Host DNA synthesis after infection of Escherichia coli with mutants of bacteriophage T4

VIROLOGY

46, 437352

Host

(1971)

DNA

Synthesis

after

Mutants :I. CASCIKO,’ Uepartlneni of

Biophysics, liniversity

Infection

of Escherichia

of Bacteriophage s. RIVA,2

University of California

AND

E.

P.

GEIDUf$CHEr?

Chicago, @h,icago, Illinois, and at San Diego, La Jolla, (‘alifornia June

with

T4

of

Accepted

co/i

Depurtment 92057

of Biology,

16, 1971

Host DNA synthesis normally ceases very soon after bact,eriophage T4 infection. However, cells infected with T4 gene 30 (DNA ligase) mut’ants resume synthesis of unstable host DNA during the subsequent degradation to acid-soluble nucleotides. Mutations in T4 genes 46 or 47 somewhat increase the stability of this E. coli DNA and permit its properties to be studied. It is double-stranded and its synthesis depends not only on the host’s polA gene product (DNA polymerase I) but also on proteins synthesized after viral infection. The unstable E. coli DNA synthesis can occur concurrently with T4 DNA replication but does not, require the products of ‘~4 late genes or of the early genes 32,41,43,44, or 45. We postulate that the E. coli DNA synthesis constitutes an ill-fated attempt to repair T4-induced degradation of host DNA and that the T4-induced DNA ligase has a role in host DNA degradation.

and DXA synt,hesis is rapidly shut off after infection with T-even bacteriophage (Cohen, 1948; Volkin and Astrachan, 1956; Astrachan and Volkin, 1958; Somura et al., 1960; Kennell, 1968). Apparently t~here are two mechanisms for shutting off host functions, one t’hat. depends on phage protein synthesis and is multiplicity-independent and a second t,hat appears to depend on the multiplicity of infection but not, upon phage protein synthesis (Somura et al., 1966). Subsequent t,o the arrest of host DNA synthesis, extensive DSA degradat,ion, under control of a number of deoxyriboEscherichia

coli

protein

’ Present address: International Laboratory of Genet,ics and E%iophysics, via Marconi, Naples, Italy. y Present address: LePetit S.p.A., Department of Rlicrobiolog~-, via I~urando 38, Milan 20158, Italy. : Present address: Department of Biology, Universit,y of California at San Diego, La Jolla CaliforlGa, 92037.

nucleases induced by phage infection, is observed (Wiberg, 1966; Koerner, 1970). The bacterial DNA in first convertfed to ;L slower sedimenting but still high molecular weight form and t,hen is subjected t$o extensive breakdown into acid-soluble mttterial (Warren and Hose, 1968; Bose nncl Warren, 1969). We have found, however, th,at3 bacteria infected with phage 7’4 gene 30 (DKA ligase) mutants sustain host, DNA synthcsis after infection. In this report, we prcsont the evidence for t#his conclusion, describe some of the properties of the ill-f:tt,ed host DNA synt,hesis, and specify some of the host, and phage protJeins t,hat( are involved. MATl~:l1IAI,S

AND

MKl’1101~6

2,. Bacterial strains. The following st,rairis h:tve been used: Bsch.erichia Coli HE (sue),

CR63 (sul+), A564 (su-, rT*,T2, G), :162S bJ+r 1 r&,T2 , T&), W3110 (thy-., pot A’, lac,,,+ ~1~~ , 7 L.m “, I:-), 1’3478-w3110 (tip ) pot A,), IS-W31 10 (tmhy--, pal :2-i-), RI I 437

438

CASCINO,

RIVA

W3110 (thy-, pol A+) and R14-W3110 (thy-, pol A+). BE and CR63 were from the laboratory collection. A564 and A625 were kindly provided by H. Revel. P3478-W3110, R6-W3110, Rll-W3110, and R14-W3110 are, respectively, a mutant in DNA polymerase I (pol A gene) and three independently isolated revertants that have normal UV resistance and DNA polymerase I enzyme levels; these were kindly provided by J. Cairns. b. Phage stocks. T4D amber and ts mutanhs are listed in Table 1. We thank W. B. Wood for providing the mutants ts P36 and ts L109 and R. Werner for the mutant amH39X-rIIA; other T4D mutants, originally obtained from R. S. Edgar or R. H. Epstein, were from the laboratory collection. All the ts mutants were backcrossed three times against wild type (mutant.: wild type input ratio 1: 5) prior to use. Multiple mutants were constructed by equal input crosses under permissive conditions and tested by liquid complementation or recombination against single mutants. All phage stocks were purified from their original lysates by low and high speed centrifugation. 16N,13C density and 3H radioactively labeled phage were further purified by CsCl density step centrifugation (0.01 M Tris .HCI, pH 7.5), dialyzed against first 1 M NaCl, 0.01 M Tris .HCl, pH 7.5 and subsequently against 0.01 M Tris .HCl, pH 7.5. Unlabeled phage were also purified in this way prior to T4 DNA preparation. c. Media. Most of the media are described elsewhere (Bolle et al., 1968a). The medium used to prepare radioactively and density labeled badteriophage was M9 containing 0.1% glucose. When 15N, 13C density labeled phages were to be prepared, [15N]NH&1 (0.1%) and [13C]glucose (0.1%) were used. d. Chemicals. [5’-3H]Uridine (Ur), [2-14C] thymidine, [methyl-3H]thymidine (TdR) and [814C]adeni’ne were purchased from Schware BioResearch. Sodium lauryl sarcosinate (sarkosyl NL-97) was purchased from Geigy Industries, Inc. Other chemicals were standard reagent grade products.

AND

GEIDUSCHEK

e. Phage-infected bacteria, DNA synthesis, RNA, RNA-DNA hybrid analysis. Bacteria were grown in M9S to 5 X lo8 cells/ ml, resuspended in fresh medium, and infected at a multiplicity of 6-8 phage/ bacterium (unless otherwise specified) in the presence of 20 pg/ml n-tryptophan. At 2 min after infection at 30” and 1 min after infection at 4042”, there were less than 1% surviving bacteria. Kinetics of DNA synthesis were determined by measuring [14C]TdR, [14C]adenine, or [3H]TdR incorporation into 5 % CLCCOOH-insoluble material after preincubation in 0.5 M NaOH overnight at 30”. Samples were filtered on nitrocellulose membranes and counted in toluene-based scintillation fluid in a scintillation spectrometer. Unless otherwise specified, 0.07 pCi/ml [14C]TdR and 5 pg/ml cold TdR were added 2 min after infection. RNA was labeled with [3H]Ur (3 &i/ml, 0.5 pg/ml). RNA-DNA hybrid formation and analysis were done as described elsewhere (Bolle et al., 1968a). f. Measurement of the rate of thymidine incorporation into DNA. A l-ml portion of the infected culture was transferred to a test tube containing 0.4 &i and 1.9 pg [ldC]TdR. After 2 min, 1 ml of 1 M NaOH was added to stop the reaction, and 5 % C13CCOOH precipitable radioactivity was measured as specified above. g. Fate of labeled bacterial DNA after infection. E. coli DNA was labeled by growing strain BE or R6 at 30” in M9S supplemented with 0.05 &i/ml and 20 pg/ml TdR to a density of about 3 X lO*/ml. Cells were washed twice with TdR-supplemented nonradioactive medium, suspended in fresh nonradioactive medium to a density of 5 X 10s/ml, and grown at 40°C for 10 min. After infecting with the appropriate mutants, l-ml samples of infected cells were pipetted at intervals into tubes containing 1 ml of 1 M NaOH, and 5% Cl,CCOOH precipitable radioactivity was measured as specified above. h. Labeled phage. Cells were grown in MY containing 0.1% glucose at 30” to a concentration of 1 X lO*/ml and infected with the appropriate mutant in presence of 20 fig/ml of n-tryptophan at a multiplicity of

0.001. [“H]TdR (10 pCi/ml and 20 pg/ml) was added 20 min before infection. Cultures were lysed witl\ CHCl, 6 hr after infection, left overnight, at i”, digested with 5 pg/ml pancreatic DNase for 60 min at 37”, and centrifuged at, low speed t,o remove debris. Further purificat8ion was done as described in (b). The specific activity obtained under t,hese conditions was approximately 1000 cpm,’ log phage. i. L!~sis OJJ’ infected cells. Two methods of lysing infected cells were used: 1. Infected cells were resuspended in 0.5 ml of 0.1 dl Tris .HCl pH 7.5, 0.01 M Ei(X, 100 pg/ml lysozyme, and incubated at, 37” for 15 min. Then EDTA, pH 7.4 was added to 0.02 dl and the incubation was continued 15 more minut,es at 37”. SDS was subsequently added at a final cone&ration of 1 %, and t’he lysate was left’ at, room temperature (for about 20 min) until lysis (CT. Selzer, personal communication). Then 0.2 mg of pronase, selfdigested for .i hr at 37”, was added, and incubation was continued for 5 hr at X7” (Iiozinski and Lin, 1965). 2. Cells were chilled in an equal volume of buffer at, 0” (0.1 Jil KCK, 0.05 dl EDTA, pH 8, 2 mg/ml lysozgme) and centrifuged. The pellet was rcsuspendcd in 0.25 original volumes of the same lgsis buffer and left, for 20 min at, 0”. Then sarkosyl was added to :I final concent~ration of 3 %. After mixing, the cells were incubated at 0” for 10 min; during the following 5 min a very gentle shaking ~-as Ijrovided until lysis occurred (Frankel, 1968). 1~. Lysis of bucteriophage. This method has been used only for lysis of int#act bact8eriophage t)o obtain the DXA used as a marker in sedimentat8ion analysis (Botstein, 196X). Equal volumes of highly purified bacteriophage (1 X lOI ml-l) and lysis buffer (0.1 M EDTA, 0.1 M Tris .HCl, pH 7.5, 0.01 ,?I NaCl, 150 pg/ml lysozyme) w(lrc mixed and lefl at 37” for 10 min, shifted to 65’ for 3 min, and sarkosyl (2 % final concentration) was added followed by very gentle mixing. Incubation at 65” was continued for ‘10 min more. I. ~Sedimentalion an,alysis. Linear 5-20 % sucrose gradients in 0.01 ill Tris .HCl, pH

7.5, 0.1’;. sarkosyl, 0.01.5 ,I/ 151)‘~1\, I *II NaCl, 0.2 AJI KC1 (ncut,rt\l) or in 0.2 A/ IWH, 0.1 ‘;;: sarkosyl, 0.015 II EDT;\, 1 31 KaCl (alkaline) were prepared at, room temperat,ure. Then 0.2 ml of cell lysate (see tion i, md hod 2) \vas layered on the gradient, with :t Z-mm diameter pipet,tc. C:ent,rifugation \\XS at 150,000 glnRx for 1.50 rnin at 15”. Fractions lvere collect)ed from the bottom of t#he centrifuge t,ubc, :md t,he amount, of labeled DNA was det~crmincd by measuring [‘“C]- and [3HITd11 as 5 “; C:ln<‘CC)C)Hinsoluble m:tt~eriaI after :ddit.ion of 5 pg of denatured DXT,2 carrier. I,arger (Z-1 ml) neutral sucrose gradients were used to pr(‘pare DNA1 fra&ons for I>XAim 11X\:_\ 11)bridizat,ion :m:tl~-sis and clxonucleasr I digestion. Lysate, 2 ml, was lay(lr(‘d ont,o these gr:tdients, ccntrifugation n-as :\I 90,000 {/ln:lE for 6 Iir at, I.;‘, :11td f’raction~ were collect.ed from the top of t,hc gradients. m. C’s Cl ~jr~licnts. For n&rnl CsC’l gradient centVrifugat8ion, 3 ml of solution XV:IJ overlaid with mineral oil and centrifuged :II, 1.2 x 105 !/X$X for 70 hr at 20”. Fractions were collected from the bot,t,om of t IIP gradient, and t Ire radioact~ivc DN.1 \\.:I.-; precipit,ated as described above. [“HJpol~ (dAT)> which served as :i density marker, was the generous gift, of S. Cozzarr~lli. n. Oth,er methods. DSA-DNA hybridiz:ltion analysis was prrformed according t 0 Denhardt (1966). All DKA was cxt,racied 5 times in the cold, using redistilled phenol saturated with 0.1 M phosphate buffer, and t,hen extensively dialyzed, first against, 0.01 M Tris .HCl pH 7.5, 1 Jf Sac:1 and then against 0.01 ,I/ Tris .HCl pH i..,. DKA WV dennturcd by h&ing in boiling water for 5 min. I;:. coli [RH]DNA :md cxonuclease 1 were kindly given to us h?. T, Cozzarelli and .\ I. Gouliau, rcspcctivoly. The exonuclease I assay was donr with 0.2 unit/ml exonucleasc I (Lehman and KUWbaum, 1964) and 0.35 pg/ml of t,h(t 14(‘labeled “unknown” DX.\ (X-D?;X) in 0.3 ml of t’he appropriate buffer. -1ft,er illcubation at 37” for 1 hr, the 5 ?, Cl&COOHprecipitable radioactivit#y was determined. DT.1 [3H]E. ccrli native and denatured served as eont,rols.

440

CASCINO,

RIVA,

AND

RESULTS

A. DNA synthesis in E. coli Infected with T4 Phase DNA Polymerase-Ligase Mutants under Nonpermissive Conditions. Several “early” viral enzymes (Epstein et al., 1963; Warner and Hobbs, 1967), among them the T4 DNA polymerase coded by gene 43 (de Waard, et al., 1965), must be synthesized before normal viral DNA replication can take place. Gene 43 am and ts mutants are unable to start phage DNA replication, although some addition of nucleotides to parental phage DNA molecules can occur (Murray and Matthews, 1969). However, we have observed that much more TdR is incorporated into acid-insoluble linkages in cells infected with a phage T4 mutant in gene 43 so long as the latter also carries a mutation in gene 30, the struct#ural gene of T4 DNA ligase (Fareed and Richardson, 1967). The results of two typical experiments are shown in Fig. 1. In these experiments, E. coli BE are infected at 40” with the double mutant tsP36 amH39X (genes 43, 30) and [14C]TdR is added 1 min ‘after infection, As Fig. la (curve 1) shows, DNA synthesis (as [‘“Cl TdR incorporation) starts at 5 min, increases until lo-12 min, and then a part of the incorporated label becomes acid soluble.

GEIDUSCHEK

The cont’rol infection with tsP36 (gene 43) shows four times less label incorporated under nonpermissive conditions (Fig. la, curve 2). In the corresponding experiment with the triple mutant tsP36 amH39X amNl30 (genes 43, 30, 46) a greatly enhanced [14C]TdR incorporation with somewhat changed kinetics is observed. As Fig. lb (curve 1) shows, the incorporation of [14C]TdR into E. coli BE that have been infected at 40” starts, as before, about 5 min after infection and reaches a maximum at about 15-20 min; then the incorporated label becomes partly acid soluble. The control experiment (Fig. lb, curve 2) carried out with the double mutant tsP36 amNl30 again shows a comparatively low incorporation, Pulse-label incorporation experiments indicate that some [14C]TdR incorporation continues beyond 15-20 min after infection, when the total accumulation of TdR in DNA is decreasing (Fig. 2, curve 1). Evidently, the cumulative incorporation of [14C]TdR described in Fig. 1 reflects DNA synthesis and degradation with degradation predominant at later times. When a gene 47 mutation (amA456), replaces a gene 46 mutation, the resulting gene 30, 43, 47 mutant produces the same characteristic [14C]TdR incorporation as the gene 43, 30, 46 mutant infection (Table 1 I/O--‘o,

bl

al

P / d I :

Minutes

FIG. 1. Thymidine

-

I

after

infection

\

\

\ 0

\ ‘0

\ ‘A.

0

incorporation into Escherichia coli BE infected with T4 mutants. (a) Curve 1: tsP36 (gene 43, DNA polymerase) amH39X (gene 30, DNA ligase); curve 2: tsP36. (b) Curve 1: tsP36 umH39X umN130 (gene 46); curve 2: tsP36 umNl30. E. coli BE, grown in M9S to a concentration of 5 X 108/ml, were infected at 40°C at a multiplicity of 8 phage/cell with the indicated mutant. [WlTdR into Cl&COOH insoluble (0.07 &i, 5 rg/ml) was added 1 min later. At various times W incorporation material was measured after overnight digestion in 0.5 M NaOH.

IIOST

l)NA

SYSTHEYIS

AFTEIC

T4 MIJTRNT

INFI~;<:‘I’IO4

111

33 and 5.5 do not, affeci, the incorporat iotl (compare part, A, lines .5-S and 4). ((1’) rIIA mutations enhance rather than lo\vc~r incorporation (part (‘, lines 1 and 2). Han-rver, one peculiar feature of this 3 TdR. incorporation should he noted. Ik\ 3 spite t,he fact that ls1’36 infected cells shop an immediat t arrest, of DNA synthesis when shifted I o any t emperat,urt~ abo~c~ 3K’C, the TdR incorporation of iht~st~mul. -2 tdple mut.ants is greater at 40 than :\I, 1’2‘. 0 IO 20 30 (Clompare part, D with parl 11, lines 1, !I, Minutes ofier infcctian and 10 and pa& B, lint> :j). FIG. 2. llate of TdR incorporation in E. coli B" The following experiment shows that. this infected with T4 mutants. Curve 1: lsP36 amN130 anomalous TdR incorporation (X-l_)Y2\ (genes 43, 46) : curve 2: lsP36 amH39X amN130 synt,hesis) is not restricted 1.0 infections itt (genes 43, 30, 46). E. coli BE were grown and infl. coli R” and i Ilat, it, is directly concc~rnrd fected at 40” as described in Fig. 1 with the two \vitl1 the functioning of the product-s of 1’4 mutants. ,it, various times [‘*C]TdR pulse incorporation during 2 min wasmeasuredasdescribed genes 30 and 46. The bacterial howl8 1’01 this experiment art’ the A.564 (s;11-j :UI~ in sectionf of Materials and Methods. A65 (sul+) strains of IS’. coli Ii12 tlt:lt, ;II’V 1, compare part, A, line 11 with lines 4 and also permissive for nonglucos31:lted T-eve~l and t llat, q)port 10). Since no differences between the pheno- phage (Revel, 196'7) normal T-l xvild-t ype phage growth :tntl types of gent 46 and 47 mutants have been DNA sgnt hesis ai, 41” (dala not sho\vn.i. reported, we have confined the rest of these The TclI< incorporation into 7’4 kl%i (~1 experiments to the effect, of the gene 46 H3SX n~N180 (genes 43, 30, 46j infected mutation. The next experiments shon the enhanced su- cells is clearly an order of magnit utlc incorporat,ion of TdR in the absence of greater than that of tlte su+ infected cells gene 43 DNA polymerase function to be a (Table 1, part I<, lines 2 and I). ,\l ihis point MY cannot distinguisll betn-ren I KO gene-specific rather than mutation-specific property and t,o occur at, 30” as well as at possibilities regarding the observed IIS. higher kmperatures. Table 1 lists someprop- synthesis: that’ it, also occurs \&en infrctiotl erties of a number of multiple mutants t.hat, is at t,he permissi\-c condit,ion for the genrh-1-8 include the T4 DNA polymerase lesion DNA polymerase but, is superimpost>tl on phage DNA synt ltesis which obliterates ii , but exhibit, sovz DNA synthesis. With only the exceptions noted in the footnotes to or t,hat ttii:: D?CA syritltesis takes plac~eortlv Table 1, the observed incorporation has the \vhen phage I)Y:zd polpmerase is inact ivattltl. general features shown in Fig. 1, curve 1: We will rclurri to this point, in srctiort IC. (a) There is a delayed onset of TdR incorporation, which reaches some maximum value and then declines. At 30”, i.e., lvith a,?~mutants in genes 43, 30 or in genes 43, 30, 46 the maximum incorporation is, The next experiments prove i hat N-DSA naturally, reached at later times than at is double-skanded and that it is not. PO40--U” (compare part A, lines 4, 9-10 valentlp connected to t,he D9h of infect,ing with part, IS, linr 3). (b) Bacteria infected phage. (:ells were infected with [SH]TdIC \vit,h gene 30-46 mutants incorporate much labeled t.4’36 amH39X arrzNl30 phage and more TdR than those infected with gene then labeled wit*h [14C]TdR and lysed 15 min 30 mutants (compare part A, line 4 with after infect,ion (at 40”), t’liat is, at the Cmt: line 3, part B, line 3 with line 2). (c) More- of maximum tbymidine incorporation. I t8\V:IS over it can be sf’en that mutations in genes immediately realized that the rcrovttry of

‘iYI_\ L

l!!?L

442

CASCINO,

RIVA,

AND

TABLE [WJTdR

INCORPORATION

IN Escherichia Specification

A)

B)

C) D)

W

1 2 3 4 5 6 7 8 9 10 11 1 2 3 1 2 1 2 3 4 1

2

tsP36 tsP36 tsP36 tsP36 tsP36 tsP36 tsP36 tsP36 tsP36 tsP36 tsP36 am4301 am4301 am4301 tsP36 tsP36 tsP36 tsP36 tsP36 am4301 tsP36 infection of A625 (suI+) tsP36 infection of A564 m-1

30

1

CELLS

of mutants

INFECTED

WITH

T4 DNA

amH39X amH39X amH39X amH39X amH39X tsA80 tsA80 amH39X amH39X

amH39X/

MUTANTS

I

and genes

46

47

55

33

Time of maximum incorporation (tin)

III

amNl30 amH39X amH39X amH39X amH39X amH39X amH39X tsA80 tsA80 amH39X

POLYMERASE

-7

I

43

coli

GEIDUSCHEK

amN130 amBL292 amBL292

amN130 amNl30 amN130 tsL109 amN130

amBL292

zmN134 zmN134

amA

amN130 rIIA rIIA

amN130 amN130 tsL109 amN130 amN130 amNl30

amN130

L

40 40 40 40 40 40 40 40 40 40 40 30 30 30 42 42 42 42 42 42 42

100 160 320 1200 350 1320 1150 1200 1200 1800 1200 75 225 800 800 1600 1000 675 1400 800 180

42

1650

b b 12 15 12 15 15 15 15 15 20 b 15 30 10 c 15 15 15 15 b

20

-

a E. coli grown at 30” in M9S (Bolle et al., 1968a) to a concentration of 5 X 108/ml were washed, resuspended in fresh medium, and infected (Materials and Methods, section e), with a number of single and multiple T4 mutants. [W]TdR (0.07 &i/ml, 5 pg/ml) was added 3 min after infection, and incorporation into 5yo Cl&COOH-insoluble material was followed. Phage mutants, temperature of the experiment, time of maximum incorporation, and amount of maximum incorporation are indicated. [Wladenine labeling of DNA yields similar results. (A) [W]TdR incorporation in cells infected with T4 mutants, all of which contain the tsP36 mutation in gene 43. (B) [W]TdR incorporation in cells infected with T4 mutants, all of which contain the am4301 mutation in gene 43. (C) Effect of an rI1 mutation. (D) Effect of raising the temperature on the maximum TdR incorporation for some ts and am mutants in genes 43, 30,46. (E) Maximum incorporation of the same T4 mutant in E. coli K12 SUI+ (A625) and su- (A564). 1, The TdR incorporation does not peak at some maximum value and decline, as in the other experiment. The listed incorporation is for the same time after infection as the appropriate comparison experiment of the same series, i.e., for Al, A2, and Bl it is 15 min and for El it is 24l min. c No maximum was observed; the listed incorporation occurs 50 min after infection.

the newly incorporated 14C radioactivity (as acid precipitable material) after lysis was very poor (see Table 2). This happened partitularly when the lysis method 1 described in Materials and Methods, section i, was

used. A similar loss of TCA-precipitable material was also observed during lysis of cells infected with tsP36 amH39X amNl30 at 30” (see Table 2) and with amH39X at 30°C (data not shown). On the other hand, paren-

HOST

1 1

2

DSA

40°C 30°C

40%

SYNTHESIS

AFTER

MITTANT

GO’:;

loo?; loo’;,. Resuspension in lysis buffer before incubatiou loo”;!

T4

20 min

30( ( al

755;

0”

1 i:;

INFECTION

2“ ( 3X’ (

22’ 38 ,

15 min at, 0” (sarcosyl)

70' ;

(1 The recovery of acid-insoluble [W]TdR, incorporated 15 min after infection of E. coli BE with ‘I’4 kP36 amH39X amN130 (genes 43, 30, 46) is shown at various steps of the lysis of infected cells using two lysis methods described in section i of Materials and Methods. E. coli BE (5 X lOB/ml) were infected at. 30” with 3H-labeled tsP36 amH39X amNl30 phage. One minute after infection, 0.5 &I and 10 rg/‘ml of [W]TdR were added, and 1 min later a portion of the culture was shifted to 40”. The tokd 1% incorporation (lOOcj; valuej was determined on an aliquot of the infected cells t)hat had t>ern pipct,ted directly into 0.5 M NaOH. Recovery of 3H-labeled T4 parental DNA at all the stages of lysis is 93-99’ ;

tal tsP36 um.H39X amS130 DNA was quantitatively recovered with this lysis method. Lysis of infected cells with our method 2 (section i) which is essentially the method described by Frankel (196s) yielded markedly better recovery (Table 2). In Frankel’s method, lysis is carried out at 0” while our lysis method 1 exposes cells at 37” during stages in which DNA becomes labile, as Table 2 shows. The fact that this instability is shared by at least a fraction of the DNA synthesized during infection with the triple mutant at 30°C (Table 2, line 2) suggests that a common species of unstable DNA might be synthesized at, 40” and 3OO.4 The nature of the chemical linkage of the infecting and newly synthesized DNA have been measured by centrifugat,ion in neutral and alkaline sucrose density gradients. As Figs. 3a and 3b show, t’he [14C]DNA synthesized at 40” is very small (6-10 S) both in neutral and in alkaline sucrose gradient. There is neither covalent (Fig. 3b) nor basepairing connection between 3H-labeled parental phage DNA and t.he newly synthesized [14C]DNA, which proves that semiconservativr phage DNA replication has not 4 The addition of an rI1 pling with the gene 43, 30, result in a differential effect the two met#hods described DNA is recovered by either,

gene mutation in cou46 mutations does not on DNA recovery by here, but more labeled method of extraction.

taken phe. Fifteen minutes after iIlfect,hJ, parental [3H]DNA has few if any double-. strand breaks (Fig. 3a) but has many singlestrand breaks (Fig. 3b). However, at later times (30 min aft,er infection) wte have observed extensive double-strand breakage (Cascino, et al. 1970). When the above experiment was repeated at 30”, i.e., under permissive conditions for phage DNA polymerase, but not ligase or the gene 46 product, a strikingly different, IY’sult was obt.ained. As Figs. 3c and 3d shorn-. two species of newly synthesized [14C]DN:2 are obtained. Relatively high molecular weight [14C]DNA is associatedwith parental [3H]DNA (Fig. 3c, peak I) and is, most probably, the product, of semiconservative phage DNA replication (see below). The low molecular weight [14C]DSA (Fig. 3c, peak II) does not overlap with input phage DNA; it could be fully replicated progeny DNA OI the same kind of small DNA that is synth+ sized at, 40”. Evidence in favor of t,he latter alternative is present,ed below. The fate of 3H input DNA is different for infection at 30” and 40”. Double-strand breaks of this DNA4 are much more frequent when replication is allowed (at 30”, Fig. ;ic) than when it is not (at 40”, Fig. 3:~). Tjiffcrences in the single-strand size distributions are much lessmarked. Hosoda and Mathews (1971) have already obserrtld n C&P COII-

444

CASCINO,

RIVA,

AND

GEIDUSCHEK bl

;I0 Fraction

20

IO

number

Fraction

30

number

d) czb*

Fraction

number

Fraction

number

FIG. 3. Parental and newly synthesized DNA in tsP36 amH39X amNl30 infection at 40 and 30”: neutral and alkaline sucrose gradient centrifugation. E. coli BE were infected at 30” with 3H-labeled CsCl purified fsP36 amH39X umN130 phage. One minute later [WJTdR (0.5 ,.&i, 5 pg/ml) was added and a portion of the culture was immediately shifted to 40”. At 15 min after infection, cells were collected and lysed (method 2, section i of Materials and Methods). (a) Neutral 5 to 200% sucrose gradient centrifugation of a sample from the 40” infection. (b) Alkaline 5 to 207c sucrose gradient centrifugation ,of the same sample as (a). (c) Neutral 5 to 200/o sucrose gradient centrifugation of a sample from the 30” infection. (d) Alkaline 5 to 20% sucrose gradient centrifugation of the same sample as (c). Sedimentation was performed as described in Materials and Methods, section (e). Sedimentation velocity increases from right to left. The positions of entire molecules, (a) and (c), or strands, (b) and (d), of mature T4 DNA are indicated by arrows ( J ). Peaks I and II in (b) and (d) are discussed in the text.

nection between replication and doublestrand scissionsin T4 DNA ligase-defective phage infection. The density-shift experiments that we describe next confirm the analysis of the previous experiments and, in particular, rule out the possibility that DNA synthesis at 40” after infection by the gene 43, 30, 46 triple mutant is due to phage DNA replica-

tion (or repair). E. coli BE were infected at 30” and 40’ with heavy, rH]TdR 13C,15Nlabeled phage. [r4C]TdR was added 1 min after infection; at 15 min the infection was stopped and cells were lysed, following method 2 of Materials and Methods, section i. Aliquots of these lysates were then centrifuged to equilibrium in neutral CsCl with added [3H]poly(dAT). The results, together

HOST

DNA

SYNTHESIS

AFTER

T4 MUTANT

INFRCTIOS

a)

z-

I-

0

I727 Density

(g/cm3

1698 1

1679 Density

(g/cm3

1

FIG. 4. Replication of input tsP36 amH39X amN130 occurs at 30’ but not at 40”. ‘“N, W, 31-f-label~d, CsCl purified phage were used to infect E. coli BE at 30’ in light 14N, %-labeled medillm. A portion of the cultlu-e was shifted to 40” 1 min later and [W]TdR (0.2 &i, 10 pgiml) was added 3 min after infection. Cells were collect,ed 15 min after infection and lysed following method 2 of Materials and Methods, section i. Neutral CsCl centrifugation was performed as indicated in section m of Materials and Methods with marker [3H]poly(dAT) (density 1.679 g/cm3). Buoyant density increases from right to left. (a) Nerttral CsCl centrifugat,ion of DNA from 40’ infection. (b) Neutral CsCl centrifugation OC DN?I from 30” infection.

lyith the experimental details, are presented in Figs. 4a and 4b. There is no detectable shift of the parental, heavy t3H]DNA (p = 1.727 mg/cm3) toward hybrid density (p = 1.713 mg/cm3) at 40” (ruling out semiconservative phage DNA replicat’ion). The [14C]DNA, on the other hand, is in a broad band because of its low molecular weight, and t)he band center is at a density (1.710 glicm3), which corresponds roughly to the buoyant densitv of E. coli DNA in neutral CsCl and issensibly different from thedensity of T4 DNA (1.698 g/cm3). When the same infected cells were lysed by method 1 of Materials and Methods, section i, the [14C]DNA was distributed throughout more than 90 % of the gradient. This is another indication of the extreme suscept’ibility to degradation of the newly synthesized DNA. The 30” lysate presents a completely different picture (Fig.

4b). Semiconservative T4 DKA replication takes place so that some parental T4 DSA has moved to the hybrid density position and an additional band of light (p = 1.69s g/cm”), fully progeny [14C]DKA also appears. It, is not possible to decide whether, some [14C]DNA with a buoymt density VI 1.710 like that’ obtained during infect,ion at 40”, is made, because of the hybrid band ai; p = 1.713 g/cm3. The very good separntiou of the heavy, hybrid and light T-l DNA peaks in this experiment is probably due IO the absence of phage D?L:A recombination, caused by Ihe absence of T4 DLA ligase and the gene 46 protein (Bernstein, 1968. I
CASCINO,

446 TABLE HYBHIDIZATION

3 OF DNA

DNA

on filters

E. coli BE DNA T4 DNA Efficiency of hybridization

[ZHIE. coli BE DNA

98%

2% 16%

RIVA,

X-DNA0 in solution [WIT4 DNA

2% 98%

‘31%

[‘“CIX-DNA

96%

4% 42%

a E. coli BE were infected at 40” with T4 tsP36 amH39X amN130 phage (genes 43, 30, 46) as in Table 1. One minute later [W]TdR (0.5 PCi and 10 pg/ml) was added. Cells were collected 15 min after infect,ion, at the time of maximum [W]TdR incorporation (see Table l), and lysed according to method 2 of Methods, section i). The lysate was subsequently centrifuged in neutral 5-207, sucrose (24 ml) and fractions of DNA, equivalent to fractions 2531 of the gradient shown in Fig. 3a were collected, purified as indicated in section n of Materials and Methods, and analyzed for ability to bind to E. coli and T4 DNA filters in DNADNA hybridization (Denhardt, 1966). Schleicher and Schuell membrane filters (B-6, 24-mm diameter, Lot No. 1004/7) were used. These data are in column 3 of the table. In control experiments 1 pg of [3H]E. coli BE DNA and [i4C]T4 DNA were hybridized to E. coli and T4 DNA filters (columns 1 and 2). The numbers reported in lines 1 and 2 are the percentages of input DNA radioactivity that hybridize to filters charged with 35 pg E. coli or T4 DNA, relative to the total radioactivity hybridized to E. coli and T4 DNA. The background of radioactivity bound to filters not charged with DNA, was subtracted. Every assay of each experiment was done in duplicate, and the numbers in the table are the averages of the results of two different experiments. Labeled and unlabeled mature T4 DNA were prepared from CsCl purified phage as indicated in Materials and Methods, section n. E. coli BE DNA, from our laboratory collection, was further purified by 5-fold extraction with phenol as in section i. [3H]E. coli BE DNA was a gift of N. Coszarelli. Hybridization efficiency is defined as the percentage of the labeled input DNA that is bound to E. coli and T4 DNA filters corrected for the radioactivity retained by filters not charged with DNA.

DNA that accumulates as the result of replication or repair. This has been unequivocally established by DNA-DNA hybridization (Table 3) : P4C]X-DNA was prepared 15 min

AND

GEIDUSCHEK

after infection, extracted, phenol purified, and tested for its ability to hybridize with T4 and with E. coli DNA. As Table 3 shows, the 14Clabel incorporated during infection at 40” hybridizes only to E. coli DNA; no significant hybridization to T4 DNA is detected. Therefore X-DNA is E. coli DNA, and the density shift experiments suggest that it is double stranded. This latter point has been established by testing the sensitivity of X-DNA to E. coli exonuclease I, an enzyme that specifically degrades singlestranded DNA (Lehman and Nussbaum, 1964). [i4C]X-DNA is insensitive to exonuclease I, but 99.5 % of this DNA is digested by exonuclease I (Methods, section n) after thermal denaturation. In a control experiment, native rH]E. coli BE DNA was also resistant to exonuclease I digestion, but 95.2 % was degradated into acid-soluble material after denaturation. At this point we conclude that, during infection with tsP36 amH39X amNl30 at the nonpermissive temperature for the T4 gene 43 DNA polymerase, [14C]TdR is incorporated int(o double-stranded E. coli DNA. This incorporation may be due either to normal replication or to DNA repair. One can discount the possibility that the observed E. coli DNA synthesis might be due to incomplet’e or slow adsorption of these particular phage mutants to the bacteria, with host functions continuing in the as yet surviving bacteria. Several facts argue against this possibility: (1) Less than 0.5 % surviving bacteria (checked for the ability to form colonies overnight at 30” on agar plates) remain 0.5 min after infection. (2) The [14C]TdR incorporation pattern is not dependent on the multiplicity of added phage (data not shown). (3) Messenger RNA labeled with [sH]Ur from 3 to 5 min after infection does not contain detectable amounts of RNA that hybridizes to E. coli DNA. (4) The peculiar physical properties of the [14C]DNA, that is, its lability during extraction and its small size, would not be expected for the product of normal DNA replication in a (small) fraction of uninfected bacteria.

HOST

DNA

SYNTHESIS

AFTER

C. Phage Gene Action Ag’ecting E. coli DLYA 8yn~the.k after Phase Infection a. Some protein synthesis is required after phage infection in order to sustain the observed TdR incorporation into E. coli DNA. If chloramphenicol is added 1 min after infection, [14C]TdR incorporation is greatly reduced. Addition of chloramphenicol at laker times leads to a reduction and modification of the TdR incorporation (Fig. 5). b. The products of T4 genes 55 and 33 are la~owr~ t,o be required for late viral protein synthesis (Bolle el al., 1968b). The DNA synt,hesis and degradation that we observe does not, however, involve the gene 55 or gene 33 products (Table 1, part] A, lines 5-S). Neither does it involve the gene 32, 41, 44, or 45 product,s, which are essential for viral DNA synt,hesis (Epstein et al., 1963) since the recombination of am or ts mutations in those genes into a gene 43, 30, 46 mutant does not, eliminate [14C]TdR incorporation int’o DNA (data not shown). c. Gene-specific suppressors of T4 DKA

2 ,1JJ+ 0

IO Minutes

+ 4; 20 after

30

. 40

T1 MUTS9T

IN’FI~;CTIOS

44 i

ligase deficiency have recently been disco\,ered (Kozinski and Mitchell, 1969; Karam, 1969; Chan et al., 1970). In particular, mutations in the rII gene are known to suppress the requirement of T4 DiYA ligase for T-k DNA replication and phage production. It. is conceivable that the rI1 mutation together with gene 43, 30 and gene 43, 30, 46 mut;ktions, might suppress E. coli DNA synthesis during infection, which depends on t’he ligase mutation. However, a direct test, of this possibility performed with mutant#s tsI’36 amH39X rIIA and tsP36 amH39X amNl30 rIIA clearly shows that the incorporation is not abolished (Table 1, part C, lines 1 and 2), but that t,he rIIA mutation decreases t,hc subsequent degradation of this X-I>;“\‘.\ (data not shown). The following experiment concerns the relationship between DNA l&se, t,hc gene 46 product, and the host DNA solubilizat,iorl that takes place after host DNA synthetiis shutoff. Exponentially growing E. coli W were labeled with [l(C]TdR for 2 gerwrat,ion~.

2 OtiI--i -10 0

IO Minutes

20 after

30

40

infectIon

infection

Fro. 5. Phagc protein synthesis is required in order to sustain TdIC incorporat,ion into X-DNA. E. coli BE grown in M9S to a concentration of 5 X lO*jml were infected with T4 tsP36 amH39X clncNl30 as indicated in Fig. 1. [14C]TdR (0.07 PCi, 5 rg/ml) was added 1 min later. At the time indicated by arrows (1 ), chloramphenicol (CM; 206 pg/ml) was added to a portion of the culture. [lK’]TdIt incorporation was measured as indicated in Fig. 1. Curve 1 (0) no CM addition; curve 2 (A), CM added 12 min after infection; curve 8 ( n ), CM added at 8 min; curve 4 (a), CM added ate 1 min.

FIG. 6. Soluhilizatiou of bacterial IIN1I, after infection with different T-4 mutank. E. coli 13” IjN.4 was labeled as indicated in section g of Materials and Methods. Bacteria (5 X lO*,‘nll) were infected at 40” wit)h the appropriate phage mutants, and then 5’;; Cl&COOKprecipit,able radioactivit,y was measured as specified in Mat(a rials and Methods, section g. The perce~~t of la beled bacterial DNA that remains acid precipitable at various times after infertion is S~OWII. Curve 1 (0): IsP36 (gene 43); curve 2 (0): tsP36 amH39X (genes 43,30) ; curve S (0) : tsP36 amN130 (genes 43,46) ; curve 4 (A) : tsP46 amH39X amNlX0 (genes 43, 30, 46).

448

CASCINO,

RIVA,

AND

Ten minutes after a chase with excess unlabeled TdR, the temperat’ure was raised to 40”, the culture was divided into four parts and separately infected with tsP36, tsP36 amNl30, tsP36 amH39X and tsP36 amH39X amN130 phage. The fate of host DNA was then followed by measuring the amount of acid-precipitable radioactivity at various times after infection at 40”. As Fig. 6 shows, the solubilization of host DNA after infection with tsP36 and tsP36 amH39X follows a pattern that is well known for the wild type: an abrupt drop in acid-precipitable DNA is observed between 5 and 10 min after infection (curves 1, and 2) (Wiberg, 1966). As already observed (Wiberg, 1966; Kutter and Wiberg, 1968) the mutation in gene 46 prevents degradation of host DNA to acid-soluble nucleotides (tsP36 amN130: curve 3). However, when infection is with the ligase mutant (tsP36 amH39X amN130; curve 4) host DNA undergoes solubilization although much more slowly than in the case of tsP36 or tsP36 amH39X mutant’s. These results raise the possibility that an exonuclease unconnected with gene 46 might be involved in degradation of host DNA.

D. Host Gene Action Affecting E. coli DIVA Synthesis after T4 Phage Infection An E. coli strain with a mutation affecting the soluble DNA polymerase (polA) having been isolated (DeLucia and Cairns, 1969), we wished to test its ability to sustain host DNA incorporation after infection with gene 43,30,46 mutants. The standard [‘“Cl incorporation experiment was performed by infecting wild-t#ype E. coli, the polA mutant, and one of the polA+ revertant strains with T4 tsP36 (gene 43) and tsP36 tsA80 tsL109 (genes 43, 30, 46) (Table 4). Evidently the TdR incorporation that correlates with the T4 gene 30 mutation occurs in the W3110 wild-type and the polA+ revertant; the polA mutant bacteria show little, if any, incorporation. The DNA synthesized in the polA revertant R6 (su-) cells infected at 40” with the T4 mutant tsP36 amH39X amN130 was analyzed for its ability to bind to E. coli and T4 DNA following the procedure described in Table 3. The results (Fig. 7) are exactly as before: the newly synthesized DNA binds exclusively to filters carrying E. coli DNA, not to filters carrying T4 DNA.

TABLE [l%]TdR

INCORPORATION Escherichia coli

IN THE WILD-TYPE, INFECTED WITH T4 [14C]TdR

Phage

mutant

tsP36 (gene 43) tsP36 tsA80 tsL109 (genes 43, 30, 46) tsP36 amH39X amNl30 (genes 43, 30, 46)

GEIDUSCHEK

4

PolA GENE

incorporation

GENE MUTANT 43 AND GENE

AND REVERTANT STRAINS 43, 30, 46 MUTANTS’

1-21 min after infection Bacterial host

(cpm/lOg

cells)

w3110 (polA+)

P3478 (poh-1

120 960

170 223

180 1360

100

110

-

-

2760

1840

1640

R6 (polA+)

Rll

(polA+)

OF

R14 (polA+)

0 [l%]TdR incorporation l-21 min after infection at 40’ with T4 tsP36 (gene 43), tsP36 tsA83 tsL109 and tsP36 amH39X amN130 (genes 43, 30, 46) mutants infecting polA wild-type host W3110, po1A1 mutant W3110-P3478 and a revertant, W3110-R6, of the polAr P3478 mutant. Cells were grown at 30” in M9S supplemented with 10 pg/ml TdR, washed and resuspended in fresh medium without TdR, and shifted to 40”. Then the culture (5 X 108 cells/ml) was divided into aliquots and infected with the appropriate T4 (8 phage/bacterium). One minute later, [14C]TdR (0.07 rCi/ml) and cold TdR (5 @g/ml) were added, and the [l%]TdR incorporation into 570 Cl&COOH insoluble material was determined as a function of time (Materials and Methods, section e). The maximum net incorporation is shown in the Table. The choice of T4 mutants for this experiment has been, in part, dictated by the weak am suppressor activity of W3110 and P3478 to which we were alerted by J. Karam and J. Cairns. However, the PolA+ revertants R6, Rll, and R14 do not contain this suppressor activity.

HOST

DNA

E. Identilication of the DNA Ge?le 50, 46 Mutants

SYNTHESIS

AFTER

Synthesized

by

Is X-DNA made only when t(he T4 soluble DXA polymerase is ina&vated, or can it

T-l

025 DNA input

05 (arbitrary

units1

FIG. 7. Hybridization of the DNA synthesized at 40” after infection of E. coli R6 (polA+ revertant) with the T4 mutant, tsP36 amH39X amN130. RF cells (the same as used for the experiment described in Table 5) as were infected at 40” with mutant phage. Labeling, lysis, sucrose gradient cent rifugation, DNA purification, and DNA-DNA hybridizat.ion were as described in Table 3. The total amount of radioactivity hybridizing to E. coli (0) and T4 (0) DNA, is shown as a function of the quantitr of input ‘Glabeled DNA (in arbitrary units). Background has been subt,racted. All experiments were done in duplicate, and the error brackets show the total range of the data. Efficiencies of hybridization ranged from 32 to 45’;. TABLE ~I)ESTIP.IC!\TIOS

OF

THE

DXA

SYKTHESIZED

Hybridization DKA

449

INFECTION

also be made concurrently wit,h phage DNA? In order to answer this question t,he following experiment was performed. RG cells infected at 40” with T4 tsP36 anzH39X amNl30 (and used for the DNA hybridization

0

MUTANT

experiment

of t,he previous

section)

weri’

shifted back to 30” 1 minute after infection. Cells were collected 13 min after infection and lysed (nlaterials and Nethods, section i, method 2); DSA n-as extracted (foot,note to Table 3) and centrifuged through a 5-20% sucrose gradient. The distribution of [14(1]DNA n-as similar to that of Fig. 3~. Fractions were collected from this gradient’ and tested for hybridization to E. di and T4 DXA (Table 5). The results show that (,l) host DKA synthesis also occurs when phage DNn is synthesized, and (2) the newly synt’hesized host DnTA is very small whereas the newly synthesized T4 DNA sediments much more rapidly and is more heterogeneous. This confirms the previous speculations about the nature of peaks I and II of Fig. 3~. Finally, n-e have examined the relative sizes of host DKA labeled before and after T4 infect,ion. Bacterial DNA (HI rain R(i) MVS labeled with L3H]TdR (Materials and .\Iet IIods, section g), cells were infected at 40” with the T4 mutants tsl’36 and tsl’:
(GENE

of radioactive

30,

46

?rlUTZNT

DS;\ (in solution) to filters

INFECTION”

with

DZX

l~untl

in filters

E. roli BE DNA T-l DK’A Efflcienry of hybridization

96:; 4s; 487~

5(‘; 95“;

70’ ; 30“;

32’.;

36’

;

65’ ; 35”; 57“ (

44’ ; 56’. ; 77’:;

4’ , 96 ; 56’ ,

(1 E. coli K6 cells were infected at 40” with T4 tsP36 anLH39X an~N130 (genes c3, 30, 46). One milrute later [I%]TdR (0.5 rCi/ml and 10 pg/ml) was added. One minute after addition of label, a portion of the infected cells was rapidly cooled to 30”. Cells were collect,ed 15 min after infection, lysed, and ccnt,rifuged in neutral 5-20yG sucrose. Fractions were collected, extracted with phenol, and analyzed, together with [3H]E. coli DNA and [14C]T4 DNA controls, for their ability to bind E. coli and T4 DNA as already described in Table 3. Data are presented for DNA fract.ions C, D, E, and F, which represent, positions in the gradient that are equivalent t’o fractions (2%29), (25-2(i), (22-23) and (15-16) of Fig. 3c, respectively. All assays are in duplicate; data presentation and background subtraction as in Tabl(a 3.

450

CASCINO,

RIVA,

infection, lysed (Materials and Methods, section i, method 2), and centrifuged in 5-20% neutral sucrose gradients. No difference was observed between the sedimentation profiles of the prelabeled and newly synthesized host DNA. Evidently, host DNA synthesized prior to T4 tsP36 umH39X amN130 or tsP36 mutant infection is subjected to the same extensive degradation even though the conversion to acid-soluble nucleotides proceeds at very different rates (Fig. 6).

AND

GEIDUSCHEK

We propose the following explanation of these findings. After infection, host DNA undergoes single strand breakage and the phage DNA ligaseis able to sealthese breaks. In the absence of phage ligase action, the single-strand breaks accumulate and cap become sites of attack by one or more eyen&eases unrelated to gene 46 product. The soluble DNA polymerase I activity, which is controlled by the E. coli polA gene (DeLucia and Cairns, 1969) persists after infection and repairs the DNA gaps produced by exonuclease action, incorporating DISCUSSION labeled thymidine along the way. T4 DNA E. coli DNA suffers two distinct insults ligase seals single-strand breaks in host after T-even phage infection: its replication DNA even in the process of its degradation, stops, and it is subsequently degraded to thereby indirectly limiting bacterial DNA acid-soluble nucleotides. The latter process polymerase I action on host DNA. also occurs in stages: an endonuclease is In order to present a more detailed model synthesized within the first minutes of infec- for the role of T4 DNA ligase in the host tibn and cuts bacterial DNA at a limited DNA shutoff mechanism, we summarize number of sites (Warren and Bose, 1968; what has recently become known about the Bose and Warren, 1969). Then a second phage-induced nucleases (Koerner, 1970) : endonuclease appears and primes E. coli endonuclease II makes single-strand breaks DNA for attack by a phage-specific exonu- in double-stranded cytosine-containing clease that is otherwise unable to solubilize DNA; endonuclease IV acts on singlehigh molecular weight DNA. The products stranded cytosine-containing DNA; exonuof the T4 genes46 and 47 are involved in the clease A, the most active exonuclease in indegradation to nucleotides although neither fected cell extracts, proceeds from the 3’-OH gene appears to code for an exonuclease terminus liberating 5’-mononucleotides; an (Wiberg, personal communication). exonuclease activity is also associated with We have shown here that the T4-induced the phage-induced soluble DNA polymerase DNA ligase is also involved in host DNA (gene 43 product). Warner et cd. (1970) have degradation (Fig. 6). Some host DNA syn- recently isolated a number of T4 mutants thesis takes place when urn or ts mutants in defective in the ability to degrade host DNA. gene 30 (the structural gene for the T4 DNA Cells infected with one of these mutants, nd ligase) are used to infect E. coli. When a 28, fail to induce the synthesis of an active mutation in gene 46 or 47 is also present, endonuclease II and host DNA accumulates even more exogenous TdR is incorporated. in fragments of about lo* molecular weight The newly synthesized host DNA is double containing very few single-strand breaks, stranded and of low molecular weight. Syn- without any further degradation. On this thesis can also take place concurrently with basis, Warner et al. have proposed a tentaT4 DNA synthesis, although its detection tive model for the sequence of events in the then becomesmore difficult,. The newly syn- degradation of host DNA in which endothesized bacterial DNA is very unstable and nuclease II controls one of t’he earlier steps is probably subject to continuous turnover in DNA breakdown. In order t’o explain our in the infected cell. The synthesis of this results, we propose that the T4 DNA ligase host DNA depends on the action of proteins is able to repair single-strand breaks in the specified by the host and viral genomesbut host DNA produced by phage-induced ennot of T4 late genes. The “residual” host donuclease II. In the absence of active T4 DNA synthesis after infection with gene 41 DNA ligase, these breaks are the sites of mutants (Oishi, 196s) conceivably might exonucleolytic attack which is antagonized have similar properties. by host DNA polymerase repair. The newly

HOST

DNA

SYNTHESIS

AFTER

synthesized host DSA is as susceptible to degradation int,o acid-soluble material as the rest of the host DNA. The exonuclease activity is not exclusively controlled by gene 46, but involves (an)other nuclease(s). This model is based on the following considerations : (1) The primary role of gene 30 T4 DXA ligase is the repair of single-strand breaks in double-st,randed DNA (Kozinski, and Rlit’chell, l!JtiS). (2) These breaks are introduced b\- T4 endonuclease II, which acts on double-stranded DNA, rnt#her than by endonucleaw IV, I\-hich works on singlestranded D?;A. In fact, Sadowslti et al. (196X) showed that T4 DS,4 ligase can repair 60 ’ ( of the breaks in native x DNA made by endonuclease II. (3) Host’ DNA c:ut be degraded int’o acid-soluble material ewn in the absence of functional gene 46 product (Fig. 6, curve 4). Therefore another nucleasr activity for host- DNA must, exist. ACKNOWLEDGMENTS This research was supported by grants of the National Institutes of General Medical Sciences (GM 15880 and GM 18386) and the National Science Foundation. E.P.G. acknowledges a research career development award of the National Cancer Institute. We are grateful to N. Cozzarelli and J. Hosoda for helpful discussion and advice and to M. .J. Hale for skillful assistance.

L., and VOL~CI?;, E. (1958). Properties of ribonucleic acid t,urnover in T2-infected Escherichia coli. Biochim. Biophys. Acta 29, 536544. BERNSTEIN, H. (19G8). Repair and recombination in phage T4. I. Genes affecting recombination. Cold Spring Harbor Symp. &ant. Biol. 33, 325331. ROLLE, A., E~TEIN, It. Ii., SALSER, W., and UEIDUSCHEE;, E. P. (1968a). Transcription during bact,eriophage T4 development: Synthesis atld relative stability of early and late RNA. .I. Mol. Biol. 31, 325-348. BOLLE, A., EPSTEIN, I<. H., SALSER, W., and (;EIDUSCHER, E. I’. (1968b). Transcription during bacteriophage T4 development: Requirements for late messenger synthesis. J. *Vol. Biol. 33, 339-362. BOSE, S. K., and WARREN, R. J. (1969). Bacteriophage-induced inhibition of host functions. II. Evidence for multiple, sequential bacteriophage-induced deoxyribonucleases responsible

ASTR.WHAN,

T4

MUTANT

INFI’CTION

4.; I

for degradation of cellular ti~oxgril-~on~~~~l~~~~! acid.J. Virol.3, 549-556. ROTSTEIN, 11. (1968). Synthesis and ntaturatiotl of phage P22. I. Identificatiotl of itttc~rmedintc:s. .I. Mol. Biol. 34, 621-641. ChScINO, A., I~IVA, s., and (~~:tDl~SCHEI<, I.:. 1'. (1970). I)NA ligation and thr coupling of T4 late transcription to replicatiotr. (‘o/r! SATIRIC/ Hrrrh~~r Symp. Quant. Biol. 35, 21:<. CHAN,

LT.

I,.,

SHIWi\IL,

d.,

alld

J’:HISi-Z

\ICI,

Ki.

(1970). Itrtergenic suppressioir of amber polynucleotide ligase mu1 at ion in bact eriophagcl ?‘1. virolog~ly 40, 40%417. COHEN, P. S. (194Sj. The syrtt hesis of l):tctrri:tl viruses. I. The synthesis of lrrtcaleic acid and protein in Escherirhla c,oli I% infected with T>r’ bacteriophage. J. Hiol. Cheer. 174, 281 ~293. D~Lucr.\, P., and c.\IRxSj .J. (1969). Isolatioll of an E. co/i strain with a mutation affecting I)N:\ polymerase. Satwe (Lodorr) 224, 1164. 116ri. DENHARDT, I>. T. (1966). A membrane-filter t c~ahnique for the detection of complementary I )X.4. Biochem. Biophys. Res. CownUn. 23, 641 (26. DE W.~AILD, A., PAUL, A. V., and LEHM is, 1. I<. (1965). The structural gene for deoxyribonucleic acid polymerase in bacteriophages T4 and X5. Proc. I\‘at. Acad. Pci. CT. S. 54, 1241-1248. EPSTEIN, 12. H., A., BOLLE, HTEINHERG, (‘. M., KELLENBERGER, F:., BOY DE IA TOUR, If;., CHEVALLEY, lt., J~:oo,zn, I<. S., Sr:s~ta~, M., DENHARDT, (:. H., and LIELAU~IH, A. (1963j. Physiological studies of conditional lethal tnutants of bacteriophage T4T). Cold Spri/78 Hrtrbor Symp. Quant. Biol. 28, 375-394. FAREEI), (;. C., and I~ICH.UWSON, C. (‘. (1967,. Enzytnatic breakage and joining of tleoxyrilm nucleic acid. II. The structural gene for polynucleot ide ligase in bacteriophage T4. I’roc.. .\'(I/ Ad. Pd. U. S. 58, MS-672. FRANKEL, F. 1:. (1968). tl:videttce for long ItNA strands in the replicating pool after ‘I’4 infrct ion, I’roc. ,Yat. Acacl. Pc,i. r’. S. .59, 131-13X. HOSOD.~, d. and M\THEWS, 12, (1971). I)NA rep11 ration i?r viva by polynucleotide-ligase defect ivc mutants of T4. II. J%ect of chloratnphetti~c,I and mutal ions in other genes. ./. .Ilol. Niol. .55. 155. KAR.\M, d. 1). (19G9). 1)NA replicatiott by phagc T4 rI1 mutants without) polvnucleotide ligase (gene 30). Biochem. Bioph ys. KPS. (‘on) m u ,I. X ~ 416322. KENNELL, I). 15. (1968). Inhibition of host prot,eirt synthesis during infect ion of Esch,erichia wli H by bacteriophage T4. I. Continued synthesis of host RNA. J. Viral. 2. 1262-1271. KOERNER, J. F. (1970). li;nzymes of nucleic acid metabolism. ‘-1 >~D,.u. Kv. Hio&enr 39, C&l- 134%.

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A. W., and LIN, T. H. (1965). Early intracellular events in the replication of T4 phage DNA. I. Complex formation of replicative DNA. Proc. Nat. Acad. Sci. U. S. 54, 273-278. KOZINSKI, A. W., and MITCHELL, M. (1969). Restoration by chloramphenicol of bacteriophage production in Escherichia coli B infected with a ligase-deficient amber mutant. J. Viral. 4, 823836. KUTTER, E. M., and WIBERQ, J. S. (1968). Degradation of cytosine-containing bacterial and bacteriophage DNA after infection of E. co% with bacteriophage T4D wild type and with mutants defective in genes 46, 47, and 56. J. Mol. Biol. 38, 395411. LEHMAN, I. R., and NUSSBAUM, A. L. (1964). The deoxyribonucleases of Escherichia coli. V. On the specificity of exonuclease I (phosphodiesterase). J. Biol. Chem. 239, 2628-2636. MURRAY, R. E., and MATHEWS, C. K. (1969). Addition of nucleotides to parental DNA early in infection by bacteriophage T4. J. Mol. Biol. 44, 233-248. NOMUR~, M., HALL, B. D., and SPIEGELMAN, S. (1960). Characterization of RNA synthesized in Escherichia coli after bacteriophage T2 infection. J. Mol. Biol. 2, 306-326. NOMURA, M., WITTEN, C., MANTEI, N., and ECHOLS, H. (1966). Inhibition of host nucleic acid synthesis by bacteriophage T4: Effect of chloramphenicol at various multiplicities of infect,ion. J. Mol. Biol. 17, 273-278. OISHI, AI. (1968). Studies of DNA replication in KOZINSKI,

AND

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