

wereadded, and theculture was grownfor

30 min at 37°C. The culture was transferred to 20°C,

L-tryptophan (40 ,ug/ml)wasadded, and the cellswereinfected

with the desired phage at a multiplicity of infection of 5.

After 20

min,

[methyl-3H]thymidine

(5 ,uCi/ml) was added,

and after22minthecellswere

superinfected

at amultiplicity

of infection of5. After 85 min at

20°C,

the infected cells,

maintainedat4°C,were centrifugedandsuspendedin

phos-phate buffer, exposed to UV irradiation, centrifuged and

suspended in unlabeled M9A, and thentransferred to42°C.

After30 minat 42°C, the cellswere

lysed

with chloroform

and treated with DNase (100

p.g/ml;

Sigma)

for 30 min at

37°C, and thephage were

purified

by differential

centrifuga-tion followed by CsCl step

gradient centrifugation.

Preparation of DNA. CsCl step gradient-purified phage

were extracted with an equal volume of redistilled phenol

(saturated with buffer and adjusted to pH 7.4) and 0.5

volume of chloroform-isoamyl alcohol (24:1, vol/vol) until the aqueous layer cleared after low-speed centrifugation. Theaqueous

layer

was

dialyzed against

endonuclease buffer

(see below).DNA solutionswerestoredat4°Cover

chloro-form.

Pyrimidine dimer assay. Pyrimidine dimersin DNAcanbe

detected as sites sensitive to

nicking by

the endonuclease

activity of phageT4 gpdenV(11,42). H-labeledDNA

(>103

cpm), purified from phages produced in temperature shift

experiments, wassuspendedin endonucleasebuffer(10mM

Tris-chloride, pH 8.0, 10mM Na2 EDTA, pH8.0, 100 mM

NaCl) and treatedwith 1 pd of

purified

T4 gp denV

(kindly

provided by E. H. Radany; activity, 1.83 pmol of

photoreleasable thymine permin at 37°C; see reference 23

for details) for 15 min at 37°C. Duplicate phage DNA

samples were treated with buffer only. The reaction was

stopped by addition of 0.2 N NaOH for 15 min at room

temperature. The DNA was then

layered

on 5 to 20% alkalinesucrose (0.15 MNaCl, 0.2 N NaOH)

gradients

and sedimented for 80 minat 45,000rpmand

4°C

in an SW50.1 rotor. Gradient samples were collected from a puncture in the bottomof the tube directlyintoACS

(Amersham

Corp.)

scintillation cocktail and counted in a Packard scintillation

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[image:2.612.116.512.70.445.2]
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I

51 102 153

L(100) 100

l

O 51 102 153

U.V

DOSE

(J/m2)

FIG. 2. Effect of UV dosageonpackagingof T4 DNA inV+and

V- infections at 9 mps. Hatched bars represent PFH, solid bars represent FH, and solid dots represent head structurescontaining

someDNA(FH plusPFH). For all microscopy data, 400to600head

structures werecounted todetermine each value (seetext). counter. The number of endonuclease-sensitive sites (ESS)

wascalculated by the method of AhmedandSetlow(1). For

controls, unirradiated T4 DNA and T4 DNAexposed toUV

irradiation after extraction were analyzed for ESS in the

same manner.

UVirradiation. Sampleswere irradiated with UV light at

254 nm, using aGE15T8 germicidal lamp (General Electric

Co.). Dosagewas measured with a IBLAKRAY UV meter

(U-V Products, J-225).

Electron microscopy. Samples were partially lysed by

addition of osmiumtetroxide(finalconcentration, 0.01%)for

2minand thenfixedwithphosphate-buffered (8) glutaralde-hyde (final concentration, 0.25%) for 55 min at 4°C. The

glutaraldehyde-fixed cell suspensions were centrifuged and

thepelletswereoverlaid with 1% buffered osmium tetroxide

for18 h at 4°C. The samples were thendehydrated, usinga

graded acetone series, and embedded in Epon 812 epoxy plastic. Thin sections were stained with uranyl acetate and

lead citrate before examination with aSiemens 1A electron microscope.

To quantitate the types of head-related structures in thin

sections, each cell profile entering the electron microscope viewing field was examined and all clearly distinguishable profiles of head-related structures were scored. For each datapoint, 400to600capsid profiles were counted.

Statisti-cal significance (99% level) was determined by chi-square

analysis. Some imprecision maybe inherentin the counting method, because structures containing very small amounts

of DNA could have been considered empty and structures

nearly filled with DNA could have been considered full head-related structures. However, in duplicate counts, i.e., thesamesample countedondifferentoccasions, the

variabil-itywas <2%.

Defective phage rescue. Phage yields from temperature

shift experiments were measured as plaques formed on

lawnsofE. coli B40, an amber-suppressing host. To

deter-mine whether defective phages (i.e., complete DNA-filled

virions with a genomic defect which rendered them

undetectable by plaque assay) were produced after UV Uf)

w r

0

H

cn0

I 0

H

CY)a

w

0

w 0~

llJ

L

100-* (31)

50-100 102J/m2

50

-I

100-(31)

50-(80)

10 20 30

51 J/m2

%

(2)

10 20 30

102

J/m2

0

(0.4)

0

pi

0 1 20 30 0 10 20 30

MINUTES AFTER

START

OF

PACKAGING

[image:3.612.53.292.67.278.2]

(SHIFT

TO

42°)

FIG. 3. Effect of UV irradiation on packaging kinetics for V+ andV-infections. Numbers in parenthesesareviablephage yields at30mpsexpressedas apercentageoftheyield foranunirradiated V+infection. Hatched barsrepresentPFH, solid barsrepresentFH, and soliddotsrepresentheadstructurescontainingsomeDNA (FH

andPFH).

irradiation, the phage yields were also plated on lawns of

amber-nonsuppressing bacteria (E. coli BE) coinfected with T4 phages having two amber mutations in gene 23

[am23(H11), am23(B17)]. These double-amber phages, al-though not competent to form a plaque, could provide

complementary gene products and thus rescue defective

phages if present in the yields from the temperature shift experiments. Thephage yieldswerealsoplatedonlawns of uninfected E. coli BE as a control for background plaques expected due to the leakiness reported for the t amber mutationat37°C (15) aswellas anyamberrevertant forma-tion. T4D+, a control for plating efficiency, and T4 am55

phages, a control for the efficiency of rescue of an amber

L)

w

I-

I--C], 0

w

w

I0

LL 0

0--

100- 90- 80- 70- 60- 50- 40-

30-

20-

IO-0-

I~

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[image:3.612.309.551.72.526.2]
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mutation inoursystem, were plated in thesame manner as

the phage yields. Plaqueswerecountedafter18hof

incuba-tionat 37°C.

RESULTS

in this study we used a previously developed electron

microscopic technique which allowsassessmentof the kinet-ics and extent of DNA packaging in vivo, independent of earlystepsinhead assembly and of the ultimateinfectivity of the phage produced (45). Growth at 20°C of cells infected with a cs2O ts2l mutant allows normal expression of T4

functions (including thets mutantgeneproductfunction) up to the point of DNA encapsidation. Normal DNA and late protein synthesis occur, leading to intracellular

accumula-tion ofempty headstructures (EH), butnoDNA-containing

head structures or viable phage accumulate (14, 45). After temperatureshift from20to42°C, the cs20blocktoprohead

function is released, DNA packaging is initiated synchro-nously, andarapid transition of EH formedat20°Ctoheads partially filled withDNA (PFH) and thentoheads complete-ly filled with DNA (FH) occurs (45). A representative

micrograph from oneexperiment (Fig. 1A) shows examples

of the head structures described above. The kinetics and levels of encapsidationcanbe measuredasdifferences in the

proportion of the threetypes of head structures(EH, PFH,

and FH) seen intracellularly after shift to 42°C and are consistent withkinetic measurementsofphage yields (6, 13, 45), i.e., the half-time for packaging is 5 min after

tempera-tureshift.Packagingand phage completion aftershiftto42°C occur in the presence ofchloramphenicol; thus, continued

protein synthesis is not required for phage maturation (14). Any head structures assembled after shift to 42°C are

blocked in maturation dueto tsgp2l at the prohead I stage

(cf. Fig.1A).Thesecore-containing, membrane-bound,

mor-phologically distinct prohead I structures cannot package

DNA and therefore are notenumerated.

Effect of UVdoseonDNApackaging. T4infections involv-ingexcisionrepair-competent bacteriophage (cs2Ots2l amt) will be referred to as V+ infections and excision repair-deficient phage infections (cs2O ts2l amt

denV,)

will be termed V- infections.

-E.

coli B40 infected with V+ phage

weregrown at20°C for 85min, exposedtovarious doses of UV irradiation, and then shifted to 42°C to initiate DNA

packaging.Anassessmentof DNApackagingwasmadeat9

min postshift (mps)to42°C by examiningthe proportionsof intracellular headstructureswhich had accumulated in thin-sectioned cells. Unirradiated V+-infected cells showed ex-tensivepackaging, with 80% of the headstructures

contain-ing some DNA: 19% PFH and 61% FH (Fig. 2). Infected cellsreceivingaUV dose of51J/m2had roughly25% fewer

DNA-containingheads: 40%PFHand 22%FH(Fig. 2).Ata UVdosageof 102J/m2therewas nosignificant changein the percentage of DNA-containing head structures, but there was asmall, statistically significantdeclineintheproportion

of FH (from 22 to 10%); at a dose of 153 J/m2 no further

impairment of packaging was observed (Fig. 2). For V-infections receiving UV irradiation, we observed

substan-tially less packaging than in corresponding

V'

infections,

and the declines in V-packagingwere more nearly

propor-tional to increases in the UV dosage used. As dosage

increased from 51 to 102to 153 J/m2, the amount of

DNA-containingheads declined from41 to 24to 15%, respective-ly,at9mps(Fig. 2).Thus,atthehighest dosage (153J/m2)at

[image:4.612.327.563.83.178.2]

9 mps, there were one-fourth as many DNA-containing heads in the V- as in the V' infection, indicating a clear differencein packagingofUV-irradiated DNA(Fig. 2).

TABLE 1. Datafrom kinetic studies'

Packaging Yield %Defective Phage UVdose (% V- FH) (% V+PFU) virions

10mps 30mps at 30 mps (due toUV)Y

V+ 0 100 100 100 0

V+ 51 54 66 31 15

V+ 102 45 70 31 NOC

V- 0 71 84 80 0

V- 51 25 60 2 48

V- 102 8 26 0.04 NO

aPackaging and yield data are compared with an unirradiated excision-competent(V+)infection.

bTakenfromTable 2. NO, No observation.

Kinetic studies. UV-induced lesions in DNA might either remain as permanent blocks to packaging or be repaired after somedelay, thus allowing DNA to be encapsidated. To test these

possibilities,

temperature shiftexperiments were

con-ducted in whichthe UV dosage was 0, 51, or 102J/m2,and

samples takenat 10and 30 mps to 42°C were examined by electron microscopy. Viable phage yieldsweremeasured at 30 mps. In irradiated V+-infected cells at 10 mps, the UV

impairment ofpackaging was evidentas a reduced level of

DNA-containingheads and anincreasedproportion of PFH,

and the decline in packaging was more evident as the UV

dosage increased (Fig. 1A and 3). However, this

dose-dependent difference in packagingwas nolonger evident at

30 mps, when packaging levels for both dosages were

essentially the same (Fig. 3). In both cases packaging

continued in the latter 20 min but complete recovery of

packaging to the unirradiated level was not observed (Fig.

1B and3). Theyieldofviable phageatbothUVdosages was

31%of that in theunirradiatedV+ infection (Table 1; Fig. 3). The UV-inducedpackagingimpairment wasgreater inthe

V- infection; at 10 mps,after a dose of51 J/m, fewer than

50% ofthe heads contained DNA and only 15% were FH

(Fig. 1C and 3). At 30 mps we observed an apparent

TABLE 2. Rescueofplaque-forming abilitytoshowpresenceof defective phageprogenyinyields from UV-irradiated(51J/m2)

infections'

Rescued back- Nonviable(%

Inputon ground (PFU) on:

Phage UV B40Rescued - Due to

PF) am23 BE background! V,

input

V+ - 300 82 31' 17 0

V+ + 300 125 29' 32 15

V- - 300 68 24' 15 0

V- + 300 229 39' 63 48

T4amSS - 300 51 0 17 0

T4+ - 300 300 301 0 0

aTheinput phagetiterswereadjustedto300 PFUonE.coliB40;

rescuedphageprogenyarethoseplaques formedonlawnsof E.coli BEseededwithadouble-ambermutant(T4amHll amB17)which provides complementing gene products. The rescue value is then adjusted fortheleakiness (see footnotec) of theamtmutation and for the 17%amber rescue efficiency observed in our experiments

(T4am55).

b Defective valueadjusted forbackground plaqueformation at-tributedtonon-UVfactors.

CPlaquesformedareattributedtoleakiness of theam tmutation expectedat37°C (15).

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recovery of some packaging(23% PFH, 48% FH); however, the yield of viable phage wasfar lower than expectedfor the amount of FH observed (Fig. 1D and 3; Table 1). The

amount of FH at 30 mpsin this V-infection irradiated at 51

J/m2was 91% that observed in the corresponding V+ infec-tion, yet theyield was only8% of theV+ value. At the102J/ m2UV dosagein a V- infection the impairment topackaging was more severe, and although packaging continued to increase with time, few FH were observed andessentially no viable phage were produced (Fig. 3).

The difference between the levels of intracellular FH and the actual yield of viable phage in irradiated V- infections

raised the possibility that DNA-filled, mature phage, defec-tive in plaque formation, were being produced. This hypoth-esis was tested in experiments designed to rescue defective progeny phages from irradiated (51 J/m2) or nonirradiated, temperature-shifted V+ and V- infections (at 30 mps). The results indeed showed that only after exposure to UV irradiation were significant numbers of defective phages produced (Table 2). Here the number of defective phage progeny rescued by complementing gene products from the double-amber helper phage are adjusted for the leakiness of the t amber mutation, as well as the 17% rescueefficiency we observed for rescue of a control amber mutation in the experiments (Table 2). It is obvious that the proportion of nonviable particles in the V- yields was far greater than in the V+ yields (Tables 1 and 2). Fully packaged nonviable phages therefore are produced in high proportion after exposure to UV, especially in repair-deficient infections, and this accounts in large part for the difference noted between DNA packaging and the yield of viable phage.

ESS in packaged DNA. The presence of pyrimidine dimers in packaged DNA can be detected by use of purified gene denV enzyme which specifically nicks DNA at the site of a dimer. Nicks in the DNA can then be detected by alkaline sucrose gradient analysis (11, 42). Packaged DNA extracted from phage produced in UV-irradiated (51 or 153 J/m2) V+ infections sedimented to the same position in alkaline su-crose gradients whether or not the DNA was treated with purified gp denV (Fig. 4B). The same sedimentation profile (peak represents head full-length T4 DNA) was observed for T4D+ phage-extracted DNA (unirradiated) treated in the same manner (Fig. 4A). Since ESS were not present in packaged DNA, we conclude that dimer-containing DNA is not encapsidated in UV-irradiated Vt infections. In con-trast, denV-treated, phage-extracted DNA from irradiated

(51 J/m2) V- infections sedimented more slowly in

denatur-ing gradients than similaruntreated DNA which sedimented to the head full-length position (Fig. 4C). The presence of ESS (two to six sites per genome length of single-stranded T4 DNA) in packaged irradiated V- DNA indicates that thymine dimers are encapsidated in repair-deficient infec-tions. The presence of phages containing thymine dimers is consistent with the large number of defective virions pro-duced in UV-irradiated V- infection. As a control for the dimer assay, DNA from V+ (and V-; data notshown) phage progeny was exposed to UVirradiation after extraction (153 J/m for

V-;

51J/m2 for V+) and then analyzed for ESS. In both cases many ESS wereobserved (40 or more per genome length ofsingle-stranded DNA), indicating the presence of many dimers (Fig. 4D).

Effect of denV deficiency on packaging. We observed a

consistent, statistically significant 15 to 20%

UV-indepen-dent decline in the amount of DNA packaging (PFH plus

FH) inall V- infections (Fig. 2 and 3; Table 1). Since denV function is deficient throughout infection in V- mutants, the

25-

20-

15-

10-

5-0

LU

n

o-LLJ

>

15-0 LUJ

10-

5-> 5-0 .0

0.

5

Cr 15-

U-0

10-z LLJ

0

5-LLJ

0l.

D

i\

I'w I \

I'\

I \

I

*.

0

I \

I 06\

0.25 0.00

B

1.00 0.75 0.50 0.25 0.00

1.00 0.75 0.50

FRACTION OF LENGTH

[image:5.612.334.531.86.636.2]

0.25 0.00 OF GRADIENT

FIG. 4. Representative ESS analysis of packaged T4 DNA. In each case sedimentation profiles in alkaline sucrose for buffer-treated (0) and purified gp denV-treated (0), phage-extracted DNAs are shown. (A) T4+ DNA from unirradiated infection. (B) DNA from V+ infection UVirradiated(153J/m2) before encapsida-tion. (C) DNA from V- infection UV irradiated (51 J/m2) before encapsidation. (D) DNA from V+ infection; after extraction from phage progeny the DNA was UV irradiated (51 J/m2) in vitro and thenanalyzedfor ESS.

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U) w D

(l)

ir cf)

w

-lJ w

w I

LL

0

0-

90- 80- 70-

60-50

40- 30-

20-10

No U.V.

51J/m2

UV.

0

l-

l

I

/+

.

0

V+ tsV

FIG. 5. Comparison of packaging at8 mpsfor unirradiated and

UV-irradiated V+, tsV(cs2O tsdenV), and V- infections. Hatched bars represent PFH, solid bars represent FH, and solid dots representDNA-containing heads (PFH plus FH).

packaging decline could result indirectly fromalack of denV

functionatanystageof T4 development. To investigate this further, a comparative shift-up experiment was conducted

withV+, V-, and cs2O tsdenV (tsV) mutant-infected cells. In thetsmutant,denV function is normaluptothetemperature

shift(34) and is then inactivated only when DNA packaging isinitiated. Since the packaging declineseenin thetsmutant

infectionwassimilartothat in theV-infection,weconclude

that absence of denVfunction during the packagingprocess

is responsible for this phenomenon (Fig. 5). In a duplicate experimentwhere the infected cellswere UVirradiated (51

J/m2) before packaging, both denV-deficient infections (V-and ts) showed the usual UV-related packaging impairment (Fig. 5).

Effect ofrifampinonpackaging.Transcription is halted at

thymine dimers in UV-irradiated DNA (35). This decline in transcription might affect the DNA encapsidation process indirectly by lowering needed mRNA levels for synthesis of packaging proteinsordirectly if RNA polymerase remained

[image:6.612.82.290.72.272.2]

boundtothe DNAatthedimer, thereby blocking packaging.

TABLE 3. Comparison of packaging of UV-irradiatedand

unirradiated V+andV-infectionsin the presence and absenceof rifampin (200 ,ug/ml)

% of head structures

Phage (JUmV) Rifampin at 20 mps

EH PFH FH

V+ 0 - 41 15 44

V+ 0 + 26 12 62

V+ 75 - 54 31 15

V+ 75 + 49 32 19

V- 0 - 37 27 36

V- 0 + 52 16 32

V- 51 - 43 33 24

V- 51 + 69 18 13

To testforthesepossibilities, packaging of unirradiated and

irradiatedDNA inboth V+ and V- infections was analyzed

in the absence and presence of rifampin, a transcription

inhibitor (31, 41). Rifampin was added 5 min before UV

irradiationtoallow timefortranscription already in progress

tobecompleted (releasingpreviously bond polymerase) and

toreduce the level of subsequentbinding of RNA

polymer-ase to DNA. Since we found no decrease in packaging of

unirradiatedDNA inthe presenceofrifampin, weconclude

that a packaging impairment is unlikely to result from

decreased mRNA synthesis (Table 3). Furthermore, we

found no support for the hypothesis that packaging is

imnpaired

by RNApolymerase bound to DNA at dimers, as

packaging of UV-irradiated DNA was not significantly

im-proved in thepresenceof rifampin (Table 3).

DISCUSSION

In bacteriophage T4 56% of UV-lethal hits have been

attributed to formation of thymine dimers in phage DNA

(24). Ourresults,based on acomparison of denV-competent

and deficient infections, showed significant differences in

packaging. These differences must be related directly to

dimerformationorrepairor bothsince denV endonuclease

is believedtobeessentially confinedtodimerexcisionrepair

(29, 32, 36, 40). Althoughwe donot exclude UV effects on

T4-infectedcells unrelated to pyrimidine dimers(see

refer-ence4), we could not assess theircontributioninthisstudy.

In temperature shift experiments where encapsidation is

normal (cs2O ts2l), packagingoccursrapidly until the

func-tionalempty proheads (made at 20°C) have been filled with

DNA. When DNA ligase is inactivated as

encapsidation

is

initiated(aftersomeinitial

packaging, presumably

tothefirst

structural blockin theDNA),thetranslocationof DNAinto

theprohead

apparently

ceasesasthere isnofurther increase

inDNA-filled heads(45). This kineticpatternfor

encapsida-tion indicates that the

ligase-deficient

blockto

packaging

is

permanent in nature.

Packaging

can only be restored

by

reestablishment of normal DNA ligase

activity

(45). The

situationissimilar for

encapsidation

in T4 gene 49-deficient

infections (20-22).

Packaging

of UV-irradiated T4

DNA,

however, shows a different kinetic pattern. The rate of

encapsidation

is

initially (10

mps)lower than in unirradiated

infections,

and

although

DNA-filledheads continueto

accu-mulate

during subsequent

incubation(to30

mps),

packaging

never reaches the level observed in unirradiated control

infections.This kineticpatternof continuedslow

packaging

in UV-irradiated infections is consistent with a transient

packaging

blockwhich diminishes therateof

encapsidation

and thusdiffers from

packaging

arrestin DNA

ligase-

and T4

gene49-deficientinfections. Differenceswerealsoobserved

in both the nature and

degree

ofthe UV

packaging

impair-mentand the structureof the

encapsidated

DNAin

V+

and

V-infections.

InV+ infectionsno

encapsidated

dimerscould be detected

even at the

highest

UV

dosage

used

(153

J/m2;

Fig. 4);

therefore, excision

repair

mustremoveall dimers before the

DNA is

packaged.

This repair process, which involves gp

denV

binding

and

subsequent

DNA structure modification

(nicking,

excision

repair,

DNA

synthesis,

and

ligation),

wouldlikelyretard theencapsidationprocess. While

packag-ing

is

presumably

halted due to the presence (discussed

below)

or

repair

orbothof dimersnearestthe

prohead,

other

dimersmoredistantfromtheprohead could

simultaneously

berepaired. Dimers could in this way

efficiently

be

repaired

before

encapsidation,

and the initial

episodes

of dimer

repair

7

tsv

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http://jvi.asm.org/

[image:6.612.66.306.589.723.2]
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would likely cause the greatest impairment of packaging. Thus, we observe asmall dose-relateddifference in packag-ing at 10 mps but that difference is no longer evident at 30 mps (Fig. 3). Since packaging is not completely recovered, UV-related effects whichcannotberepairedmayremainasa permanent block to DNA translocation into some of the

proheads. The small proportion of defective but rescuable

phage progeny produced in irradiated V+ infections could notbe attributed toencapsidated dimers and may represent the contribution oferror-prone repair processes, i.e., pack-aging ofmutant T4 DNA.

In irradiated V- infections, where pyrimidine dimers are notexcised(Fig. 4), DNA encapsidation was slower than in

irradiated V+ infections (Fig. 2). The rate of V- packaging

was inverselyproportional toUV dosage, indicatingamore direct relationship between increasing numbers of dimers

and impairment of encapsidation than wasobserved for V+

infections(Fig. 2). The impairmenttopackagingof irradiated

V- DNA is transient sincedimersareultimately encapsidat-ed (to yield defective phages). The nature of this packaging impairment remains unclear. Results of experiments with

rifampinargue against the hypothesis that RNApolymerase bound atdimers is theprimary causefor delayin encapsida-tion. Dimer-specific binding of otherproteins could explain the delay in packaging, but there is no direct evidence

favoring this hypothesis since E. coli gp uvrA and -B and mutantT4gp denVdo notbind to dimers after T4 infection

(37, 38, 44). Whereas the binding of other proteins such as

photoreactivating enzymes (39) or other photosensitizing

proteins (12) remains conceivable, two explanationswe find moreattractivearethat(i)thepyrimidinedimer itselfmaybe a DNA structural modificationwhich impedes, butdoes not arrest, packaging or (ii) dimer-induced postreplication or recombinational repair processes delay encapsidation but are less efficient than excision repair.

Role of gp denV in T4 morphogenesis. The denV gene

productisawell-characterizedproteinwithbothglycosylase and apurinic, apyrimidinic endonuclease activity and is

thoughttofunction specifically in excisionrepairof pyrimi-dine dimers in DNA (26, 29, 32, 40). We observed a consistent 15 to 20% UV-independent decline in DNA

packaging and progeny phage yield in denV-deficient T4

infections. Controls forbackgrounddimerformation(dueto roomlight;datanotshown)orforaden Vrequirementbefore

encapsidation (ts denV experiments) were negative. Thus,

denVfunction during packaging appears to be required for

wild-type levels of encapsidation of unirradiated T4 DNA. Mosiget al. (28) also inferredthat denVendonuclease may affectpackagingintheabsence ofUVdamage, asmutations

in gene denV partially compensate for certain gene 17 (linkage-packaging protein) ts mutations. Genetic evidence that gp denV (possibly in conjunction with gp uvsX) is involved inremoval ofheteroduplex loops in T4 DNA has beenprovided (2, 3). Ifheteroduplex loopsoccur

infrequent-ly in T4 concatemericDNA(perhaps as aresult of

recombi-nation), thenadecreasedefficiency orfailure in removal of

these loops in denV-deficient infections could explain the observed low level of interference with packaging. This situation would be analogous to T4 gene49-deficient infec-tions where unresolved branches in DNA formed during recombination block the packaging process. If this

UV-independent decline in DNA packaging in denV-deficient infections does indeed result from unresolved

heterodu-plexes in DNA, then the DNA structural requirements for packaging may be more precisely defined; namely, small

non-base-paired regions resulting from dimerscanbe

encap-sidated,

but

larger

heteroduplex

regions

cannot be

translo-cated into the

prohead.

Implicationsfor DNA

packaging.

Packaging

canbeviewed

as aDNA transition from a

dispersed, structurally

diverse

molecule,coatedwithDNAmetabolism

proteins

to a

capsid-encased,

highly

condensed,

intact

duplex

molecule free of

bound

proteins. By

inference,

encapsidation requires

elimi-nation of DNA structural modifications

(incurred

during

replication,

repair,

recombination,

and

transcription)

to

pro-duce an intact

duplex

molecule,

as wellas the concomitant

releaseof DNA-bound

proteins.

Itfollowsfrom this

hypoth-esis that factors which

permanently

perturb

duplex

DNA

structureshould blockDNA

encapsidation.

Defective

pack-aging

inDNA

ligase-

andgene49-deficient infections appear

to represent such blocks to

encapsidation (13,

16,

26, 45).

Factors which

temporarily

modify

DNA

duplex

structureor

perturbations

in the

duplex

which

impede

butdo not arrest

packaging might

be

expected

toaffecttherateof

encapsida-tion. This may be the case for UV-irradiated

V+

infections

where dimer-stimulated

repair

processes

transiently

modify

DNA structure but lead to reestablishment of the normal

DNA

duplex

and for irradiated V- infectionswhere dimers

are

ultimately

encapsidated

yet there is a more

significant

effecton the rate of

packaging.

ACKNOWLEDGMENTS

We thank S. Adhya and S. Garges for critical reivew of this

manuscriptandE.Radanyand D.Yarosh for adviceondimerassay

procedures.

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http://jvi.asm.org/

Figure

FIG.1.fromoccurred.reducedpackagingshowing Thin sections through UV-irradiated (51 J/m2) excision repair-competent (V+) and -deficient (V-) T4-infected E
FIG. 2.V-representsomestructures Effect of UV dosage on packaging of T4 DNA in V+ and infections at 9 mps
TABLE 1. Data from kinetic studies'
FIG. 4.tion.thenencapsidation.eachphagetreatedDNAsDNA Representative ESS analysis of packaged T4 DNA
+2

References

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