in vivo cleavage of cytosine-containing bacteriophage t4 dna to

Vol. 48. No. 1JOURNAL OF VIROLOGY, OCt. 1983, p. 18-300022-538X/83/100018-13$02.00/0Copyright c 1983, American Society for Microbiology

In Vivo Cleavage of Cytosine-Containing Bacteriophage T4DNA to Genetically Distinct, Discretely Sized Fragments

KARIN CARLSONI -23* AND JOHN S. WIBERG'Depalrtment of Radiation Biology and Biophysics, School of Medicine and Dentistry, Unihersity of Rochester,

Rochester, New York 14642I; Depairtnent of Microbiology, Faculty of Pharmacy, University of UppsalaBiomediccal Center. S-751 23 Uppsala, Sweden2: and Institute of Medical Biology,. University of Tromnsij,

N-9001 Tromnsii, Norwa N*

Received 24 January 1983/Accepted 23 June 1983

Mutants of bacteriophage T4D that are defective in genes 42 (dCMP hydroxy-methylase), 46 (DNA exonuclease), and 56 (dCTPase) produce limited amounts ofphage DNA in Escherichia coli B. In this DNA, glucosylated 5-hydroxymethylcy-tosine is completely replaced by cytosine. We found that this DNA rapidlybecomes fragmented in vivo to at least 16 discrete bands as visualized on agarosegels subjected to electrophoresis. The sizes of the fragments ranged from morethan 20 to less than 2 kilobase pairs. When DNAs from two of these bands wereradioactively labeled in vitro by nick translation and hybridized to XbaI restric-tion fragments of cytosine-containing T4 DNA, evidence was obtained that thetwo bands are genetically distinct, i.e., they contain DNA from different parts ofthe T4 genome. Mutational inactivation of T4 endonuclease II (gene denA)prevented the fragmentation. Three different mutations in T4 endonuclease IV(gene denB) caused the same minor changes in the pattern of fragments. Weconclude that T4 endonuclease II is required, and endonuclease IV is involved toa minor extent, in the in vivo production of these cytosine-containing T4 DNAfragments. We view these DNA fragments as "restriction fragments" since theyrepresent degradation products ofDNA "foreign" to T4, they are of discrete size,and they are genetically distinct. Thus, this report may represent the first, directin vivo demonstration of discretely sized genetically distinct DNA restrictionfragments.

Despite the widespread use in vitro of class IIrestriction endonucleases-which producelarge, specific fragments of DNA by breakage atspecific base sequences (43)-few, if any, exam-ples exist in which such fragments have beendemonstrated in vivo. Kutter and Wiberg (28)reported that upon mutational inactivation of T4genes 46 or 47, which appear to control a DNAexonuclease (34, 42), host DNA or phage cyto-sine-containing DNA (Cyt-DNA), produced by aT4 mutant also defective in gene 56 (dCTPase)(55), is no longer converted to acid-soluble prod-ucts. Neutral sucrose gradient analysis revealedthat the host DNA instead was degraded to anaverage M, of about 106 (corresponding to about1.5 kilobase pairs [kbp]), whereas the T4 Cyt-DNA had an average Mr of about 107 (about 15kbp). The authors noted the disparity in sizebetween degraded host and phage Cyt-DNA andsuggested that an endonuclease attacking only atspecific sequences might be responsible. Somecircumstantial evidence in support of this modelwas offered.Dharmalingam and Goldberg (13) used the

same strategy (inactivation of an exonuclease) toreveal three other examples of the generation invivo of large DNA fragments as primary prod-ucts of restriction endonuclease cleavage; theymutationally inactivated Escherichia coli exonu-clease V (coded by the recB and recC genes) (1).Most of the conversion of the following threephage DNA species to acid-soluble material wasprevented, and relatively large phage DNA wasdemonstrated as a small number of broad peaksin alkaline sucrose gradients: (i) unglucosylatedT4 DNA in an rgl+ host, (ii) T4 DNA lacking theT4 gene 2 protein at either end, and (iii) unmodi-fied X DNA in an hsdRK' and hsdMK' host.More recently, a similar example was providedby Ishaq and Kaji (23), who used alkaline su-crose gradients to show Rts-1 restriction of T4phage that contain glucosylated hydroxymethylCyt-DNA (glc HmCyt-DNA) in E. coli lackingexonucleases I and V.

In some of these cases, the identity of theenzyme(s) involved in degradation is known: E.coli endonuclease RK (gene hsdRK), which isinvolved in fragmentation of unmodified X DNA

18

SPECIFIC CLEAVAGE OF T4 CYT DNA IN VIVO 19

TABLE 1. Phage mutations usedGene Mutationa Relevant phenotype" Source" and reference

42 amN55 x 5 dCMP hydroxymethylase- This laboratory42 amN122 x 5 dCMP hydroxymethylase- This laboratory

46 amB14 x 5 46-47 nuclease- This laboratory46 amN126 46-47 nuclease- This laboratory56 amE51 x 5 dCTPase-; Cyt This laboratory56 amE114 x 5 dCTPase-; Cyt This laboratory

denA S112 x 5 endonuclease 11- This laboratory (19)denA SP4 x 5 endonuclease lI- This laboratory (19)

denB D2a2 x 5 endonuclease IV- Vetter and Sadowski (51)denB D2a23 x 5 endonuclease IV- Vetter and Sadowski (51)denB saA&9 endonuclease IV- (12)

" The suffix "x 5" indicates that the original mutant was backcrossed five times to remove potential additionalmutations present in the original isolate. saA&9 is a deletion extending from DI into ac; all others are presumablypoint mutations. The two mutations in gene 42 and the two in gene 56 map at different sites within the respectivegene (unpublished data), as do those in gene denA (19), and gene 46 (57).

b Phenotype in nonpermissive host strains. Cyt indicates that phage DNA made in nonpermissive hostscontains cytosine instead of hydroxymethylcytosine.

' T4 amber mutations were from R. S. Edgar, R. H. Epstein, or W. B. Wood and were backcrossed at theUniversity of Rochester, Rochester, N.Y.

(38), is a type I restriction enzyme, i.e., itrecognizes specific sites but cleaves randomly(43); T4 endonucleases II (47) and IV (51) de-grade host and phage Cyt-DNA (8, 19, 26, 46-48, 53). In vivo sequence-specific cleavage bythese enzymes has not been demonstrated, andin none of the examples cited above (13, 23, 28)was discrete size or sequence specificity ofdouble-stranded fragments, indicative of in vivorestriction, demonstrated.The present study concerns the fate ofT4 Cyt-

DNA, produced after infection with mutantsdefective in genes 42 (dCMP hydroxymethylase)(56), 56, and 46. The defects in genes 42 and 56assure that no hydroxymethylcytosine, but onlycytosine, can enter newly synthesized phageDNA. The product of gene 42 may play anadditional role in DNA replication, resulting insomewhat reduced DNA synthesis by 42- mu-tants when DNA synthesis is not dependentupon dCMP hydroxymethylase activity (11, 36).Gene 46 (together with gene 47) controls anuclease (34, 42) which, in addition to beinginvolved in degradation of host and phage Cyt-DNA (28, 54), is also involved in metabolismand recombination of T4 DNA (2, 4, 6, 7, 14, 16,21, 22, 28, 54). We show that newly synthesized,native phage DNA in cells infected with T442-46-56- phage appears in discretely sizedfragments, demonstrable as at least 16 separatebands upon electrophoresis in agarose gels. Theformation of these discrete bands is preventedby mutations in T4 gene denA (endonuclease II).Mutations in T4 gene denB (endonuclease IV) do

not block formation of bands, but they do causea significant change in the electrophoretic pat-tern. By hybridization analysis, some fragmentswere localized to specific regions of the geneticmap.(A preliminary report of this work has been

presented [Abstracts, Cold Spring Harbor Labo-ratory Bacteriophage Meeting, Cold Spring Har-bor, N.Y., 1982, p. 4]).

MATERIALS AND METHODSStrains of bacteria and bacteriophage. E. coli B (14)

is nonpermissive for amber mutants of T4. E. coliCR63 (14) and K803 (60) are permissive due to thepresence of amber suppressors supD and supE, re-spectively, and were used for propagation of phage.Phage mutations used in this study are listed in Table1. Multiple mutants were constructed and identified asdescribed previously (59). The presence of denA ordenB mutations in multiple mutants containing 56-mutations was confirmed by complementation assaysin liquid culture as follows: denA-, lack of ability torescue phage production in mixed infection of E. coli Bwith denA-,55amC64X5 in the presence of 0.01 Mhydroxyurea (19); denB-, high levels of DNA synthe-sis in mixed infection of E. coli B with denBD2a23 x 5,56- or with denBD2a2xS,56- (8, 51). T4 Cyt-O is apentuple mutant that makes viable phage containingfully cytosine-substituted T4 DNA when grown on E.coli B834 (9).Growth media and chemicals. The glycerol-Casa-

mino acids (GCA) medium of Fraser and Jerrel (15)was used for preparation of phage stocks and forlabeling. For plating, tryptone agar (10) was used.Sodium dodecyl sulfate (SDS) and agarose were fromBio-Rad Laboratories (catalog no. 1610301 and

VOL. 48, 1983

20 CARLSON AND WIBERG

1620100, respectively). Pronase was from Calbiochem(catalog no. 53702). and hydroxyurea was from SigmaChemical Co. (catalog no. H8627). Restriction en-zymes were from New England Biolabs or fromBoehringer-Mannheim Corp.. and [ '4C]thymidine(['4C]dThd) was from Amersham Corp. (catalog no.CFA 532). Other chemicals were reagent grade.

"4C-labeling and extraction of DNA. E. coli B wasgrown at 37°C to 3 x 108 to 5 x 108 viable cells per mlin GCA and was infected at 37°C with a multiplicity ofinfection (MOI) of 8 to 10 PFU per cell. Thesevariations in cell concentration and MOI did notsignificantly affect the phenomena studied here. Lessthan 5% of the cells remained viable 5 min afterinfection. Four minutes after infection, a mixture of['4C]dThd. unlabeled dThd. and deoxyadenosine(dAdo) was added, yielding final concentrations of 2 to5 Fg of dThd per ml (0.1 to 1 p.Ci/ml) and 50 to 360 ,ugof dAdo per ml (5. 37). Details are noted in the figurelegends. The initial concentration of dAdo was relatedto the duration of the experiment; higher concentra-tions of dAdo permit longer incorporation of dThd, butat lower rates (unpublished data). Samples were with-drawn at different times after infection and chilled onice in two volumes of ice-cold 0.15 M NaCI-0.05 MNa2H2EDTA (pH 8.0). DNA was extracted by amodification of the SDS-pronase-phenol method (25),in which, after SDS and pronase treatment, the sam-ples were extracted with a mixture of one volume ofwater-saturated phenol and one volume of chloroform.The aqueous phase was transferred to 2-ml Eppendorffcentrifuge tubes and precipitated with four volumes ofcold ethanol. After 18 h at -20°C, the precipitate wascollected by centrifugation at 12,800 x g for 15 min at4°C. The pellet was suspended in about 100 til of 10mM NaCI-10 mM Tris hydrochloride (pH 7.4)-i mMNa2H2EDTA. The recovery of trichloroacetic acid-insoluble material during extraction was 70 to 90%.About 103 to 10i cpm per slot were used for gelelectrophoresis; the lowest amount was sufficient todetect the specific fragments. Gel analyses of materialfrom different stages of purification (after SDS lysis,pronase treatment, and extraction with organic sol-vent) showed fragmentation patterns similar to thoseof the purified material (after ethanol precipitation).

Gel electrophoresis. Horizontal slab gels (18 by 24 by0.3 cm) contained 0.7% agarose in 160 mM Tris acetate(pH 8.1)-80 mM sodium acetate-8 mM Na.H.EDTA-72 mM NaCI and were electrophoresed for 18 or 40 to48 h at a constant current of 100 mA per gel submergedin the same buffer.

Autoradiography. For autoradiography, gels werefixed by immersion for 30 min at room temperature in96% ethanol and then overnight at -20°C in freshethanol. The gel was then placed on a sheet of Milli-pore HA nitrocellulose membrane or Whatman 3MMfilter paper, covered with plastic wrap. and dried on aHoefer gel drier with vacuum, but no heat, untilvirtually all liquid was removed. The gel and itssupport were wetted on a water saturated pad ofWhatman 3MM filter paper for about 30 sec. Then, thegel was baked at 60°C under vacuum for 40 min. Thegel, now bonded to its support, was removed andmounted for autoradiography with Kodak SB-5 X-rayfilm or Wicor XRP X-ray film from CEWA-verken,Strangnas. Sweden. The autoradiograms werescanned in a densitometer built by the Electronics

Shop, University of Uppsala Biomedical Center. Sizesof DNA fragments were estimated as described previ-ously (9, 50).

Hybridization. Nick translation (31), blotting (49),and hybridization were carried out as described previ-ously (9).

Terminology. DNA bands are seen as stained orradioactive material separated from other bands ongels. DNA fragments are discretely sized, geneticallyunique molecules. Thus, one band may contain severaldifferent fragments of the same size.

RESULTSDiscrete fragments of T4 Cyt-DNA. Figure 1

shows that the Cyt-DNA produced after in-fection with mutant 42amN55 x 5,46amB14 x 5,

L i

LT.

A

cj,

(3 ( C-. .4 -3(X

12

12.4-8.4--

3 5 -

2L-

FIG. 1. Fragmentation of T4 Cyt-DNA. E. coli Bgrowing in GCA at 37°C was infected at a cell densityof 3 x 108 per ml with T4 42amN55x5,46ainB14x5,56amE51xS phage (MOI, 10). ['4C]dThd to 2 ,ug/mland 1 ,uCi/ml and dAdo to 250 ,ug/ml were added 4 minafter infection, at which time 1.8% of the cells sur-vived as colony formers. Samples were withdrawn atdifferent times, and DNA was extracted and subjectedto gel electrophoresis together with a size standard(Bglll digest of labeled Cyt-O DNA); 15,000 (10-minsample) to 33,000 (30-min sample and later) cpm pergel slot. After electrophoresis, the gel was fixed inethanol, dried, and autoradiographed. (A) Electropho-resis for 45 h. (B) Electrophoresis for 21 h. Numbers atleft represent sizes, in kbp, derived by interpolationfrom the known sizes (shown in Fig. 2) of the referenceBglll fragments of T4 Cyt-O DNA shown in the leftlane.

J. VIROL.


56amE51 x5 was extensively fragmented start-ing between 10 and 20 min after infection. Inaddition to discrete bands, a fairly high back-ground was observed in the form of a smear inthe high-molecular weight range. There was alsoa diffuse band of low-molecular-weight materialmigrating ahead of the fastest-moving, discreteband (Fig. 1B). This suggests that there is somerandom degradation of the DNA in parallel withspecific cleavage. About 16 bands of 20 kbp orsmaller were observed. Bands corresponding tofragment sizes larger than 20 kbp were difficultto enumerate because of the high background inthis region of the gel. The discrete fragmentsbecame progressively obscured by random deg-radation beyond 40 min after infection. That thefragments likely arose from higher-molecular-weight intermediates was shown by pulse-chaseexperiments as follows. E. coli B growing inGCA was infected with bromodeoxyuridine-labeled, CsCl- purified, 42-46-56- phage(MOI,10), and [1 CJdThd (to 1 ,uCi/ml), togetherwith unlabeled dAdo (to 50 ,ug/ml), was added 4min after infection. At 8.5 min, the culture wasrapidly filtered (Millipore HAWP 047 00),washed with warm GCA, and suspended inwarm GCA containing 5-fluorodeoxyuridine (10jig/ml) and uridine (20 jig/ml). Control experi-ments showed that this addition of 5-fluorodeox-yuridine (and uridine) immediately stoppedDNA synthesis (monitored as uptake of 32p, intoalkali-stable, trichloroacetic acid-insoluble,DNase-sensitive material). Samples were re-moved at intervals and extracted and banded inCsCl. DNA from the light band (yielding a peakof radioactivity at a density of 1.71 to 1.72 g/ml)was subjected to gel electrophoresis; bothstrands of this DNA were synthesized afterinfection and consisted of phage DNA contain-ing thymine (no 5'-bromouracil) and cytosine(no hydroxymethylcytosine). Results (notshown) were very similar to those shown in Fig.1: immediately after resuspension (9.5 to 10 min)most DNA was large, whereas later samplesshowed progressively increasing amounts offragments. Since no new DNA was synthesizedbeyond 10 min, these fragments must have aris-en by breakage of large DNA. Results similar tothose shown in Fig. 1 were observed when adifferent 42-, 46-, or 56- mutation was substi-tuted, i.e., when the infecting phage was42amN122 x 5,46amB14 x5,56amE51 x 5; 42am-N55x5,46amN126,56amE51X5; or 42am-N55x5,46amN126,56amE114x5 (data notshown).The discrete fragments were seen only with

phage Cyt-DNA. Figure 2 shows an analysis ofphage DNA labeled after infection with42+46-56+ phage, which contains and synthe-sizes glc HmCyt-DNA. This DNA remains large

0)

0 ~~~~~~C)3>, cZ C CD C) CD uL C-j(_ - CN mn -3 C~ CL) --

1 7.5 -14I -

10.6-

79-

55-

4.4-

3 3-

FIG. 2. Test for degradation of phage HmCyt-DNA. E. coli B was infected with 46amB14X5, andDNA was labeled and analyzed as described in thelegend to Fig. 1, with similar levels of survivingbacteria and amount of sample per gel slot. Electro-phoresis for 45 h. Numbers at left denote sizes (in kbp)of Cyt-O BglII fragments.

(.30 kbp) throughout infection. Similarly, when5'-bromouracil phage (containing glc HmCyt-DNA) was used to infect in dThd-containingmedium where newly synthesized phage DNAwas labeled with ['4C]dThd after infection,once-replicated DNA (containing 5'-bromoura-cil plus glc HmCyt in one strand and thymineplus cytosine in the other) was not fragmented(data not shown). Figure 3 shows an analysis ofprelabeled host DNA from cells infected with42-46-56- phage. Degradation of host Cyt-DNA was more extensive than degradation ofphage Cyt-DNA (Fig. 3 versus Fig. 1), which isin agreement with observations of Kutter andWiberg (28) upon infection with 46-56- phage.By 60 min after infection, most of the prelabeledhost DNA migrates as material smaller than 10kbp, whereas most phage DNA at this time islarger than that.

Involvement of T4 endonucleases II and IV infragmentation of phage DNA. T4 endonucleasesII and IV in vitro specifically degrade Cyt-DNA(45, 46) and are implicated in degradation of host(19, 47, 53) as well as of phage (8, 26, 48) Cyt-DNA upon phage infection. To determine

VOL. 48, 1983


denB' phage (Fig. 1). Notably, the backgroundin the high-molecular-weight area was lower(Fig. 5A), and the heterogeneous, low-molecu-lar-weight material (Fig. SB) was absent.The differences between the fragmentation

patterns seen in the presence or absence ofendonuclease IV are further illustrated in Fig. 6,which shows densitometer tracings of autoradio-grams like those shown in Fig. 1A and 5A. In thedenB+ sample (upper tracings), at least 16 bandsformed distinct peaks. Assuming one fragmentper band, the bands 20 kbp and smaller account-ed for at least 140 kbp of DNA. There were alsodiscrete bands larger than 20 kbp. The heights ofsome of the peaks (e.g., 12 and 3.5 kbp) suggest-ed that the corresponding bands contain morethan one fragment. In the sample from thedenB- infection (lower tracings), 22 bandssmaller than 20 kbp formed distinct peaks, andtogether these accounted for at least 136 kbp ofDNA. Here also, some bands may contain morethan one fragment. In the region below 20 kbp,

C.r

FIG. 3. Test for degradation of host Cyt-DNA. E.coli B was grown for two generations in the presenceof 0.5 1Ci of ['4C]dThd per ml and 100 ,ug of dAdo perml. At a cell concentration of 3 x 108 cells per ml, cellswere centrifuged at 4,300 x g for 10 min and suspend-ed in the same volume of GCA (at 37°C) containing 100,ug of unlabeled dThd per ml. The cells were infectedwith an MOI of 10 of 42amN55x5,46amB14x5,56amE51 x5 phage. Samples were withdrawn at differ-ent times, and DNA was extracted and electrophor-esed for 48 h. Sizes of the reference Cyt-O Bglllfragments are indicated in Fig. 2.

whether these nucleases are involved in thediscrete fragmentation of phage Cyt-DNA justdescribed, E. (coli B was infected with42-46-56- phage in which either gene denA(endonuclease II) or deniB (endonuclease IV)was mutationally inactivated. Newly synthe-sized DNA was labeled and analyzed by gelelectrophoresis. Figure 4 demonstrates that verylittle, if any, fragmentation occurred when theinfecting phage was denAS112 x 5,42-46-56-.This result implies a major role for endonucleaseII in generation of the T4 Cyt-DNA fragments.Results similar to those shown in Fig. 4 wereobtained with denASP4 x 5 ,42-46-56- phage(data not shown); denASP4 maps at a differentsite than denAS112 (19). Figure 5 shows thatwhen the infecting phage was denBD2a23 x 5,42-46-56-, extensive fragmentation was ob-served, but the fragment pattern differed some-what from that observed with the corresponding

FIG. 4. Test for fragmentation of denA 42-46-56-Cyt-DNA. E. coli B was infected with T4 denAS112x5,42amnN55x5,46amB14x5,56amE51x5 phage as de-scribed in the legend to Fig. 1. ['4C]dThd to 2 ,ug/mland 1 ,uCi/ml and dAdo to 250 ,ug/ml were added 4 minafter infection, at which time 0.3% of the cells sur-vived as colony formers. Samples were withdrawn andanalyzed as described in the legend to Fig. 1; electro-phoresis was for 46 h. Sizes of the reference Cyt-OBglll fragments are indicated in Fig. 2.

J. VIROL.

r.1 .%

I

-,", mm NA- .--


A B

.~~~~24.-8-9 v

2 4

FIG. 5. Fragmentation of denB-42-46-56- Cyt-DNA. E. coli B was infected with denBD2a23x5,42amN55x5,46amB14x5,56amE51x5 phage as de-scribed in the legend to Fig. 1. A total of 0.2% of thecells survived as colony formers at the time of isotopeaddition. Labeling and analysis was carried out as forFig. 1 on the same gels. (A) Electrophoresis for 45 h.(B) Electrophoresis for 21 h.

several bands differed between these two sam-ples. Notably, the 7.1- and 3.5-kbp bands fromthe 42-46-56- infection were missing in thedenB-42-46-56- infection; instead, bands at10.6, 8.9, 6.7, 4.8, 4.1, 2.7, and 1.7 kbp wereobserved.Fragmentation patterns similar to those

shown in Fig. 5 were seen also when the in-fecting phage was denBD2a2x5,42amN55x5,46amB14 x 5,56amE51 x 5 or denBsaA9,42am-N55 x 5,46amB14 x 5,56amE51 x 5. We did notobserve a fragment with altered mobility result-ing from the saA9 deletion, suggesting that thisdeletion falls within a large fragment. The threedenB-42-46-56- strains differed markedly,however, in the amount of stable phage DNAaccumulated after infection. In data not shown,infection with 42-46-56- or denA-42-46-56-phage yielded about ,the same level of DNAsynthesis: about 20 phage-equivalent units percell at 60 min, measured by uptake of 32p, inGMC medium (10). Infection with denBD2a-23 x5,42-46-56- or denBsaA9,42-46-56-phage yielded somewhat less, about 10 phage-equivalent units per cell at 60 min, and infectionwith denBD2a2 x 5,42-46-56- yielded consider-ably more, 60 to 100 phage-equivalent units per

cell ,at 60 min. Analogous results were obtainedwhen DNA synthesis was monitored by uptakeof labeled dThd in GCA medium. The reason forthis variation is not understood and is currentlybeing investigated. Degradation of phage Cyt-DNA in the absence of 46 or 47 function startsfairly late (Fig. 1 and 5) and does not proceed toacid-soluble products (28; confirmed in the pres-ent study). Therefore, no differences in net DNAsynthesis between the 42-46-56-, den-A-42-46-56-, and denB-42-46-56- mutantswould be expected, unless some mutation af-fects earlier events in DNA metabolism. Back-crosses to 46amB14x5 (data not shown) showedthat none of the denB-42-46-56- strains carrieda das mutation; das mutations (20, 34) partiallyrelieve the DNA arrest (14, 54, 58) due to gene46 deficiency (20).

Specificity of fragmentation. Hybridization ex-periments were performed to determine whetherthe discrete bands obtained upon electrophore-sis of DNA from 42-46-56--infected cells con-tained genetically unique fragments. The probeswere obtained by elution from wet, ethidiumbromide-stained gels of 42-46-56- Cyt-DNA,since autoradiography required an absolutelydry, baked gel from which DNA recovery wasdoubtful. Only a few bands were discernible instained gels, notably the 12-kbp band and a bandin the region corresponding to 3.5 kbp of DNA(see Fig. 1). The latter will be referred to as 3.5-kbp DNA, although its identity to the 3.5-kbpband seen in Fig. 1 has not been rigorouslyproven. These two DNA probes were mappedby hybridization to XbaI restriction fragments ofT4 Cyt-O DNA blotted onto nitrocellulose fil-ters.

In addition to the discrete bands of intracellu-lar DNA seen in Fig. 1 and 5, a significantbackground ofDNA is seen, probably composedof randomly sized fragments from random loca-tions in the genome. Thus, both the 3.5- and 12-kbp probes undoubtedly contain significantamounts of this background material. The largerthe XbaI restriction fragment to which hybrid-ization of the probe is attempted, the higher isthe probability that this background material willfind a homologous sequence. Thus, we havechosen not to accept as specific (although it maybe) the hybridization of either probe to XbaIfragments 1 to 7 (see Fig. 8); since neither probehybridized to XbaI-8, yet did hybridize to cer-tain smaller XbaI fragments, we accept as highlysignificant the presence or absence of hybridiza-tion to the smaller fragments starting with XbaI-8.

Restriction endonuclease XbaI was chosen forthe hybridization studies, since it provides smallfragments from a large part of the genome. Thepositions of the XbaI cleavage sites and frag-

VOL. 48, 1983


FIG. 6. Densitometer tracings of 42-46-56- and denB-42-46-56- T4 Cyt-DNA fragments. Autoradiogramsfrom gels showing analysis of samples similar to those shown in Fig. 1 and 5, obtained 30 min after infection of E.coli B with 42-46-56- (upper panel) or denBD2a23x5,4246-56- (lower panel) were scanned in a densitometer.Migration was from left to right. Fragments smaller than 1.8 kbp migrated out of the gel. The three tracings ineach panel correspond to three different sensitivity settings on the densitometer. Numbers within the panelsdenote estimated fragment sizes (kbp) for the peaks (average of 2 to 6 determinations; standard deviation, 0.02 to0.13 for upper panel, 0.06 to 0.42 for lower panel).

ments of Cyt-O DNA are shown in Fig. 7, basedon data of Kutter et al. (27) and our analysis ofCyt-O DNA.Cyt-DNA isolated 40 min after infection with

T4 42-46-56- phage was subjected to gel elec-trophoresis, and the 12- and 3.5-kbp DNA bandswere extracted from the gel, labeled, and ana-lyzed. Figure 8 shows the hybridization patternsobtained. Both probes hybridized rather strong-ly to the high-molecular-weight fragments andshowed discrete and different hybridization to

the small Xbal fragments. The hybridization toonly certain, small fragments indicates that ge-netically unique sequences are present in theprobe. Since both probes may contain more thanone fragment, it is possible that some of thehybridization to large fragments is also specific,i.e., that the probe may contain unique se-quences from regions where XbaI yields largefragments.The 12-kbp DNA probe. DNA from the 12-kbp

band hybridized prominently to XbaI bands 9

J. VIROL.

SPECIFIC CLEAVAGE OF T4 CYT DNA IN VIVO

grapiOt . $49.21 oo 7

FIG. 7. Genetic and restriction map of T4, modified from Yee and Marsh (61), with permission. Positions ofall XbaI cleavage sites but one are those determined by Marsh and collaborators (33, 61) for Cyt-DNA carryingthe denB-rII deletion H23B. The positions of cleavage sites determined for other enzymes (Sall, Kpnl) differ, onthe average, by 0 to 500 base pairs between Cyt-O (9) and the H23B-deleted strains (33, 61). These differencesmay be experimental rather than real; the zero point used in calibrating the H23B map was not precisely known,since it lies under the deletion, whereas the Cyt-O map used a zero point within a sequenced region including theorigin. Carlson (9) has suggested that this origin be defined as the first A * T base pair in the rlIB translationalstart codon. The Xbal site at 165.6 kbp was located by our analysis of Cyt-O DNA (containing no knowndeletions and having a genome length of 166.5 kbp) (9). Cyt-O DNA yields 21 Xbal bands (cf. Fig. 8; Xbal band21 has migrated out of that gel; band 2 is a triplet, and band 9 is a doublet). The positions of the 24 Xbal fragmentsfrom Cyt-O DNA are shown here. The numbering of some fragments differs from that derived from Cyt-DNAdeleted in the denB-rII region (33, 61), but agrees with that of Kutter et al. derived from the Cyt-DNA of acomplete genome (27). except that (i) their fragments 2.1, 2.2, and 2.3 are our fragments 2a. 2b, and 2c.respectively; and (ii) their fragments 9, 10.1, and 10.2 correspond to our fragments 9a, 9b, and 10, respectively.Our fragment 9b is about 0.5 kbp larger than their fragment 10.1, which arises from the same region of the map;we cannot explain this discrepancy. On the innermost two circles are indicated those Xbal fragments that dohybridize (thick sectors) the respective probes (12 or 3.5 kbp) and those that do not (thin sectors). *, One or bothfragments hybridize. The text and the legend to Fig. 8 explain the placement of these sectors.

and 11 and to an incompletely cleaved fragmentbetween XbaI-8 and -9 (Fig. 8) and did nothybridize to the XbaI fragments 8, 10, and 12through 17. Since XbaI fragments 9a and 9b areonly 6.2 kbp, hybridization of the 12-kbp probeto XbaI-9b would imply that it also hybridized to

the 14-kbp fragment XbaI-2c and possibly toXbaI-21 (Fig. 7). Similarly, hybridization of the12-kbp probe to XbaI-9a would imply that it alsohybridized to either the 11-kbp fragment Xbal-5or to the 14-kbp fragment XbaI-2b, or to both(Fig. 7). The hybridization of the 12-kbp probe

VOL. 48, 1983 25


1 -2a,b, c_]

3-4-5-6-l7-8-

9a, b-i10-

11-I12-13-i

14-15-116-17--

.I

.0 -,

c'Jix

N~

'it

I 0 + -

;. _ ,

I w

18j

19,20-I

FIG. 8. Specificity of fragmentation of 42-46-56-DNA. The 12-kbp band and a band from the areacorresponding to 3.5 kbp from 42-46-56--infected E.coli B 40 min after infection (see Fig. 1A) were elutedand labeled with 32P by nick translation. For compari-son, T4 Cyt-O DNA was similarly labeled. UnlabeledCyt-O DNA was cleaved with Xbal restriction endo-nuclease, electrophoresed, and the denatured frag-ments blotted onto a nitrocellulose membrane filter.This was cut into strips, and the labeled samples weresonicated, heat denatured, and hybridized to adjacentstrips. Upon conclusion of hybridization, the stripswere autoradiographed. Numbers at the left denoteXbal bands in order of the distance migrated. The + or- to the right of various bands indicates ourjudgmentas to whether specific hybridization occurred (+) ornot (-), considering the relative intensities of thevarious bands and the probable background of nonspe-cific DNA (in the 12- and 3.5-kbp probes) that wasonly partially digested in vivo; see text for details.

to XbaI-11 (4.4 kbp) implies that it also hybrid-ized to XbaI-4 (12.2 kbp). Thus, the 12-kbpprobe hybridized to at least two separate geneticregions.The 3.5-kbp DNA probe. In addition to hybrid-

izing to an incompletely-cleaved XbaI fragmentbetween bands 10 and 11, the 3.5-kbp probehybridized to XbaI-15 (2.8 kbp). Because of itssize, this probe probably also hybridized toXbaI-2c. It did not hybridize significantly to the10 XbaI fragments 8, 9a, 9b, 10 through 14, 16,and 17.The 12- and 3.5-kbp probes clearly hybridize

to different, discrete regions. The differencesobserved indicate that the bands of intracellularDNA represent different segments of thegenome, and thus, it is likely that the fragmenta-tion involves a sequence-specific process.

DISCUSSIONThe experiments described here show that T4

Cyt-DNA in vivo is cleaved into a limited num-ber of discretely sized fragments, that T4 endo-nuclease II is required for this fragmentation,and that T4 endonuclease IV is involved to aminor extent. DNA from a 12-kbp band from42-46-56--infected cells was mapped to two orthree separate regions on the T4 chromosome,and DNA from a 3.5-kbp band was mapped to athird, distinct region, showing that the cleavageinvolves some sequence-specific interaction.The nature of this interaction is not known. Thesite specificity discussed below should thereforebe understood in the broadest possible senseuntil additional data permit more definite con-clusions concerning the mechanisms.

Role of T4 endonuclease II. It is difficult tojudge whether the obligatory role of endonucle-ase II in generation of the discrete T4 Cyt-DNAfragments is direct or indirect. In vitro, the 250-fold purified enzyme shows at least a 10-foldgreater activity on native than on denaturedDNA and introduces predominantly single-strand breaks (nicks) into Cyt-DNA. It does notattack HmCyt-DNA. At high concentrations ofenzyme, some double-strand breaks (cuts) aremade. The enzyme makes a limited number ofbreaks in DNA from phage A, and the averagelimit product sedimented in alkaline sucrosegradients is about 1,000 nucleotides (45). Al-though all four deoxynucleotides are found atthe 5'-phosphate terminus of these fragments,dGMP and dCMP predominate.We propose several models to explain our

results. In model A, endonuclease II only nicksin vivo. Then, the opposite strand must bebroken, perhaps by the combined action of anexonuclease (not that controlled by genes 46 and47, of course) to produce gaps and a single-strand-specific endonuclease that breaks oppo-site the gap. Site specificity could reside in anyof these three enzymes, but it would most likelyreside in the first. The single-strand-specificendonuclease would appear not to be endonucle-ase IV, since denB mutations had only minor

J. VIROL.


effects on formation of the T4 Cyt-DNA frag-ments (Fig. 5 and 6).

In model B, endonuclease II does not nick invivo but always breaks both strands. This is thesimplest model and implies that the nickingactivity is somehow repressed in vivo. Onepossibility is that, also in vivo, endonuclease IIpredominantly nicks, but the nicks are repairedvery quickly and only double-strand breaks re-main. Another possibility is that some otherprotein or factor associates in vivo with endonu-clease II, or the DNA, to change the specificityof the enzyme. Kemper et al. (24) recentlyprovided an example of enzyme specificity al-tered by a protein factor, perhaps analogous tothis case. They found that the activity of highlypurified T4 gene 49 endonuclease (endonucleaseVII) (35) on single strands can be completelyinhibited by the addition of helix-destabilizingproteins from T4 or E. coli, whereas the reactionwith rapidly sedimenting DNA is markedly stim-ulated. They suggested that the targets on rapid-ly sedimenting DNA are not gaps, but recombi-national crossover junctions, and this wasshown to be so (35).

In model C, endonuclease II is not involveddirectly in the degradation of T4 Cyt-DNA.Instead, the enzyme generates so many nicks inthe host DNA that the bulk of the DNA ligase isdiverted in an attempt to repair these nicks.Nicks created in the T4 Cyt-DNA by some othermechanism may then remain long enough forfragmentation to proceed as in model A. Ananalogous role of endonuclease II was suggestedby Warner to explain the observation that adenA mutation partially suppresses a T4 gene 30(DNA ligase) mutation; this effect requires anactive host DNA ligase (52). Kutter et al.(26) showed that the DNA made in 56-denB-infections is smaller than that made in56-denA-denB- infections on both neutral andalkaline sucrose gradients; this argues that endo-nuclease II somehow causes some damage to T4Cyt-DNA in the 46+47+ condition. They in-voked Warner's model (52) by suggesting thatthe denA mutation spared DNA ligase to repairnicks in the T4 Cyt-DNA that were produced bysome other endonuclease.For all models, the site specificity might re-

side in the endonuclease itself, in factors associ-ated with it in vivo (sequence recognition), or insome features of the DNA that might be lost invitro (e.g., small regions of denaturation whereboth DNA strands might be nicked simulta-neously, or start or stop sites for replication ortranscription).

Role of T4 endonuclease IV. Highly purifiedendonuclease IV is free of exonuclease contami-nation and displays a 200-fold greater activity onsingle-stranded DNA than on duplex DNA (44).

Endonuclease IV, like T4 endonuclease II, doesnot act on HmCyt-DNA but does act on Cyt-DNA (46). The limit digest on single-stranded KDNA is 25% acid soluble and consists of oligo-nucleotides with an average chain length of 50nucleotides (44). The products contain exclu-sively (single-stranded K DNA) (44) or predomi-nantly (+X174, fd, or M13 DNA) (3) 5' terminaldCMP. All four nucleotides are present at the3'-termini (3, 44), but dTMP may predominate(3). Endonuclease IV in vitro cleaves the single-stranded DNAs of bacteriophages fd, fl, M13,and 4X174 into various discrete fragments de-monstrable by polyacrylamide gel electrophore-sis (30). In unpublished work, E. Kutter (person-al communication) also observed discrete bands(eight) of fd DNA after digestion with low levelsof purified T4 endonuclease IV.

Endonuclease IV is implicated in degradationof T4 Cyt-DNA to acid solubility, since muta-tions in the denB (=D2a) gene allow synthesis ofstable Cyt-DNA in 56- infections (8, 51). Thus,it was somewhat surprising that denB mutationsdid not prevent fragmentation of the Cyt-DNAmade by 42-46-56- phage (Fig. 5 and 6). Onepossible explanation is that in a 56- or 42-56-infection, nicks provided by endonuclease(s) arequickly enlarged to gaps by the 46 and 47exonuclease, and these gaps present a single-stranded substrate for endonuclease IV. If thesenicks were provided only by endonuclease II,stable DNA synthesis would be expected in a42-56-denA- or 56-denA- infection; this is notthe case (26). Thus, it appears that in a 56- or42-56- infection (i) nicks are introduced into T4Cyt-DNA by endonucleases other than endonu-clease II, or (ii) nicks and gaps appear as prod-ucts of recombination or discontinuous replica-tion or both.By comparing DNA fragmentation in cells

infected with denB+ or denB- phage, both alsodefective in genes 42, 46, and 56 (Fig. 6), a fewbands in the 1- to 10-kbp range were seen only inthe presence of endonuclease IV, but manymore were seen only when this enzyme wasdefective. Thus, a limited number of the specificbreaks in 42-46-56- Cyt-DNA in vivo dependupon the presence of endonuclease IV. Thebroad smear below 2.4 kbp that is seen withdenB+ (Fig. 1B) but not denB- (Fig. 5B) infec-tions suggests that the fragments of 42-46-56-Cyt-DNA are subject also to limited nonspecificdegradation in the presence of endonuclease IV.Different models may be proposed to explain theendonuclease IV-dependent fragmentation. Inmodel A, endonuclease IV acts only on single-stranded DNA in vivo, and we would postulatethat the fragments of denB-42-46-56- Cyt-DNA contain single-stranded areas that are tar-gets for endonuclease IV. Some, but not all, of

VOL. 48, 1983


these gaps are sequence specific. It is unlikelythat the gaps are products of recombination,since gene 46 deficiency, at least in a 42+56+background, prevents molecular recombination(6). The gaps may be results of endonucleaseplus exonuclease activity, or the result of dis-continuous DNA replication or both. In modelB, endonuclease IV in vivo also makes double-stranded scissions. This is the simplest model,but it is unclear how this would relate to theknown base or sequence specificity of endonu-clease IV, which was determined on single-stranded DNA (3, 44). The same possibilitiesdiscussed for endonuclease II recognition inmodel B may apply here also.

Specificity of in vivo fragmentation. The elec-trophoretic patterns illustrated in Fig. 6 do notshow the smooth decrease of areas under indi-vidual peaks with decreasing fragment lengththat is observed upon complete in vitro restric-tion cleavage of T4 Cyt-DNA (cf. Fig. 1C inreference 9). Such behavior (Fig. 6) would beexpected if (i) the in vivo cleavage is incomplete(i.e., if the same genetic regions appear in frag-ments of different sizes) or (ii) there is more thanone fragment in some bands. We consider itlikely that both factors contribute. The 12-kbpband was shown by hybridization to containDNA from at least two separate genetic regions.There are several discrete bands larger than 20kbp (Fig. 1, 5, and 6) and about 16 (denB+) or 22(denB-) bands that are 20 kbp or smaller. If oneassumes unique fragments in all bands, thesebands account for more than the genome equiva-lent of T4. Thus, it is likely that some of thesebands are composed of incompletely cleavedfragments.Two additional arguments support the no-

tion of specific but incomplete cleavage. (i)Other hybridization experiments (data notshown) indicate that isolated 12- and 3.5-kbpDNA, as well as a 2.3-kbp, cloned fragment ofT4 DNA, hybridize to discrete subsets of thebands from 42-46-56- or denB-42-46-56- in-fections. (ii) In experiments described by Hanggiand Zachau (18), T4 Cyt-DNA produced by56-denA-denB-alc or by 42-46-56- mutantswas cut by restriction enzymes and used as atemplate for the in vitro synthesis of T4 dihydro-folate reductase, which is coded by thefrd gene(17, 62). The yield of in vitro translatable (active)fragments from the 42-46-56--produced Cyt-DNA was found to be much lower than fromthe largely intact Cyt-DNA made by a56-denA-denB-alc mutant. The sizes of theactive fragments that were recovered were iden-tical whether DNA from the triple mutant orfrom the quadruple mutant was cleaved by re-striction enzymes. Our results make it probablethat a limited number of T4 genes are specifical-

ly inactivated by the in vivo production of theCyt-DNA fragments in 42-46-56- infections.Thus, the results of Hanggi and Zachau (18)suggest to us that the T4frd gene contains one ofthe targets for the enzyme(s) that produce theCyt-DNA fragments in E. coli B infected with42-46-56- phage and that the residual templateactivity is due to incomplete digestion in vivo.The activity causing cleavage at each site is

very reproducible since all experiments showedthe same proportion of material in each band.Our present data do not provide any informationconcerning the recognition mechanism. Cleav-age may be at, or directed by, a specific se-quence in the manner of restriction endonucle-ases, or it may depend upon some structuralfeature of the DNA, possibly associated withreplication or transcription or both. Nonethe-less, we believe it is appropriate to view thefragments of T4 Cyt-DNA (Fig. 1, 5, and 6) asrestriction fragments because: (i) they are degra-dation products of a DNA that is foreign to T4because it lacks the glucosylated hydroxymethylgroup that normally modifies cytosine in T4DNA and (ii) they are of discrete size and aregenetically distinct, both of which indicate non-random cleavage. Thus, our observation mayrepresent the first, direct in vivo demonstrationof discretely sized, genetically distinct restric-tion fragments. Lee and Sadowski (29) recentlydescribed two site-specific cleavages in vivo inT7 DNA that were demonstrable when the T7gene 6 exonuclease was mutationally inactivat-ed; however, there was no indication that thisrepresented a restriction phenomenon. Further-more, Roberts noted in a compilation of restric-tion enzymes that "all endonucleases cleavingDNA at a specific sequence have been consid-ered to be restriction enzymes although, in mostcases, there is no direct genetic evidence for thepresence of a restriction-modification system"(43).

In comparison to phage Cyt-DNA, host DNAin 42-46-56--infected cells yielded smaller frag-ments that were not discernible as separatebands upon gel electrophoresis. The differencesin size of the limit product between T4 Cyt-DNAand host DNA were noted also by Kutter andWiberg (28), who suggested that a nucleasespecific for cytosine-rich clusters (41) might beresponsible. The different base composition ofT4 (35% G+HmC, 65% A+T [32]) and E. coli(50% G+C, 50% A+T [32]) suggests how cleav-age dependent upon sequence-dependent fea-tures of the DNA would result in more extensivedegradation of E. coli DNA than T4 DNA. If, onthe other hand, the specific cleavage of T4 Cyt-DNA depends upon special structural featuresof the DNA caused by its active replication ortranscription (see above) or both, DNA lacking

J. VIROL.


such features, such as the host DNA after infec-tion, could be degraded in a different, perhapsmore random fashion. In E. coli, any definedsequence of given length would be equally prob-able, on the average. In T4 DNA, a sequencecontaining predominantly G * C pairs would be,on the average, less frequent than a sequence ofthe same length containing predominantly A * Tpairs, an expectation borne out by several stud-ies of the occurrence of various restriction sitesin T4 DNA (9, 10, 33, 39, 40), and also lessfrequent, per unit length, in T4 DNA than in E.coli DNA. It is perhaps not surprising that thehost DNA from cells infected with T442-46-56- phage yielded only a broad smearwith no evidence of discretely sized fragments,since the E. coli chromosome is about 20 timesmore complex than that of T4. Judith Munro(Ph.D. thesis, University of Rochester, Roches-ter, N.Y., 1971) showed that the 106-dalton hostDNA from 42-46-56--infected cells is not ho-mogeneous in size. By recentrifuging the leadingand trailing edges of the peak from a neutralsucrose gradient, she found that they migrated attheir original, different rates, corresponding tosizes of 1.5 x 106 and 0.2 x 106 daltons, respec-tively. Experiments are under way to determinewhether site-specific cleavage is involved in theT4 degradation of host DNA, by asking whethercloned fragments of the E. coli genome hybrid-ize only to a limited region of the smear.

ACKNOWLEDGMENTS

J.S.W. thanks Peggy Spear and Gary Wilson for earlyassistance with DNA gels. K.C. thanks J.S.W. and Ola Skoldfor kind hospitality at the University of Rochester and Univer-sity of Uppsala, respectively, Aud 0vervatn and CharlotteSjunneskog for excellent technical assistance, and S. Hjertenfor the use of the gel drier and densitometer. We are grateful toRichard Roberts for valuable discussion about restrictionenzymes, and we thank C. Linder for criticism of the manu-script.

This paper is based on work performed partially undercontract number DE-AC02-76EV03490 with the U.S. Depart-ment of Energy at the University of Rochester Department ofRadiation Biology and Biophysics, and has been assignedreport no. UR-3490-2243. Work in Uppsala and Tromso waspartially supported by grant no. 13.17.14-082 from the Norwe-gian Research Council for Science and the Humanities (toK.C.) and by grant no. 05950 from the Swedish MedicalResearch Council (to Claes Linder).

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in vivo cleavage of cytosine-containing bacteriophage t4 dna to

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