Proc. NatI. Acad. Sci. USAVol. 73, No. 5, pp. 1576-1580, May 1976Biochemistry

Bacteriophage T5-induced endonucleases that introduce site-specific single-chain interruptions in duplex DNA

(agarose gel electrophoresis/DNA affinity chromatography)

STEPHEN G. ROGERS AND MARC RHOADES

Department of Biology, The Johns Hopkins University, Baltimore, Maryland 21218

Communicated by Matthew Meselson, March 3, 1976

ABSTRACT Four site-specific endodeoxyribonucleases havebeen partially purified from extracts of bacteriophage T5-in-fected Escherichia coli by gel filtration and affinity chroma-tography on single- and double-stranded DNA. The enzymes

were detected and characterized by agarose gel electrophoresisof alkali-denatured digestion products. None of the four is foundin uninfected cells. In the presence of a divalent cation, all fourendonucleases make ligase-repairable, single-chain interrup-tions at specific sites in the duplex DNA of several bacterio-phages (X, T7, and T5) and a mammalian virus (adenovirus 2).These activities are not stimulated by ATP. None of the four isactive on single-stranded DNA. The fragments produced by eachenzyme from ligase-repaired T5DNA do not correspond to thosederived from mature T5 DNA. Each of the enzymes is able tocleave the intact strand of T5 DNA.

Bacteriophage T5 possesses a linear, nonpermuted DNA (1) thathas a molecular weight of approximately 76 X 106 (2) andcontains a large (8%) terminal redundancy (3). In addition, T5is unique in several respects. There is a two-step transfer process

during which only 8% of the genome is initially injected intothe host (4). Most significant is the occurrence of at least fourto five single-chain interruptions in the DNA (5, 6). The in-terruptions occur only in one strand and can be repaired by theaction of DNA ligase (7). Although the interruptions are thoughtto occur at genetically determined sites (5, 6), recent studieshave indicated considerable heterogeneity in the locations ofthese scissions. Hayward and Smith (8, 9), using gel electro-phoresis of denatured T5 DNA, were able to detect over 40discrete classes of single-strand fragments that occur in variousproportions. These classes are altered in a deletion mutant ofT5 that lacks one of the interruptions (7, 9), which suggests thatDNA site recognition is important in determining the locationof the interruptions.On the assumption that T5-induced proteins were responsible

for the interruptions, we prepared extracts from T5-infectedcells and examined them for site-specific endonuclease activity.This paper reports the discovery and partial characterizationof four such activities found in T5-infected cells.

MATERIALS AND METHODSBacteria and Bacteriophage Strains. All bacteria and bac-

teriophages have been described previously (10).Nucleic Acids. T5 DNA was isolated and ligase-repaired as

described by Rhoades and Rhoades (3). The DNA of Xb2 was

prepared by phenol extraction after purification of the phageby two cycles of centrifugation in CsCl step gradients. Single-stranded Xb2 DNA was prepared as described by Rhoades andThomas (11). Phage T7 DNA was the gift of Mr. G. Burdick.Adenovirus 2 DNA was the gift of Dr. T. J. Kelly, Jr. All DNApreparations were dialyzed against 0.01 M Tris.HCl at pH 8.0,0.1 mM EDTA, and stored at 0-40.

Affinity Columns. Single-strand DNA-agarose (0.3 mg ofDNA per ml) was prepared by the method of Schaller et al. (12)

from T5 DNA and Bio-Rad agarose. Native DNA-cellulose (1.8mg of DNA per ml) was prepared by the method of Alberts andHerrick (13) from salmon testes DNA (Worthington) and AvicelTG-105 cellulose (FMC Corp., American Viscose Division).Enzyme Assays. The T5-induced 5'-exonuclease was assayed

using heat-denatured DNA under the conditions of Frenkel andRichardson (14). Exonuclease III was assayed under the con-ditions of Richardson (15). All assays were performed on 32plabeled T5 DNA. Acid-soluble products were measured by themethod of Rhoades and Meselson (16).

Standard Assay for Site-Specific Endonucleases. Standardreaction mixtures (0.03 ml) contained 33 mM Tris-HCl at pH8.6, 1 or 10 mM MgCl2, 0.3-0.45 .ug of DNA, and 2-10 ALI ofenzyme. Assays on crude extracts or Bio-Gel fractions wereperformed in 33 mM glycylglycine-NaOH (pH 8.4), 3 mMsodium p-hydroxymercuribenzoate (Sigma), 1 or 10 mMMgCl2, and 0.3-0.45 ,g of DNA. The reaction mixtures wereincubated for 1 hr at 370 and stopped with 5 ,l of 2% sodiumdodecyl sulfate, 0.1 M EDTA (pH 8.0) followed by incubationat 370 for io min. The digestion products were routinely de-natured by addition of S ,l of freshly prepared 1 M NaOH.Horizontal 0.7% agarose slab gels were formed from agarose(Sigma) prepared by refluxing in Tris-phosphate buffer (8).Each gel was 15 X 9 X 0.7 cm thick and contained slots formedwith a Plexiglas comb with 1.5 X 3 mm teeth. Wicks were cutfrom Whatman 3 MM paper. Following the loading of up to27 samples, electrophoresis was begun at 120 V, constant volt-age. After 20 min, the buffer was removed from the slots whichwere then filled with 0.7% agarose in Tris-phosphate. Elec-trophoresis was resumed at 120 V and continued for 6-7 hr atroom temperature. At the end of a run, the gel was stained in0.5 Mg/ml of ethidium bromide (Sigma), and a photograph wastaken using the method of Sharp et al. (17) with Polaroid PN105film.

Purification of the Endonucleases. Escherichia coli 1100,an endonuclease I-deficient, thiamine-requiring K12 strain (18),was grown at 370 with vigorous aeration to 109 cells per ml inTris-maleate glucose medium (19) supplemented with 1 ,g/mlof thiamine. CaCl2 was added to 1 mM, and T5 phage wereadded at a multiplicity of 5. After 25 min, the infected cellswere quickly chilled on ice and concentrated by centrifugation.The pellet was frozen and stored at -200.

Extracts were prepared by resuspending 3 g of infected cellsin 12 ml of buffer A (20mM Tris-HCl at pH 7.6, 1.0 M NaCl,10mM 2-mercaptoethanol). This and all subsequent steps wereperformed at 0-4°. The resuspended cells were disrupted bysonication and centrifuged at 27,000 X g for 15 min. The su-

pernatant was then centrifuged in a Spinco 40 rotor at 35,000rpm for 3 hr. The resultant supernatant (10 ml, 15 mg of proteinper ml) was then chromatographed on a Bio-Gel A-0.5m(200-400 mesh) column equilibrated in buffer A. The flow ratewas 18 ml/hr, and 4 ml fractions were collected. This step re-

1576

Proc. Natl. Acad. Sci. USA 73 (1976) 1577

20

23

4_567

I2

1041 1 _161

17 __

1920 _2122 _3

FIG. 1. Digestion products from Xb2 DNA. Standard reactionmixtures contained 0.45 ;ig of Xb2 DNA and appropriate amounts ofeach endonuclease. Reactions were stopped and the products wereanalyzed as described under Materials and Methods. Slots 1 and 23contain denatured T5 DNA. Slot 2 contains a control mixture withno endonuclease and shows Xb2 duplex, r strand, and 1 strand, startingfrom the origin. Slots 3 through 6 contain the products made by En-zyme I after incubation for 15, 30, 45, and 90 min. Slot 7 shows theproducts after doubling the Enzyme I concentration at 45 min andcontinuing incubation for a total of 90 min. Slots 8 through 12, 13through 17, and 18 through 22 contain the analogous series for En-zymes II, III, and IV, respectively.

moved DNA and partially fractionated the endonucleases.Three patterns of site-specific endonuclease activity, assayedas described above, were eluted in a broad peak from 0.7 to 0.9column volumes. The activity designated Enzyme I was foundat the leading edge of this peak. The activity designated En-zyme III was found in the last fractions of the peak. Anotheractivity, designated Enzyme II, overlaps and nearly obscuresboth Enzymes I and III. Therefore, the broad peak was dividedinto five pools (12 ml each) for separation on DNA-agarosecolumns.

After extensive dialysis against buffer B (20 mM Tris-HClat pH 7.6,50mM NaCl, 1 mM EDTA, 10% vol/vol glycerol),each pool was loaded on a separate DNA-agarose column (1 X3 cm), equilibrated in buffer B. After the column was washedwith two volumes of buffer B, the activities were eluted witha 100 ml linear gradient formed from buffer B and 0.8 M NaClin buffer B. The flow rate was 10 ml/hr, and 1 ml fractions werecollected. This step resolved each of the activities into separate,but overlapping peaks. Enzyme I eluted at 0.20 M NaCl fromDNA-agarose columns 1, 2, and 3. Enzyme II eluted at 0.27 MNaCI from columns 2, 3, and 4. Enzyme III eluted at 0.37 MNaCI from columns 3, 4, and 5.A fourth activity, designated Enzyme IV, which was not

discernible in the Bio-Gel fractions, was eluted at 0.47 M NaClfrom DNA-agarose columns 1 and 2. Enzyme IV was notpresent if the sonication was performed in 0.2 M KC1 ratherthan 1.0 M NaCl. Enzyme IV fractions, free of other site-spe-cific endonucleases, were concentrated by dialysis againstbuffer C [20 mM Tris-HCI at pH 7.6, 1 mM MgCl2, 0.1 mMEDTA, 0.01% Triton-X 100 (Packard), 50% vol/vol glycerol]and stored at -20°.

DNA-agarose column fractions containing Enzyme II or IIIwere pooled separately and dialyzed against buffer B. Bothenzyme pools were then chromatographed on DNA-cellulosecolumns (1.5 X 2.0 cm) equilibrated in buffer B. After washingwith two column volumes of buffer B, a 100 ml linear gradientformed from buffer B and 1.0 M NaCI in buffer B was appliedto the column. Flow rates were 3-4 ml/hr, and 1 ml fractionswere collected. Enzyme II eluted at 0.33 M NaCl, and EnzymeIII eluted at 0.43 M NaCl. Peak fractions containing only En-zyme II or only Enzyme III were pooled separately, dialyzedagainst buffer C, and stored at -200.

DNA-agarose fractions containing Enzyme I also containedan exonuclease activity. The pooled fractions were dialyzedagainst buffer D (20 mM Tris-HCl at pH 7.6, 10% vol/volglycerol) and loaded on a 1.5 X 10 cm DEAE-cellulose(Whatman DE-52) column equilibrated in buffer D. Afterwashing with two column volumes of buffer D, a 100 ml lineargradient formed from bufferD and 0.8 M NaCl in buffer D wasapplied to the column. The flow rate was 10 ml/hr, and 1 mlfractions were collected. Enzyme I was distributed between thepass-through and fractions up to 0.1 M NaCl. The exonucleasedid not elute until 0.3 M NaCl. Fractions containing EnzymeI were pooled, made 1 mM in EDTA, and loaded on a 1.5 X 2.0cm DNA-cellulose column equilibrated in buffer B. This col-umn was operated as were those for the Enzyme II and III pools.Enzyme I eluted at 0.21 M NaCl. Peak fractions, free fromEnzyme II, were pooled, concentrated by dialysis against bufferC, and stored at -200.The inability to assay and identify each enzyme indepen-

dently in the early stages of the purification prevents deter-mination of overall recovery. The final yield of protein in eachcase was too low to be measured. All four endonucleases haveshown no noticeable loss of activity after 3 months of storage.

RESULTS

Production of Specific Single-Chain Fragments. Fig. 1shows the electrophoretically separated single-chain productsfrom Xb2 DNA after treatment with purified fractions of eachof the T5-induced endonucleases. Slot 2 contains a reactionmixture with Xb2 DNA and no enzyme. The minor band closestto the origin is native Xb2 DNA. The major bands are the 1 andr strands of Xb2 DNA, the r-strand being closest to the origin(20).Each endonuclease is selective as to which strand is initially

attacked. Enzymes I and IV degrade the r strand first; EnzymeIII degrades the I strand first; Enzyme II degrades both. Inaddition, each enzyme makes a characteristic, reproducible setof fragments from Xb2 DNA. The presence of one set of frag-ments, coupled with the failure to obtain other sets over a widerange of digestion, is taken as evidence of homogeneity in eachof the most highly purified endonuclease preparations.With increased time of incubation or readdition of the en-

zyme, there is an increase in the amount of the smaller frag-ments and a concomitant decrease in the amount of the largerfragments as judged by their intensities in the photographs. Ascan be seen in slots 3 through 6, increased time of incubationwith Enzyme I results in a series of digestion products thatgradually shift toward the smaller fragment sizes. Slot 7 showsthat readdition of enzyme gives complete degradation of-ther strand. If incubation is continued for times longer than shownhere, most of the DNA is found in the smaller fragments.However, we have not been able with any of the endonucleasesto obtain a set of digestion products that are completely resistantto further degradation. it is possible that there are several classes

Biochemistry: Rogers and Rhoades

1578 Biochemistry: Rogers and Rhoades

FIG. 2. Duplex digestion products from Xb2 DNA and digestionproducts from denatured Xb2 DNA. Slots 1 through 5 contain 0.45 ggof undenatured Xb2 DNA after incubation for 2 hr under standardconditions with no enzyme, Enzymes I, II, III, or IV, respectively. Slots6 through 10 contain the products of a 2 hr incubation of denaturedXb2 DNA (0.40 ,g) in the presence of no enzyme, Enzymes I, II, III,or IV, respectively. The quantity of each endonuclease used is thesame as that in Fig. 1. Slot 11 contains denatured T5 DNA.

of sites that vary in affinity for the endonucleases or in sus-

ceptibility to hydrolysis.The resolving power of the assay is greatly enhanced by the

strand separation obtained with Xb2 DNA as substrate. Thisseparation of complementary strands indicates that base com-position or secondary structure, in addition to molecular weight,influences electrophoretic mobility. The quantity of DNA ina band also appears to affect mobility. The greater the amountof DNA, the greater the distance the fragment moves. Thiseffect is illustrated by the Xb2 1 strand in Fig. 1. As the 1 strandis degraded by either Enzyme I (slots 2 and 3) or Enzyme IV(slots 21 and 22), its size apparently increases. Close inspectionof some of the product bands in Fig. 1 reveals other, less pro-

nounced, examples of this phenomenon.The endonucleases produce predominantly single-chain

scissions in duplex DNA. Fig. 2 shows an analysis of un-

denatured Xb2 DNA after digestion with each of the enzymes.None of the four endonucleases produces simultaneous cleavageof both strands of Xb2 DNA, with the possible exception ofEnzyme III. Slot 4 shows that a low level of discrete, duplexfragments is present after treatment with Enzyme III. Thesecould be due either to the proximity of single-chain interrup-tions in complementary strands or to the presence of a smallamount of a Type II restriction endonuclease (21), althoughthere is no other evidence for such an activity.

General Properties of the Endonucleases. Although theassay employed in this study does not give quantitative esti-mates of enzyme activity, it is possible by comparison of theintensities of product and substrate bands to assign relativeactivities within a series of experimental points. This methodwas used to determine pH optimum, co-factor requirements,and inhibitor effect for each endonuclease.

In the standard assay, with Xb2 DNA as substrate, all fourenzymes show a broad pH optimum with no distinguishabledifferences within the pH range from 8 to 9 in 33mM Tris-HCl.All four absolutely require a divalent cation. Greater activityis achieved in 1 mM (versus 0.1 or 10 mM) MgCl2 for EnzymesI, II, and III and in 10 mM (versus 5 or 20mM) MgCl2 for En-zyme IV. Activity is reduced with corresponding concentrationsof CaC12 or MnCl2.

There is no stimulation of the activity of any of the four by100 ,gM ATP. None of the four is affected by tRNA (15 nM) or

p-hydroxymercuribenzoate (3 mM). The latter finding greatlyfacilitated the initial detection of the endonucleases, since the

23

4

5

6

7

9810

1 2 ---'---

2

3

4 A

56 o

8 [C

9

10

12

+FIG. 3. DNA ligase repair of the digestion products from Xb2

DNA. Individual reaction mixtures (0.18 ml) containing 2.25 gg ofDNA and an appropriate amount of each enzyme were incubated for30 min. The reactions were stopped by addition of EDTA to 20mMfollowed by phenol extraction and extensive dialysis against 70mMTris-HCl at pH 7.6, 0.1 mM EDTA. Aliquots of each endonucleaseproduct were made 7 mM MgCl2, 1 mM 2-mercaptoethanol, 67 gMATP. T4 DNA ligase was added and the mixtures were incubated for30 min at 37°. Electrophoresis was carried out as described underMaterials and Methods. In both parts of the figure, slots 3, 5, 7, and9 contain the products of Enzymes I, II, III, and IV, respectively. Slots4,6,8, and 10 contain the same series after treatment withDNA ligase.Slot 11 contains T5 DNA treated with the same dose ofDNA ligaseunder identical conditions. Slots 1 and 12 contain denatured T5 DNA.Slot 2 contains denatured Xb2 DNA. In (a) the endonuclease digestionwas carried out under the standard conditions. In (b) digestions werecarried out with one-half the dose used in (a) in the presence of 3mMp-hydroxymercuribenzoate as described under Materials andMethods. The T5 DNA used was T5st (0) in (a) and T5st(+) in (b).

T5-induced 5'-exonuclease is 95% inhibited at this concentrationof the sulfhydryl reagent. (

All four endonucleases are free from detectable single- ordouble-strand exonucleases under the conditions of the T5-induced 5'-exonuclease, exonuclease III, or standard endonu-clease assays. The absence of significant exonuclease or phos-phatase contamination is further indicated by the ability ofDNA ligase to repair the single-chain interruptions introducedby the endonucleases (see Fig. 3).The presence of site-specific endonuclease activity in unin-

fected E. coli cells was examined by purifying extracts throughthe DNA-agarose column step as described in Materials andMethods. No site-specific endonuclease activity was detected.

Activity on Denatured DNA. Slots 6 through 10 in Fig. 2present the results of treatment of denatured Xb2 DNA witheach enzyme. None of the four endonucleases can form discretefragments by their action on a single-stranded substrate.However, Enzyme IV contains a nonspecific endonucleaseactivity able to utilize denatured DNA (slot 10). The origin ofthis activity has not been determined, but it does not interferewith the analysis of Enzyme IV on duplex DNA.

IProc. Natl. Acad. Sci. USA 73 (1976)

Proc. Natl. Acad. Sci. USA 73 (1976) 1579

Ligase Repair of the Scissions Produced by the Endonu-cleases. Fig. 3 shows the results of T4 DNA ligase treatmentof Xb2 DNA after extensive digestion (Fig. 3a) or limited di-gestion in the presence of p-hydroxymercuribenzoate (Fig. Sb)with each of 'the endonucleases. The latter experiment wasperformed in an attempt to achieve complete repair by inhib-iting possible undetectable exonuclease contaminants. As canbe seen in both parts of Fig. 3, most of the DNA is found at theposition of intact 1 and r strands after treatment with DNA li-gase. Consequently, the interruptions produced by the en-donucleases, like those found in mature T5 DNA, consist ofadjacent 3'-hydroxyl and 5'-phosphomonoester groups (22).Native T5 DNA was treated with ligase under identical con-ditions, and repair was nearly complete, as shown in slot 11 inboth parts of Fig. 3.

Fig. 3a demonstrates that even the smallest fragments pro-duced by each endonuclease can be repaired into intact strands.This indicates that the scissions produced early and late in thereaction are identical. Fig. Sb shows that repair is more nearlycomplete after limited digestion in the presence of p-hydrox-ymercuribenzoate, but this result may only be due to the smallernumber of interruptions that need to be sealed. In neither ex-periment was repair 100% for any of the four endonucleases,with the possible exception of Enzyme I. The failure of theproduct of Enzyme III to be completely repaired is not unex-pected in light of the double-stranded fragments mentionedabove.

Activity of the Endonucleases on Various DNA Substrates.Specific sites for endonuclease cleavage exist in DNA isolatedfrom several bacteriophages and a mammalian virus. Fig. 4shows the digestion products of Xb2, T7, T5, and adenovirus 2DNA. With the exception of Enzyme IV on adenovirus 2 DNA,each endonuclease makes a unique set of single-strandedfragments from each of the DNA species.

All four endonucleases produce specific fragments from li-gase-repaired T5st(0) DNA (Fig. 4). None of these correspondsexactly to the fragments found in mature TMst(0) DNA. Invitro, all four enzymes act at sites within unrepaired T5st(0)DNA and at sites in the normally intact strand of T5 DNA. Thelatter observation is apparent for Enzymes I and II in slots 24and 25. Although this finding is not obvious for Enzymes III orIV in Fig. 4, increasing the doses of these enzymes results in thedestruction of the intact strand of T5 DNA. A more detailedstudy of the fragments produced from T5 DNA will appear ina subsequent publication.

DISCUSSIONThe presence of site-specific endonuclease activity inducedafter T5 infection has been predicted in light of the increasingknowledge of the structure of T5 DNA. We now report that atleast four such activities are found in T5-infected cells.The separate identities of the four endonucleases were ini-

tially revealed by their fractionation by gel filtration and af-finity chromatography on single- and double-stranded DNA.The differences in single-chain fragments produced by eachendonuclease on four species of DNA, however, provide thestrongest evidence for four separate enzymes. These four en-donucleases are clearly distinguished from the only other re-ported T5-induced endonuclease (23), which makes double-strand scissions on X DNA, T5 DNA, and T2 DNA.The production of discrete, single-chain fragments from

duplex DNA indicates that the four endonucleases recognizespecific sites, presumably specific nucleotide sequences, withinthe DNA. These endonucleases are thus analogous to Type IIrestriction endonucleases (21). The recognition and cleavage

2 _3

6

879

16

1213 _14 _15 a16 fz

1920

21 di22 =23 I242526 a27 __

FIG. 4. Digestion products from various species of DNA. Ap-propriate amounts of each endonuclease were added to standard re-action mixtures containing 0.45 ,g of each DNA. After incubation for30 min, the reactions were stopped and the products were analyzedas described under Materials and Methods. Slots 2 through 6 containthe products formed by incubation of Xb2 DNA with no enzyme,Enzymes I, II, III, or IV, respectively. Slots 7 through 11, 12 through16, 18 through 22, and 23 through 27 contain analogous series withT7 DNA, adenovirus 2 DNA, ligase-repaired T5st (0) DNA, and un-repaired T5st(0) DNA, respectively. Slots 1 and 17 contain denaturedT5st(+) DNA. Strand separation is apparent for Xb2 and T7 DNA,but the strands of adenovirus 2 DNA migrate as a wide continuousband (slot 12). Under these conditions, no strand separation is ob-tained with ligase-repaired T5st(0) DNA.

promoted by both types of endonucleases takes place in thepresence of a divalent cation without ATP. The importantdifference is that the T5 endonucleases cleave only one strandof the duplex DNA, suggesting that the sites recognized by theT5 enzymes do not possess a 2-fold rotational symmetry as dothe restriction endonuclease sites.

There are at least two other endonucleases having propertiessimilar to the T5 enzymes described here. The qX174 gene Aprotein, which is required for initiation of DNA replication (24),has been shown to be an endonuclease that makes a single in-terruption in the "plus" strand of kX174 replicative form DNA(25). Unlike the T5 endonucleases, which have no activity onsingle-stranded DNA, this enzyme cleaves viral OX174 DNAand viral S13 DNA. The T4-induced endonuclease isolated byAltman and Meselson (26) makes a limited number of single-chain interruptions in duplex A DNA and T4 DNA. This en-donuclease has a pronounced preference for sites in the "minus"strand of OX174 replicative form DNA, but shows no activityon viral q5X174 DNA (27).We do not know whether any of the T5-induced endonuc-

leases described here are responsible for the natural interrup-tions in T5 DNA. All make ligase-repairable scissions resultingin specific fragments of T5 DNA. Furthermore, three of theenzymes are present in infected cells at 15 minpost-infection(S. Rogers, unpublished results), when the interruptions appear

Biochemistry: Rogers and Rhoades

1580 Biochemistry: Rogers and Rhoades

to be re-introduced into T5 DNA (28). None of the enzymes,however, generates single-chain fragments identical in size tothose found in T5 DNA, and all are able to cleave sites in theintact strand. Thus, if one of these endonucleases is responsiblefor the natural interruptions, its activity must be regulated invivo. It must be emphasized that the initial screening for en-donuclease activity with Xb2 DNA precludes the detection ofendonucleases specific for T5 DNA.

Endonucleases that act at specific sites could perform anumber of functions in T5 bacteriophage development. Threeof the endonucleases are present 10 min' after infection (S.Rogers, unpublished results) when DNA synthesis has just begun(29) and may play an initiator role in replication. The en-donucleases may have a role in recombination, generating thebasic structures from which single-strand'exchange could occur.Site-specific endonucleases could function in DNA maturationby creating unit length T5 molecules from the concatemericstructures found early in infection (30). The role of each of thefour enzymes in T5 infection may be ascertained by the isola-tion of mutants for each of' the endonucleases. Mutant studiesmay also aid in determining if the endonucleases are responsiblefor the natural interruptions in T5 DNA.

This research was supported by National Science Foundation GrantGB-20460. S.G.R. was supported by a Training Grant (HD-139) fromthe National Institutes of Health. This is Contribution no. 855 from theDepartment of Biology, The Johns Hopkins University, Baltimore, Md.

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