JOURNALOFVIROLOGY, Feb. 1971, p. 260-266
Copyright © 1971 AmericanSociety forMicrobiology Vol.
7,No. 2 PrintedinU.S.A.
Properties of
Bacteriophage
T4
Mutants
Defective
in
Gene 30
(Deoxyribonucleic
Acid
Ligase)
and
the
rII Gene
J. D. KARAM AND B. BARKER
Divisionof Genietics,Sloani-KetteringInstituteforCancerResearch, New York,New York 10021 Received forpublication6November 1970
InEscherichia coliK-12strains infectedwithphageT4whichis defective ingene
30 [deoxyribonucleic acid (DNA) ligase] and in the rIIgene (productunknown), near normal levels of DNA andviable phagewere produced. Growth of such T4 ligase-rIl doublemutantswasless efficient in E. coliBstrains which showthe
"rapid-lysis" phenotype of rII mutations. In pulse-chase experiments coupled with tem-perature shifts and with inhibition of DNA synthesis, it was observed that DNA
synthesized by gene 30-defective phage is more susceptible to breakdown in vivo when the phage is carrying a wild-type rII gene. Breakdown was delayed or in-hibited by continued DNA synthesis. Mutations ofthe rII genedecreased but did
not completely abolish the breakdown. T4 ligase-rII double mutants had normal sensitivitytoultraviolet irradiation.
The enzyme deoxyribonucleic acid (DNA) ligasecatalyzesthe repairofsingle-strand breaks in DNAduplexes (5, 11,12, 27, 32).InEscherichia coli and inphage T4-infected E. coli, the repair of DNA single-strand breaks may constitute an essential step common to the processes of DNA replication (15, 16,21,23-26, 29), recombination (1),repairafterultraviolet(UV)orX-ray irradia-tion (6, 28), and repair after nucleolytic attack (19, 20). Gene 30 is the structural gene for the DNA ligase of phage T4 (9). Under restrictive conditions, the infection of E. coli with amber (am) or temperature-sensitive (ts) mutants of T4 gene30 resultsin limited DNA synthesis and in verylow phageyields (14, 21). This indicates that the gene 30ligaseisneededfor phagegrowth al-thoughaDNAligaseisalsopresent inuninfected E. coli (11, 12, 27). However,theneed for the T4 gene30ligasecanatleast be partiallyovercomeif chloramphenicol is added to infected cultures at early times after infection, suggesting that the phageligaseisused in the repair of DNA damage resultingfrom the action ofendonucleases which aresynthesizedafterT4infection (19, 20).
It wasrecentlyobserved thatamandtsmutants of T4 gene30become viable under the restrictive conditions when the phage rII gene is mutated (4, 8, 17). The effects of rII mutations on the rescue ofT4 gene 30 mutants are analogous to thoseof
chloramphenicol
(4). It is possiblethat, in both cases, intracellular endonuclease activityis reduced and the amount ofhost DNA ligase
presentissufficientto supportthe needs ofphage DNAreplication and other repair. In thisreport,
we describe the defects of T4 ligase-rIl double mutantsandtheir effectsonin vivoDNA synthe-ticprocesses.
We have observedthat E.coliK-12strains sup-port very efficient growth of T4 ligase-rIl double mutants. Under restrictive conditions, some de-fects inphage DNA synthesiscanbedetected, but theseseem to have smallor no effectsonphage production and sensitivity to UV irradiation. E. coli B strains also support the growth of T4 ligase-rII double mutants, buttheabsence of lysis-inhibition coupled with thedefects inDNA syn-thesis result in shorter growth cycles and lower phage yields. T4 rIl mutations decrease, but do not completely abolish, damage to phage DNA resultingfrom thegene30ligasedeficiency.
MATERIALSAND METHODS
Phagestrains. The T4mutations used arelisted in Table 1. Thegene 30 mutants weregenerously
sup-plied by J. Hosoda, the rll mutants byS. Champe, andthegene34 and 43mutantsby R.Edgar. The map locations oftherlI mutations used in this studywere
reported byBenzer (2) and Benzer andChampe (3). Double and triple mutants were constructed by ge-netic recombination and characterized by comple-mentation and recombination tests and by crossing backto wildtypeand reisolatingthe singlemutants.
All phagescarryingam mutationswere grown onE. coliCR63; other strains were grown on E. coli BB.
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Bacterial strains. E. coli CR63 and K37strr are
permissive for T4 am mutants (su+l, ser), and E.
coliBB, S/6, K38strr, and K38(X)strr are restrictive
(su-) hosts. The strr strains are resistant to 200 .Ag
ofstreptomycin per ml. E. coliK38strr and K37strr
were obtained by curing the corresponding
X-lyso-genic strains with Xi434 phage which was generously
supplied, with instructions for curing, byM.
Gottes-man. E. coli K38(X) and K37(X), the
streptomycin-sensitive ancestors of K38strr and K37strr, were
obtained from N. Zinder via J. Speyer. They are
strains S26 and S26Rle, respectively, ofGaren et al. (10). E. coli CR63 and S/6 were from R. Edgar
and BBwasfrom F. Stahl.E.coli S/6 isaderivative
ofE.coli B (7).
Media. The growth medium used was M9S (3).
M9S was supplemented with thymidine-methyl-3H(3H-TdR) inexperimentsonDNAsynthesis. The
3H-TdR was purchased from theNew England Nu-clearCorp., Boston, Mass. Nonradioactive thymidine (TdR) was purchased from Schwarz BioResearch,
Inc., Orangeburg, N.Y.
Tris(hydroxymethyl)aminomethane-UV buffer
con-tained 0.001 M MgCl2, 0.085M NaCl, and 0.001%
gelatin in 0.01 M Tris (pH 7.5). Other media and
methods for growth of bacterial and phage strains andfor phage assayswere as described by Steinberg
andEdgar (31).
Burst size. Burst size (phage production per cell)
measurements weremade inM9Sat30 Casfollows.
Log-phasecells (108in 1 ml) were infectedata mul-tiplicity of 10 with the phage in question. After 8
min of adsorption, T4 antiserum was added (final
concentration k = 1/min) to remove unadsorbed
phage. At 13 min after infection, the cultures were
diluted 103-fold in M9S, and the infective-center titers were determined. The diluted cultures were
lysed with CHCl3 and assayed for burst titers after 2 hr of incubation. Phage assays were made on E. coli CR63.
UV irradiation. The procedure was similar tothat used by Speyer andRosenberg (30). The source of UV light was a filtered Mineralight V-41 lamp (Ultraviolet Products, Inc., San Gabriel, Calif.). Samples (0.2 ml) ofphage (at 107/ml) in Tris-UV
bufferwereplaced in duplicate in wells of depression plates (model 96 U-CV, Linbro Chemical Co., Inc., New Haven,Conn.) and irradiated at a dose rate of
5 ergs per mm2 per sec, incident to the suspensions. Four samples were irradiated at a time. The wells containing the samples were placed on a turning Spray-Fisher dish turntable (Fisher Scientific Co., Springfield, N.J.) during irradiation to allow identical exposure of all phage suspensions. The irradiation wascarried out in subduedlight, but all other manipu-lations preceding the platings for survivors were
carried out under normal laboratory fluorescent lighting. Platings were done under conditions of yellow light from a G.E. Gold fluorescent lamp which does not cause photoreactivation (13). The concentration of agar in the toplayerforplatingwas
0.5% instead of the usual 0.65% (31). This "softer" agar permits faster diffusion of phage on the plates andallows for better visibility of plaques when phage development isdelayed because of UV damage.The plates wereincubated at 30 C in total darkness.
RESULTS
Plating properties of T4 ligase-rIH double mu-tants.Table 2 shows that theT4ligase-rlldouble mutantsamH39X/rUV375 and amE605/rUV375 platedathigh efficiencyon E. colistrains which restrict amH39X, amE605, and other T4 am mutations. These double mutants also failed to growon X lysogens, indicating that ligase muta-tions do not, in turn, suppress the rII mutant phenotype. The plaques formed by ligase-rll
TABLE1. Phage T4 mutants Gene Mutantdesignation
30 amH39X, amE605
34 amB265
43 amB22, tsL56
rll rUV375(rIIBochre),r205(rlIAmissense
or frameshift), r638(rIIB deletion), rl605(rIIA-rIIB deletion)
TABLE 2. Effect ofT4 rll mutations oi theplatingefficienicy ofgenie30 mutants Plating efficiency(%C)aonE.coli
Mutant
K37strr K38strr BB S/6 K38(X)strr
amH39X... 100 5 X 10-1 4 X 10-1 2 X 10-4 10-3
amH39X/rUV375... 100 94 90 0.1 <10-6
amE605... 100 5 X 10-2 5 X 10-1 10-4 5 X 10-4
amE605/rUV375... 100 84 104 3 X 10-2 <10-6
amB22... 100 3X10-6 9X10-6 3X10-6 2 X10-6
amB22/rUV375... 100 7 X 10-6 4 X 10-1 4 X 10-6 <10-6
amB265... 100 4 X 10-5 6 X 10-5 6 X 10-5 4 X 10-5
amB265/rUV375... 100 5 X 10-5 8 X 10- 6 X 10-5 <10-6
rUV375... 100 108 92 61 8 X 10-6
aPlating efficiencies weredetermined relative to K37strr.
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double mutants on E. coli BB and K38strr are slightly smaller than wild-type plaques, and on E. coli K37strr they are indistinguishable from wild type. On E. coli S/6, however, all of the ligase-rIl double mutants which wehave tested produced very small plaques which were often barelyvisible. The apparent lowplatingefficiency ofamH39X/rUV375 andamE605/rUV375 onE. coliS/6 (Table 2) isprobablyduetoinaccuracies in plaque counts rather than to the
inability
of thesedoublemutantstoadsorbtoS/6orto prop-agate in these cells; the transmission coefficient ofamH39X/rUV375-infected E. coli S/6 isnear unityand is about thesameasthatofS/6infected with rUV375.TheresultsinTable 3 show the effectiveness of rIImutationsinovercomingthe need for the gene 30 ligase. Ligase-rll double mutants gave phage yields near control values when grown in the am-restrictinghost, E. coliK38strr. On the other hand,theburst sizesonE. coli S/6werelower and probably account, in part, for the
tiny
plaques produced by these double mutants on this bac-terial strain. In this experiment(Table 3)
and in several similar experiments, the appearance of viable intracellular phagebeganatabout 15 min after infection in allcultures, irrespective
of the bacterial host or phage mutant used. However, viable phage yields wereconsistently
lower in amH39X/rUV375-infected E. coli S/6 cultures, although these experiments were carried out underconditionswhich donotpermit lysis-inhibi-tion ofinfected E. coliK38strr.Thus,
itappears thatligase-rll phagematuration ismoredefective in E.coliS/6. [image:3.498.267.457.63.336.2]DNA synthesis byligase-rIl double mutants in E.coliS/6.Figure1 comparesthe DNAsynthesis profilesfor E. coliK38strr andS/6 after infection with rUV375, amH39X/rUV375, and amH39X. Thedifferences between rII,
ligase-rll,
and ligaseTABLE 3. PhageproductiontbyE. coli infectedwith T4 ligase-rIl doublemutants
Phageproduction inE. coli Mutant
amH39X 200 0.25 0.1 0.1
amH39X/rUV375 109 80 10 0.06
amH39X/r1605 154 177 20 0.04
amH39X/r205 218 146 23 0.1
amH39X/r638 118 114 33 0.07
amB265 290 0.06 0.02 0.2
amB265/rUV375 172 0.01 0.01 0.03
rUV375 136 92 120 0.02
50h
40
30
en
-30
c .E
a2
c.!
° 201
Al
rUV375"} omH39X/rUV375
o omH39X
- Eco/lK38sir' EcollS/6
o4V/J I fi fII
0 10 20 30 40 50 60 70 80 90
Timeafterinfection(min
FIG. 1. DNA synthesis after infection of E. coli K38strr and S/6 with ligase-defective T4. Log-phase
cells (108 in I ml) were infected at 30 C with 109
particles (in 0.1 ml) of the phage in question. At 6
min after infection, the cultures were diluted 10-fold
into M9S containing 3H-TdR (10 pCi of3H per ml
at aspecificactivity of 20pCiof 3H per,AgofTdR). Theincorporation of 3H-TdR into DNA was measured bythemethod previously described (17, 18). Infective-ceniter titers were determined on samples treatedwith anti-T4 serum (k = I/min) at 6min after inifection. Samples were lysed with CHCl3 at 180 miii after
in-fection andtiteredforprogenyphageyields.
mutants are similar to what was previously ob-served with E. coliBBstrr (23). DNA synthesis in in the cells infected with amH39X/rUV375 oc-curred intwostages: anearly stage of slow synthe-sis followed by a second stage of rapid synthesis. The length of the early slow DNA synthetic period obtained with amH39X/rUV375-infected E. coliS/6 is very similar to that obtained with the E. coliK38strr host. Differences between the twoinfected bacterial hosts begin to show at later times after infection and are primarily due to earlierlysis of the E. coli S/6 cultures.
The experimentshown in Fig. 1 was intention-ally carried out under conditions which would permitlysis-inhibition.Wefound that, in M9S at 30 C and at cellconcentrations of about 108 per ml, the addition of 10 rII phage particles per cell resulted in theinfection of more than 99% of the cells in 6 min. However, a substantial fraction of
262 J. VIROL.
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[image:3.498.57.249.501.650.2]the added phage (40 to 50%) was still not ad-sorbed by this time. The 10-fold dilution into 3H-TdR mediumsloweddownthe rateof adsorp-tion which then continued for at least 50 min, causing lysis-inhibition of the infected E. coli K38strrcultures.Weobserved that cell lysis in the amH39X/rUV375-infected S/6 culture began at about40min after infection and was nearly com-plete by 80 min. In the corresponding K38strr culture, the release of phage from infected cells began at about 90 min after infection and lysis was notcomplete until about 180min after infec-tion. The burst sizes in this experiment were measured at 180 min after infection were as follows: forE. coli host K38strr, rUV375, 144; amH39X/rUV375, 120;amH39X, 0.5; for E. coli hostS/6,rUV375, 100;
amH39X/rUV375,
25.Thus, despite the early slow DNA synthetic activity, amH39X/rUV375-infected K38strr cells producedenoughDNAtoresult in ahighphage yield.AcombinationofearlyslowDNAsynthesis andearly cell lysis seemed tobethe causeforthe lower phage yields in the S/6 culture. Weak growth ofligase-rII double mutantswasalso ob-served with allof severalderivatives of E. coli B which showtherapid-lysis phenotypeofrII muta-tions. The two-step DNA synthesis profile (Fig. 1) has beenobserved withalltheligase-rIl double mutants which we have tested, including cases where rII deletions were used. Thus, the early slowDNAsyntheticactivity isindependentof the quality of the mutatedrIIgene product as is prob-ablycausedbythegene 30ligasedeficiency.
Stability of replicating DNA in cells infected with T4ligase-rIHdoublemutants.The T4 gene 43 (DNA polymerase) mutation tsL56 was
intro-duced into amH39X/rUV375, amH39X, and
rUV375phages.The presenceof thismutationdid not alter
qualitatively
the shapes oftheprofiles shown inFig.1 whengrowthwas at30C.Ashift to 42 C stopped DNA synthesis rapidly. It has beenobservedthat, afterDNAsynthesis starts at 30 C in tsL56-infected cells, a shift to 42 C and maintenance atthistemperatureresultin gradual lossoftrichloroaceticacid-insoluble radioactivity fromphageDNA (18). Weassume here thatthis solubilization of counts reflects the suspectibility ofphage DNA tonucleolytic
attack in vivo and suggests thepresence ofsingle-strand
breaks and gaps inreplicatedphage DNA.T4 mutants carrying various combinations of tsL56,amH39X, andrUV375 wereused to infect E. coli K38strr and werepermitted tobegin syn-thesizing DNA at 30 C. An 8-min pulse of 3H-TdRwasgiventotheinfectedculturesatthe per-missive temperature to label DNA within the period corresponding to the slow rate of DNA synthesisin cellsinfected with
ligase-rII
mutants.Afterthepulse,thecultureswerediluted into ex-cessnonradioactive TdR medium at 42 C, and the fate of the countspreviously incorporatedat30C wasfollowed(Fig. 2). The following observations canbe made with respect to the effect of therII mutation: (i) DNA made by amH39X/rUV375 was more stable than DNA made by amH39X-(rll+),and(ii)DNA madebyamH39X/rUV375/
tsL56 was more stable than DNA made by
amH39X/tsL56(rII+). Thus, the rII mutation re-sulted in increased stability of replicated phage DNA.Figure2also showsthat,aftertheshift-up
100 < - o ~ ~ >- I
90
(I-'
30-
0~ 0
Cr,
c
70
00
60
C
50
(3)E
40 -4
30 (4)
ec 20 I0
00 5 10 15 20 25
Time Qt 420
(min)
FIG. 2. Stability of DNA replicated by ligase-defective T4. Log-phase E. coli K38strR cells (108min
1 ml) were infectedat30 C with 109particles (in 0.1 ml) ofthe phage in question and incubated withi
oc-casional mixing. At 12 min after infection, 0.2 ml of each infected culture was transferred to 20 jiliters of
3H-TdR mediumi containing2 IACi of'Hat a specific activityof27.8jACiof'HperjAgof TdR.After8miin ofincubation at 30C with 'H-TdR, 0.15 ml ofeach
incorporating culture was transferred to 42 C by diluting in 0.6 mlof prewarmedM9S containing200 ,ugof TdRperml. Theamountof trichloroacetic acid-insoluble 3Hwasdeterminedon samples taken within
30secafterdilutionat42 C(zero time onthe graph) and at subsequent 5-mi,z intervals. The amounts of trichloroacetic acid-insoluble 3H present at zero time were asfollows: (1) amH39X/rUV375, 2,617
countsl
min; (2) am H39X/rUV37S/tsL56, 1,252 counts/min; (3) amH39X, 1,574 counts/mini; (4) amH39X/tsL56, 1,236counts/mitn.
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[image:4.498.254.440.213.474.2]KARAM AND BARKER
to 42 C, DNA madebyamH39X/rUV375/tsL56 was unstable whereas DNA made byamH39X/ rUV375 was quite stable. In the experiments withamH39X/rUV375andamH39X,DNA syn-thesiswaspresumablynothaltedbythe tempera-ture shift since both ofthese phages carry the wild-type allele for gene 43. However, 3H-TdR incorporationstopped because ofthechasewith nonradioactive substrate. In cells infected with phagecarryingthe tsL56mutation,DNA synthe-sisand thepotentialtoincorporate 3H-TdRwere stoppedby the chase at 42 C. Other ts mutations in gene 43 andin genes 32 and 45leadto effects similar to those oftsL56
(unpublished
data). So the difference in DNA stabilities betweenamH39X/rUV375/tsL56 and amH39X/rUV375
indicatesthat thecontinuation ofDNAsynthesis is necessary to maintain the stability of phage DNA in cells infected with
ligase-rII
double-mutants.Figure3comparesthestabilities ofDNAmade by amH39X/rUV375/tsL56 and rUV375/tsL56 (normalgene 30
ligase)
during therapidphase of DNAsynthesis. Theexperiment is verysimilarto the onedescribedinFig. 2;ashorterpulse period110
60 _
50 40 _
(I) +/rUV375/fsL56 (2) amH39X/rUV375/tsL56 30_
20 10
0
L
5 10 15 20 25 30
Time at42C (min.)
FIG. 3. Stability ofDNA replicated by amH39X/
rUV375/tsL56 late after infection. The conditions
were asdescribedinFig. 2,exceptthat the pulse with
3H-TdR wasfor2 min beginning at 38 mitt after
in-fection. The amounts oftrichloroacetic acid-insoluble
3H present atzero time were 3,738 counts/min for
amH39X/rUV375/tsL56 and 5,096 counts/min for
rUV375/tsL56.
(2 min) wasusedhere to compensate, in part,for thedifferenceinthe ratesof DNA synthesis
dur-ing
early and late times afterinfection. Figure 3 shows that DNA madeatlater times after infec-tionin theabsenceof the gene 30ligase(amH39X/
rUV375/tsL56) was more susceptible to break-down thanDNA made
by
thephage carrying anormal gene 30
(rUV375/tsL56).
Thismeansthat phageDNAreplicationis affectedbythe gene 30 ligase deficiency throughout the phagegrowth
cycle
and that the transition fromslow torapid
synthesis is probably not a reflection ofincreased DNArepair activity duringlate times after infec-tion.
UV sensitivityof T4ligase-rIHdouble mutants. Figure4 comparesthe UV sensitivities of rUV375 andamH39X/rUV375.The twophageshave very similar survival curves, implyingthat the gene 30 ligase isnotessential for the invivo repair of UV-irradiatedDNAwhenthe rII geneismutated. In other experiments (notshown),wehaveobserved that rll mutantsareno moresensitive to UV irra-diationthan wild-typephage.
DISCUSSION
In E. coliinfected withphageT4 rII mutants, the phage DNA ligase (gene 30) is not essential for DNA replication and phage production (4, 14). T4 ligase-rIT double mutants are restricted in E. coli K-12 (X) and grow poorly in E. coliS/6 (a derivative ofE. coli B). The poor growth of
0 50 100 150 200 250 300
UV
dose (ergs/
mm2)
FIG. 4. UVsensitivity ofT4ligase-ril double mu-tants. Phage survival after UV irradiation was
de-termined by the method described in Materials and
Methods.
a)
:U
c
0%
.C .c)
0) 0
c
0
C-) 0Il
264 J. VIROL.
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1000
90
80
70
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[image:5.498.60.249.343.580.2] [image:5.498.261.453.397.618.2]thesedoublemutantsonE.coliB strains
acombinationof defectiveDNAsynthesis
cell lysiswhichoccursbeforetheaccumulation
large amounts of intracellular phage particles.
E.
colistrains which do notshow thephenotype ofrII mutations (e.g., K38strr
BB) produce near normal yields of
infection with T4 ligase-rIl double mutants
spiteabnormalitiesinDNAsynthesis. Inpulse-chaseexperiments,wehaveshown
DNA synthesized by gene 30-defective
more susceptible to breakdown in
phageis carryingawild-typerII gene.
ments (Fig. 2) alsoshowthatthecontinuation
DNA replication after infection stabilizes
againstdegradation despite the gene
the doublemutants. This suggests that
plication involvesthe repairofsingle-strand
aswellas breaks. Therepair may be
by host-derived or phage-induced enzymes,
cludinganalternateligaseactivity. If
stitution doesoccur,thenitmust be
since the DNAsyntheticcapacityofcells
with ligase-rIT double mutants is very
that ofcells infectedwithphagecarrying
ligase gene (Fig. 1). Also, the survival
irradiated phage, which presumably
ligase activity (28), isnotalteredwhen
defective (Fig. 4).
Ithas beenobserved that, insu- cells
with T4 am gene 30 mutants, the
chloramphenicol at early times after
leadstotheinhibitionofphage DNA
andtoproductiveDNAreplication
effects of chloramphenicol, however, somewhat different from thoserIIof
(16), since this antibioticinhibitsall
thesis (DNA synthetic aswellasdegradative
zymes) whereasrII mutationsprobably
somefunctions.TheeffectrIIof mutations
rescueofligase-defectiveT4maybetwofold:
decrease endonuclease activity, directly
directly, and (ii) to make host repair
(possiblythe host ligase) more accessible
DNAreplicationandrepair.
ACKNOWLEDGMENTS
WethankL.F.CavalieriandB. Rosenbergnumnerous
discussions.
This work was supported by Public grant
CA08748 from theNationalCancerInstituteAto.nic
Commissioncontractno.AT(30-i)-910.
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19. Kozinski,A.W.1968.Molecular
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22.
Newmnan,
J.,andP.Hanawalt.ligase in T4DNAreplication.J.Mol.
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KARAM AND BARKER
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