Processing of bacteriophage lambda DNA during its assembly into heads

J. Hoi. Biol. (1975) 91, 166-174

Processing of Bacteriophage Lambda DNA during its Assembly into Heads MICHAELSYVANEN

Department of Biochemistry Stanford University School of Medicine Stanford, C&if. 94305, U.S.A. (Received 26 June 1974, and in revised form 10 September 197’4) DNA purified from bacteriophage A added to a cell-free extract derived from induced h lysogens can be packaged into infectious phage particles (Kaiser & Masuda, 1973). In this paper the structure of the DNA which is the substrate for and head assembly is described. The active precursor is a in vitro packaging multichromosomal polymer that contains covalently closed cohesive end sites. Neither circular or linear DNA monomers nor polymers with unsealed cohesive ends are packaged efficiently into heads. The unit length monomer is packaged when it is either contained in the interior of a polymer (both of its ends are in cos sites) or when it has a free left end and a COBsite on its right. The monomer unit with a free right end is not a substrate for packaging. A procedure is given for the purification of A DNA fragments that contain either the left or the right cohesive end. The fragments are produced by digesting h DNA with the site-specific Escherichia coli R1 endonuclease ; the left and right ends are separated by sedimentation through a sucrose gradient. These fragments are used to construct small polymers that have a unit length h monomer with (1) a free left end and a closed right end, (2) a free right end and a closed left end, or (3) both ends closed in co9 sites.

1. Introduction Packaging of DNA into heads is a problem central to the understanding of the assembly of large DNA containing bacteriophages. Bacteriophage A is a very useful system for studying this process because DNA packaging into infectious particles has been accomplished in a cell free extract (Kaiser I%Masuda, 1973). The bacteriophage X chromosome, as it is found in phage, is a double-stranded DNA molecule of 30 million molecular weight with complementary single strands 12 bases long at each end (Wu & Taylor, 1971). After infection the DNA is not detected in this form but is found either as circles or as end-to-end polymeric structures (Young & Sinsheimer, 1968). Single chromosomes are cut from the polymers late in infection and this cutting requires the action of seven phage genes required for head synthesis (Mackinlay & Kaiser, 1969). Thus, the packaging of DNA during virion assembly is coupled with the cutting of the DNA to make the cohesive ends. The unique site on a X DNA polymer where this cutting occurs is called ws. A cos site can be made in vitro by annealing the single strand cohesive ends and then covalently sealing them with Escherichia coli DNA ligase (Gellert, 1967). 12

165

M. SYVANEN

166

In the present work I have to in vitro packaging and head a necessary part of the in vitro structural requirements are of

studied the structure of the DNA that is the precursor assembly. I have asked whether the cutting of DNA is DNA packaging reaction and, if so, what the minimum uncut DNA to be packaged in vitro.

2. Materials and Methods (a) struins Cell-free extract8 were prepared from the heat-inducible lysogen W3101 (hcIts857 Sam7). The phage hAam Barn1 imm434 eIta.56 was the 8ource of DNA. Titers of phage ,% were determined by plating on Ymel and of himm434 in the presence of h on Ymel (X). C64 end- 1 wa8 obtained from Dan Ray. (b) Cell e&ruct This wa8 prepared

according

to Kaiser

& Masuda

(c) Preparation Linear

(1973).

of DNA

himm434

DNA was prepared by extracting a purified phage stock with water (distilled over nitrogen) and then by dialysing against 50 mM-KCl, 10 mM-Tris, 0.1 mM-EDTA, pH 8.0. The circular DNA monomers were isolated as superwith purified Aimm434 in the presence of coils from the strain C64 end- 1 after infection chloramphenical according to the procedures of Wang (1969).

saturated phenol

(d) Preparation

of h DNA fragments produced by cleavage with RI endonwleaae E. coli R, endonuclease (Hedgpeth et al., 1972) cleave8 &mm434

The site-specific DNA into 6 pieces. The left cohesive end is contained in a fragment 0.46 the length of X while the fragment with the right cohesive end is 0.07 the total length (Allet et al., 1973). The difference in sizes between these 2 pieces of DNA allows their separation by zone sedimentation through a sucrose gradient. Purified E. coli R, restriction endonuclease was graciously provided by Ron Davis. 10 mM-MgCl,, pH 7.5) To 1 ml of linear himm434 DNA (A,,, = 1.0 in 100 mu-Tris, the R, enzyme at 37°C was added in 2-pg amounts every 4 min until 8 pg had been added; 10 min after the last addition the reaction was stopped by making the mixture 5 mMEDTA and by heating at 65°C for 10 min. The digestion mixture W&B cooled quickly with layered upon an 8% to 24% sucrose step gradient ice to 20°C. The mixture was immediately in an SW41 polyallomer centrifuge tube and centrifuged for 6 h at 38,000 revs/min at 20°C. After the run the tube was pierced through the bottom and 45 twelve-drop fractions were collected. The absorbance at 260 nm was measured in each fraction to monitor the distribution of DNA. 2 disperse peaks of DNA were evident, one of about 20 S and In the sucrose gradient the other of 26 S which skewed toward faster sedimenting material. This fa&er sedimenting DNA is presumably incompletely digested h DNA. Fraction8 in the slower in the 26 S peak were pooled, made sedimenting half of the 20 S peak and all the fraction8 0.4 M in NaCl and the DNA was precipitated with 67% ethanol at - 20°C. The 26 S material, which contains the 0.46 X length fragment, was again heated, fractionated through a sucrose gradient and precipitated with ethanol. The ethanol precipitate8 were resuspended in 0.1 M-KCl, 5 mM-EDTA, pH 8.0. The purity of the 2 DNA samples wae controlled by electron misroscopic examination for which they were prepared according to Davis et al. (1971). The distribution8 of lengths of the 26 S fragment and the 20 S fragment are given in Fig. 1. As can be seen the 26 S material contains DNA of the 0.46 h length piece, which has the left cohesive end of h (Allet et al., 1973). No DNA of the other R, cleavage product8 of A is evident. The lower panel of Fig. 1 shows that the 20 S material contains DNA pieces of 4 different lengths. 32% (by weight) of the molecules are of 0.07 /\ lengths; this is the fragment with the right

PROCESSING

OF

167

h DNA

cohesive end. The 20 S material does not contain DNA longer than 0.15 A lengths; therefore it is probably free of DNA containing the left cohesive end. The presence of the other internal R, cleavage products in the 20 S preparation will not interfere with the following constructions.

(e) Construction of partial

X DNA polymers

Molecules of X DNA were prepared which contain one complete monomer plus part of another monomer covalently linked through the cohesive ends to give co8 sites. Tho preparations of the 26 S and 20 S R, cleavage products were used for this purpose. The overall pathway for construction of these molecules is given in Fig. 2.

I

26Sfraction

4

0

0.10

0.30 0.40 of A)

0.20 Lengthcfraction

FIG. 1. Distribution of lengths of or 20 S fraction (lower panel). The prepared according to Davis et al. aration. Bacteriophage PM2 DNA

h DNA R1 restriction fragments in the 26 S fraction (top panel) lengths of the DNA were measured from &ctron micrographs No DNA longer than 0.6 h lengths w&s seen in the 26 S prepoircles were used as the length standard.

Left

RI RIRIRI RI R 1 I I I I

A

Right

D!gestwithRIenryme separate fragments

/

\

A

‘9

26Sfragment Anneal -.- A

to unit length

20s monomer

f rogment

-.-.- R - .-.-.-.

._._._._._. -.-.._._ and seal with

ligase

I

!

Left

0.50

-.-. -.-.-.!?s-!.-.-.-ti-.-.~

.-.k.._._._._._ .-.-.-.EC& -.-.-.-.-.I AnnealtoZOSfrogment seal

wth

Anneal Ilgase

to26S

.-._._.-

.__

R,ght

fragmeni /

\

-.-.-. RcosA

-R ms’n -.-. - .-.-.-. --.-.-.-.-.-.-.-.-.

’

III

FIQ. 2. Outline for the synthesis of the partial polymers. Beginning at the top, X434 DNA4 is digested with E. wli R1 endonuclease and the 20 S and 26 S fragments are separated. These are then annealed to full length r\434 monomers through the cohesive ends (designated by ) and then covalently sealed with ligase to give structures I, II or III. The locations of genes A and R are shown for reference to the genetio map.

M. SYVANEN

168

Full length &mm434 DNA at A2s0 = 1.0 was heat’ed at F5”C for 10 mill to m(llf iur> annealed cohesive ends. Five ~1 of this solution were addod either to 50 1~1of t11c: ?(I 5 preparation (A,,, = 0.55), to 50 ~1 of the 26 S preparation (9 260 = 0.42) or, as a control. to 50 ~1 of buffer which contained no DNA, at 48°C. The buffer contained 0.13 or-l addition of EDTA to 9 IIIM and by heating at 65°C for 5 min. The 20 S preparation when reacted with full length monomers should give a molecule with a free right end and a cos site at the left, i.e. structure (II) in Fig. 2. \Vith the 26 S preparation the final product will contain the unit longth monomer with a free left end whose right end is in a cos site, i.e. structure (I). When whole length monomers are :nmoalcd in the absence of any fragments, the predominant structure formed should be monomer circles; the concentration of whole length DNA in any of t,hese annealitlg mixturtas w+ls too low to form bimolecular or higher aggregates (Wang & Davidson, 1966). Tlie DNA and the ratio of linear in the 3 preparations was examined in the electron microscope, molecules of about unit length to circular forms was measured. The monomer prepara,tion that had not been reacted with any fragment had 5776 of its molecules as circles. The 430/ linear molecules may have had damaged ends that could not bc joined by the DXA ligase. On the other hand, when the preparations that were submit.ted to tllch proc~ctlur~, to construct molecules (I) and (II) were examined in the electron microscope, only 99; of the molecules in each sample was circular, the remainder were linear molecldes of about unit length. This shows that the cohesive ends of the unit length monomers lvcr‘o blockc~1 by exposure to either the 20 S or 26 S DNA preparation. A partial polymer that contained no cohesive ends and two cos sites, structure (Ill), Fig. 2, was constructed 2 ways. In one, (I) was annealed and joined to the 20 S fragment ; in the other (II) was annealed and joined to the 26 S frqment.

3. Results The extract time

when

The phage

for in vitro DNA

phage

production

assembled

packaging is occurring

is derived

from

at maximal

in vitro can be distinguished

a heat-induced

rate

from

(Kaiser

those

which

lysogen

& Masuda, derived

at a 1973).

from

the

Phago particles made Crb vitro that contain himm434 DNA will make a plaque on a h lysogen, whereas phage with h DNA will not. The aim of this paper is to determine the structure of the &mm434 DNA that packages into phage. induced

h lysogen

by adding

&mm434

DNA

to the extract.

(a) DNA

polymers

are precursors

to packaging

In Table 1, it, is shown that molecules which are found in phage particles, the linear monomers with cohesive ends, are not an efficient substrate for head assembly. DNA whose ends are cohered to give covalently closed circular monomers are even less efficient in the assembly of phage. The best substrate tested is a polymer formed by the joining of several molecules through their cohesive ends (Syvanen, 1974). In Table 1 the linear monomers were prepared by selectively melting the cohered ends of the high activity annealed polymeric DNA. When the cohesive ends of these linear monomers

PROCESSING

169

OF h DNA

were allowed to reanneal the high activity returned. It may be concluded then that, the precursor DNA to the head packaging reaction is contained in a polymer and that the conditions used to melt and reanneal the cohesive ends do not interfere with the high activity. (b) DNA polymers have covdently

closed cos sites

The enzyme E. coli DNA ligase is present in the packaging extract. Because this enzyme will covalently seal the cohered h ends, it was of interest to see if such closure TABLE

Polymeric

1

DNA is preferred substrate for in vitro DNA packaging and head assembly x434 PhqUeB per 0.1 /~g DNA

r\434 DNA Annealed polymers (1) Linear monomers (2) Reannealed linear monomers to give polymers Circular monomers (4)

14,000 60 9800

(3)

3

(1) The annealed polymer DNA was derived from phage particles and stored at a concentration of Aaso = 6.2. (2) The linear monomers were prepared by heating the annealed monomer DNA at 65°C for 10 min immediately before it was added to the extract. (3) The DNA from (2) at a concentration of AZBO = 5.2 w&s incubated at 46°C for 2 h. (4) Circular monomers were isolated from infected cells according to Wang (1969). The cohesive ends are covalently closed in this preparation. The plaques from the circular monomers arise after the 434 DNArecombines in the extract (Syvanen, 1974). Concentration of exogenous circular DNA in the extract is 2 pg/ml.

Untreated O-

DNA *

0.5 NMN concentration

I.0 (mu)

,

FIG. 3. Effect of nicotinamide mononucleotide on in vitro DNA packaging and head assembly. The concentration of NMN added to the extract is shown. The inam plaques are normalized to 100% when no NMN is added to the extract for either the annealed polymeric DNA, untreated with DNA ligase (-A-A-) or for the DNA sample which had been reacted with NAD and DNA ligase (-O-O--). The NMN was added to the extract at 0°C 6 min before any DNA was added. For the untreated DNA, 100% corresponds to 24,000 plaques/O.1 pg DNA. For the ligated DNA, 100°/c oorresponds to 74,000 plaques/O.1 Irg- DNA. The difference in maximal activity is due to variations in the extract. When the untreated and ligated DNA are added to portions from the same extract no difference in their activity is observed (see Table 2).

hl . S Y \’ A N 15N

170

was necessary for packaging. The extract ligase was therefore blocked with nicotinamide mononucleotide (NMN), a known inhibitor of this enzyme (Olivera & Lehman, 1967). As can be seen in Figure 3, NMN causes a pronounced inhibition in the number of plaques when annealed polymeric DNA (designated as untreated DNA) is used as the substrate. On the other hand, when this DNA is reacted with NAD and purified E. coli ligase prior to its addition to the extract (designated as ligated DNA), the NMN does not cause the pronounced inhibition of the packaging reaction. Thus, NMN does not inhibit the assembly reaction per se. This result shows that it is necessary for the annealed polymeric DNA to react with DNA ligase. This reaction may occur either in the extract or prior to its addition. It is most likely that the ligase covalently seals the cohesive ends. To show that the ligated DNA used in Figure 3 did have sealed ends it was examined in the electron microscope. The DNA was made 40% in formamide prior to attaching the DNA to the electron microscope grid, a treatment which dissociates cohered ends but not the rest of the DNA duplex (Davis et al., 1971). When the DNA which had not been reacted with DNA ligase was examined, all of the molecules appeared as unit length monomers, as expected. With the ligated DNA, on the other hand, 11 of 24 molecules measured were greater than unit length. In another test the DNA preparation was heated at 65°C for 10 minutes, a treatment which dissociates the cohered ends of h DNA. This causes inactivation of the annealed DNA preparation in the DNA packaging assay but the ligated DNA should be unaffected by such treatment, which is the observed result as is shown in Table 2. When the two preparations are both heated and added to an extract that contains NMN the ligated DNA is still active whereas the unligated sample now has only 0.1 y. of its original activity (Table 2). These results show that the ligase did in fact seal the cohesive ends and the most straightforward explanation is that this covalent closure is a necessary requirement for a DNA to be packaged. (c) Packaging To further series of partial

from polymer

DNA

is asymmetric

charact.erize the DNA structure that is precursor to head assembly a polymers was constructed. These were molecules which contained one TABLE

Precursor

2

activity of polymeric DNA which had been reacted or not with NAD and E. coli DNA Eigase

Conditions for DNA packaging Normal protocol Heat DNA Add NMN to extract Heat DNA and add NMN to extract

434 plaques per 0.1 pg of DNA when polymers are Ligated Not reacted with ligase 12,200 14,400 11,200 9500

17,500 200 800 28

To 60 ~1 of extract is added 0.6 pg of either the ligated or untreated DNA. When heated the DNA is incubated at 65°C for 10 min immediately before it is added to the extract. The extract is made 0.6 mm in NMN, where indicated, 5 min before the DNA is added.

PROCESSING

OF

h DNA

171

complete Ximm434 monomer plus a fragment of A DNA that had either one or the other end. These are the molecules shown in Figure 2 which are designated as structures (I), (II), or (III). Their synthesis is given in Materials and Methods and outlined by the scheme in Figure 2. The relative activities of these different DNA molecules in the DNA packaging and head assembly assay are shown in Table 3. The monomer that contains either a left cohesive end with a cos site on the right (structure (I)) or one that has cos sites on both the left and the right (structure (III)) are dicient precursors for heads assembled in extracts. However, the partial polymer with the right cohesive end, structure (II) packages no more e&Gently than the linear monomer. This resdt demonstrates an asymmetric property for the DNA precursor in the in vitro DNA packaging reaction. A unit length monomer with a free left end in a polymer will package whereas one with a free right end will not. TABLE

Packaging

h434 DNA

of DNA

preparation

Linear monomer Circular monomer 20 S Ri fragment 26 S R, fragment Structure (I) Structure (II) Structure (III) (1) Structure (III) (2)

3 is asymmetric h434 plaques per 0.1 /~g DNA 310 40 0 4 7800 460 5400 1950

To 50 ~1 of extract 1 to 5 ~1 of DNA were added. The linear monomers were prepared by heating at 66°C for 10 min h DNA isolated from phage. This preparation of DNA gave 20,000 plaques/O.1 rg prior to melting. The 20 S and 26 S DNA are from the same samples used in the preparation of structures I, II and III. In the calculation of the relative activities for structures I, II and III the oonoentration of unit length DNA is used, that is the concentration of the 20 S and/or 26 S DNA present in these mixtures is subtracted. (1) Struoture III was oonstruoted by annealing structure I with 20 S R1 fragment and (2) st~cture III was aonstructed by amealing structure II with 26 S R1 fragment (see Fig. 2).

The specific activity of DNA molecules I and III in Table 3 is reduced about fivefold over the activity of the annealed and aggregated DNA shown in Table 1. An explanation for the low activity in those two preparations is that much of the DNA has been damaged by the repeated manipulations required in the synthesis of these partial polymers. Then, one may argue that the molecule with the free right end was damaged more than the one with the free left end, thus accounting for the differences in the activities between structures I and II. This is probably not the case since when structure II is reacted with the left ended 26 S R, fragment structure III is obtained. As is shown structure III prepard in this way has four times the activity of its parent molecule structure II. This shows that in the structure II preparation the free right ends are still competent.

172

hf. SYVBNEN

Another possible experimental artifact that may account for the much lower activity of molecules that contain a free right end is that right ends are degraded rapidly by nucleases in the extract whereas left ends are resistant to degradation. This possibility has been ruled out. If structure II is annealed to a 26 S fragment (but not covalently sealed by ligase) its right end is protected. When this molecule is tested in the DNA packaging reaction (in the presence of NMN, so the ligase endogenous to the extract does not seal the ends) it has no more activity than structure II. NMN in the extract does not block the precursor activities of structure I or 111 (data not shown). I would like to acknowledge Ray White who suggested the use of the purified R, fragments to construct the partial polymers used in these experiments.

4. Discussion My major findings on the precursor structure of bacteriophage h DNA that will give viable phage particles in vitro are summarized as follows. (1) The mature h DNA molecule isolated from infectious particles, the linear monomer, is incompetent to package in in vitro head assembly. The active substrate from which a X monomer is packaged is a polymer held together through the annealed, single-strand ends (Table 1). It therefore appears that the free single-strand ends of the linear monomer are formed when DNA is packaged. (2) The high activity polymers must have covalently closed cos sites before they will package. This means that not only is the DNA cut to produce cohesive ends at the time of packaging but that this cutting is obligatory to packaging. (3) The packaging of monomers from polymers is asymmetric. It is shown that a unit length monomer may be packaged from a polymer where, either both of its ends are contained in cos sites, or where it has a left cohesive end with its right end in a cos site. The molecule with a free right end packages less efficiently. Thus, either just a cos site on the right or two cos sites are cut during packaging. It is not surprising that DNA is packaged from polymers during in vitro head assembly. In the infected cell if X head assembly is blocked by mutation in any of the steps prior to DNA packaging, the h DNA will accumulate as polymeric structures (Mackinlay & Kaiser, 1969). In pulse-chase experiments it has been shown that polymers are immediate precursors to cut DNA (Smith & Skalka, 1966). These results imply that A DNA polymers are precursor to packaging and that cutting occurs during head assembly. Szpirer $ Brachet (1970) showed that a h DNA monomer could not be packaged unless it contained two cos sites. Stahl et al. (1972) and Enquist & Skalka (1973) showed that infectious phage were not made in cells which accumulate X DNA as circular monomers, even if the proper proteins were present. However, if a DNA recombination pathway were available which allowed circular monomers to recombine with themselves to produce polymeric structures, then this DNA became packageable. On the other hand when a circular X monomer contained a tandem duplication of the cos region, Feiss $ Margulies (1973) showed that the DNA between the two cos sites would package. These results have been summarized to say that an infectious monomer is packaged between two cos sites. This conclusion is supported by the data given here (Table 3) (also Freifelder et al., 1974). R ecently Hohn & Hohn (1974) have

PROCESSING

173

OF h DNA

shown that monomeric units from polymeric X DNA can be packaged into particles in lysates from infected cells. The fact that a DNA molecule with a free left end and a cos site on the right will package (while one with a free right end will not), suggests that packaging of DNA into head protein occurs from the left toward an uncut cos site on the right. The asymmetry in packageable DNA does not necessarily establish this polarity of packaging. However, it is consistent with other observations made on h. Emmons (1974) has analyzed the segregation pattern of a tandem duplication mutant of X that has two cos regions. He was able to conclude that h DNA monomers were frequently packaged beginning from a free left end going to the right. Some of the monomers were packaged from within two cos sites, in an analogous manner to that shown here. From studies on completed particles, Padmanabhan et al. (1972), Chattoraj & Inman (1974) and Thomas (1974) have shown that the right cohesive end of the DNA molecule is poised at the head-tail junction. Since the DNA injects through this opening it is presumed that the right end of the DNA leaves the head first. If the right end leaves first during injection then during assembly the left end is likely to enter first. This is entirely consistent with Emmons’ (1974) analysis and my own results presented here. These results fit the following model: the head proteins recognize a site near the left end of the DNA molecule and the molecule is packaged until the cos site on the right is reached. The Ter nuclease (Yarmolinsky, 1971) then cuts to encapsulated produce the cohesive ends and the complete monomer is irreversibly into head protein. One conclusion from these considerations is that the substrate for the Ter nuclease is more complicated than the specific DNA sequence contained in cos. The cos site is cut only when the unique DNA sequence in cos is properly aligned wit.hin a specific stereochemical environment defined by a eapsid protein uncut DNA assembly complex (see accompanying article by Kaiser et al., 1975). When two cos sites must be cut to package a monomer from the interior of a polymer (see structure 111, Table 3) it is clear that each cos site will have a different spatial relation t,o the capsid-DNA assembly complex. Wang & Brezinski (1973) have proposed that two cos sites are cut when they are aligned along an axis of 2-fold symmetry.

Even though

the overall head protein-DNA complex is asymmetric, there are local 2-fold symmet,ries of major capsid protein in the shell. The icosahedral model (Caspar $ Klug, 1962) for the protein arrangement in h heads contains dimers of the h E protein (Williams & Richards, 1974). Thus it is suggested here that during packaging of DNA two cos sites and two molecules of Ter nuclease align with one E dimer. These elements taken

alone define a local 2-fold

axis of symmetry

normal

to the surface of

the capsid. The cos sites are then cut from this structure. I would like to thank Dale Kaiser for his support and useful suggestions made in the course of this work. I am grateful to Drs Scott Emmons, Ray White, Dave Freifelder and Richard Calendar for helpful discussion during the research and preparation of this article. This investigation was supported by a Fellowship from the California Division of the American Cancer Society and by Public Health Service grant no. PHS-A-I-04509-13. REFERENCES

Allet, B., Jeppesen, P. G. N., Katagiri, K. J. & Delius, H. (1973). Nature (London), 241, 120-123. Caspar, D. L. D. & Klug, A. (1962). Cold Spring Harbor Symp. Quant. Biol. 27, l-24. Chattoraj, D. K. & Inman, R. B. (1974). .7. Mol. Biol. 87, 11-22.

174 Davis,

M. SYVANEN

R. W., Simon, M. & Davidson, N. (1971). In Methods in Enzymology (Grossman, L., ed.), vol. 21, pp. 413-428, Academic Press, New York and London. Emmons, S. W. (1974). J. Mol. Biol. 83, 511-525. Enquist, L. W. & Skalka, A. (1973). J. Mol. Biol. 75, 185-212. Feiss, M. & Margulies, M. (1973). Mol. Gem. Genet. 127, 285-295. Freifelder, D., Chud, L. & Levine, E. E. (1974). J. Mol. BioZ. 83, 503-509. Gellert, M. (1967). Proc. Nat. Acad. Sci., U.S.A. 57, 148-152. Hedgpeth, J., Goodman, H. M. & Bayer, H. R. (1972). Proc. Nat. Acad. Sci., U.S.A. 69, 3448-3452. Hohn, B. & Hohn, T. (1974). Proc. Nut. Acud. Sci., U.S.A. 71, 2372-2376. Kaiser, D. & Masuda, T. (1973). Proc. Nut. Acad. Sci., U.S.A. 70, 260-264. Kaiser, D., Syvanen, M. & Masuda, Y. (1975). J. Mol. Biol. 91, 175-186. Mackinlay, A. G. & Kaiser, D. (1969). J. Mol. BioZ. 39, 679-683. Modrich, P. & Lehman, I. R. (1973). J. BioZ. Chem. 248, 7502-7511. Olivera, B. M. & Lehman, I. R. (1967). Proc. Nut. Acud. Sci., U.S.A. 57, 1700-1704. Padmanabhan, R., Wu, R. & Bode, V. C. (1972). J. Mol. BioZ. 69, 201-207. Smith, M. G. & Skalka, A. (1966). J. Gen. Physiol. 49, no. 6, part 2, 127-135. Stahl, F. W., McMilan, K. D., Stahl, M. M., Malone, R. E. & Nozu, Y. (1972). J. Mol. BioZ. 68, 57-67. Syvanen, M. (1974). Proc. Nat. Acad. Sci., U.S.A. 71, 2496-2499. Szpirer, J. & Brachet, P. (1970). Mol. Gen. Genet. 108, 78-92. Thomas, J. 0. (1974). J. Mol. BioZ. 87, l-10. Wang, J. C. (1969). J. Mol. BioZ. 43, 263-272. Wang, J. C. & Brezinski, D. P. (1973). Proc. Nat. Acad. Sk., U.S.A. 70, 2667-2670. Wang, J. C. & Davidson, N. (1966). 15, 111-123. Williams, R. C. & Richards, K. E. (1974). J. Mol. BioZ. 88, 547-550. Wu, R. & Kaiser, A. D. (1967). Proc. Nat. Acud. Sci., U.S.A. 57, 170-174. Wu, R. & Taylor, E. (1971). J. Mol. BioZ. 57, 491-511. Yarmolinsky, M. B. (1971). In The Bacteriophage Lambda (Hershey, A. D., ed.), pp. 97-111, Cold Spring Harbor Press, New York. Young, E. & Sinsheimer, R. (1968). J. Mol. BioZ. 33, 49-59.