J. Mol. Biol. (1985) 186, 759-771

Processive Action of Terminase During Sequential Packaging of Bacteriophage a Chromosomes

Michael Feiss, Jean Sippy and Greg Miller

Department of Microbiology University of Iowa, Iowa City, IA 52242, U.S.A.

(Received 12 April 1984, and in revised form 1 ,4ugust 1985)

Bacteriophage L chromosomes are packaged in a polarized, sequential fashion from a mult’imeric DNA substrate. Mature chromosomes are generated when terminase introduces staggered nicks in the cohesive end sites (cos sites) bounding a chromosome. Packaging is polarized, to the initial and terminal cos sites for packaging a chromosome can be defined. To initiate packaging, terminase binds to cos at cosB, and subsequently cuts at COSS. To terminate packaging of a chromosome, a functional cosB is not required at the terminal cos. To explain this finding, it was proposed earlier that terminase scans for the terminal COSLV. rather than any subsequent cosB, during packaging.

In the work described here we performed helper packaging experiments to see whether processive action of terminase occurs during sequential packaging of L chromosomes. The helper packaging experiments involve trilysogens; strains carrying three prophages in tandem. Infection by a hetero-immune helper phage results in packaging of the repressed prophage chromosomes, since the prophage structure is analogous to the normal DNA substrate. Two chromosomes can be packaged from between the three cos sites of the prophages of a trilysogen. Both chromosomes are packaged even when the central cos is cosB-. Our interpretation of these data is that (1) terminase is brought to the central cos by packaging; (2) f 11 o owing cleavage of the central cos, the terminase remains bound to the distal chromosome; and (3) terminase acts to begin packaging of the distal chromosome. The frequency at which terminase reads across the central cos to initiate packaging of the distal chromosome is in the range from 0.3 to 0.5 in our experiments. Reading across cos was found not to be greatly dependent on the state of cosB, indicating that cosB binding is only needed for packaging the first chromosome in a packaging series. A multilysogen was constructed in which the initial cos was cos+ and the distal cos sites were all cosB-. The initial and downstream chromosomes were found to be packaged. This result indicates that terminase that is brought to the central cos by packaging is not only able to initiat,e packaging of a downstream chromosome, but, can also scan and terminate packaging of the downstream chromosome.

A model is presented in which processive action of terminase is the basis for sequential packaging of 1 chromosomes.

1. Introduction

The bacteriophage I chromosome is a 48.5 kbt linear duplex DNA molecule (Sanger et al., 1982). At t,he 5 ends of the molecule are complementary single-stranded segments, 12 bases in length, called cohesive ends. Upon injection of a L DNA molecule into a cell, the cohesive ends rapidly anneal, circularizing the molecule, and the nicks are sealed by host ligase. At early times in the growth cycle, bidirectional replication generates a poll of unit

t Abbreviations used: kb, lo3 base-pairs; cos, cohesive end site; eosN and cosB, the terminase nicking and binding sites, respectively; 21, lambdoid phage 21.

length circles (Schniis & Inman, 1970). At late times after infection, the rolling circle mode of replication predominates and produces mult,ichromosomal lengths of DNA called concatemers (Takahashi. 1974; Bastia et aZ., 1975). Concatemeric DNA is the substrate for DNA packaging during virus assembly, in contrast to monomer circles, which are not (for reviews. see Skalka, 1977; Furth & Wickner, 1983).

At the time of DNA packaging, the infected cell contains both bacterial and phage DNA, and the phage DNA is specifically recognized for packaging. The recognition step is the binding of a specific site on the 1 DNA by terminase, an enzyme encoded by the Nul and A genes of 2. The site of action of

759 0 1985 Academic* Press Inc. (London) Ltd.

760 M. IGiss, J. Sippy and 0. Miblrr

cod (a) - c7ff ____w3 N&A Ott R cosi

Off u --

N+8+ N+B+

I w

(b)

(cl

COS~ZI cod --- ---

N+ B- N+ B+

t \/ A w

--- - --

N+ B+ N+ E-

Cd) --__w7 lr- -- N+ B+ N+ 6’- N+ B+

P

cash. c42r cm+ 21

(e) -- --

N’ f3+ A,+ 8- P

n/r e-

I I

Figure 1. Tandem prophage structures for helper packaging studies. (a) Prophagr structure of a i. dilysogen. Tht~ :! prophages are bounded by att sites. The cos sites are drawn to indicate the nicking (:V) and binding (H) sites. Thr arro\b shows (1) the chromosome that is packaged when a helper phage provides a /( packaging system. and (2) thqa polarit? of packaging. (b) Prophage structure of a dilysogen in which the initial cos is from phage 21 (coG#J%~) and the terminal sitr is cosl. The filled box indicates cosB of phage 21. which differs in specificity from cosB of i.. and hence is COSK for thr i packaging system. The crossed-out arrow indicat,es the inability of a helper phage with d packaging specificity to package the chromosome, because of the cosB defect. (c) Prophage structure of a dilysogen in which the initial site is r~sl and the terminal site is ~0421. The arrow indicates the chromosome that is packaged by a i.-specific- helper. a r,esult from previous studies that indicates that cosB is not required to terminate packaging (Feiss et al.. 1979: Feiss N: LVidnrr. 1982). (d) Prophage structure of a trilysogen in which the central site is ~0~421. The left arrow shows the first chromosome that can be packaged by a I-specific helper; it is analogous in structure to the packageable c’hromosom~~ in (c). The right arrow indicates the chromosome that could be packaged by a i,-specific helper if the terminase that cleaves the central cos to terminate the left chromosome also initiates packaging of the right chromosome. The question mark indicat,es that packaging of the right chromosome is an experimental question. (e) Prophage structure of a trilysqyn it1 which the initial cos is cosl and the distal sites are ~0~421. The left arrow shows the first chromosome that c-an hr packaged hy a i-specific helper. The right arrow indicates t,he chromosome that could be packaged by a i-specific helper’ if t,he terminase that cleaves the central cos initiates, sc’ans and terminates packaging of t,he downstream chromosome. The question mark indicates t,hab packaging of the right chromosome is an experimental question.

terminase. called co9 includes the cohesive end sequence, and during packaging terminase introduces the staggered nicks that generate the cohesive ends. It has recently been found that’ COR consists of a terminase binding site (co&) as well as the nicking site (cosN), as shown in Figure l(a) (Miwa 8: Matsubara, 1983; Feiss et al., 1983). The binary complex of terminase with ;1 DNA has been detected in vitro by Becker et aE. (1977). The binary complex, called complex 1: was shown t,o bind a prohead to generate a t’ernary complex called complex IT (Becker et aZ., 1977). Terminase is thus multifunctional, it bonds cosB, nicks COSLV and binds proheads. The point during packaging at which nicking of the initial cos occurs is at present unclear. Terminase is required for the packaging of nicked 1 DNA; thus one obvious

function for t,erminase during pa,ckaging oft nicked DXA is for prohead binding t,o form complex 11 (Hohn, 1975: Becker & Gold! 1978).

The packaging process is asymmetric-, prot*eedirrg from the Nul-A (left) end of the DNA to the R-Ra (right) end (Emmons, 1974: Feiss & Bublitz. 1975: Sternberg & Weisberg, 1977). The basis fol asymmetric packaging is presumed to lie in the asymmetric structure of COR (Feiss et rcl., 1983). Packaging is also processive, so that a /‘OS cleaved to terminate the packaging of one chromosome also serves as the initial cos for packaging the next (Emmons, 1974; Feiss & Bublitz. 1975). The level of processivity was est,imated at two t)o thrt~ chromosomes per packaging sequence by Emmons (1974).

Because packaging is asymmetric. it is possible t’o

Processive Action of I Terminase 761

define an initial cos at which packaging of a particular chromosome starts, and a terminal cos at which packaging terminates. We have reported helper packaging experiments in vivo to ask about the role of cosN and cosB at the initial and terminal positions (Feiss et al., 1979). In a helper packaging experiment, a host carrying two tandem, repressed prophages is infected by a hetero-immune helper phage. The helper phage carries out lytic growth, providing packaging components. The tandem prophages remain repressed, and are a passive packaging substrate. A chromosome may be packaged from between the cos sites of the two prophages, since the structure is analogous to a chromosome in a concatemer (Fig. l(a)). Helper packaging experiments are done under recombination-blocked conditions, to prevent, recombination between the helper and the prophages. When a chromosome is packaged from tandem double prophages, it carries the Nul arm of the first prophage and the R arm of the second prophage. When t’he prophages are marked genetically. the orientation of the prophages can be determined from the genetic constitution of the helper-packaged chromosome.

Lambdoid phage 21 has head genes that are analogous to those of 1 in structure, position and function (Siegele et al., 1983). Phages L and 21 have diverged sufficiently so that the recognition specificities of the head gene products are different. For example, phages 1 and 21 will not package each other’s DNA molecules (Hohn, 1975; Feiss et al., 1979). We have reported helper packaging experiments in which ~0~421 was used in the initial and terminal positions to assess the role of cosB in packaging (Feiss et al., 1979: diagrammed in Fig. I (b) and (c)). When the helper phage provided the 1 packaging system, it was found that co&21 was functional in the terminal position. Thus, cosq521 is cosN+ as a substrate for L t’erminase. When ~0~421 was present in the initial position, packaging did not occur. We have subsequently found that L t,erminase does not bind ~0~421, so with respect to 2 terminase ~0~421 is cosN+ cosB- (Feiss & Widner, 1982). The ability of ~0.~421 to function in the terminal position was explained by scanning: that is. the terminase that cleaves the terminal cos binds to the initial cosB and scans a chromosome during packaging until the terminal cosN is encountered. Hence there is no requirement for a functional cosB to terminate packaging at the terminal cos. This suggested that scanning provides a way for terminase to encounter cosN that is an alternative to cosB binding.

We have now ext’ended the helper packaging study to ask whet’her terminase that has scanned for and cleaved a COSN’ cosB- terminal site can function in initiation of packaging of the eosB- chromosome. Can scanning result in a bypass of the cosB+ requirement so that packaging of the cosN+ cosB- chromosome occurs? The experiment requires the construction of a trilysogen with the cos sit’es arranged cosl-+cos~#~2l -+cosA, as diagrammed in

Figure l(d). In the Figures of this paper, structures will be oriented so that packaging goes from left to right. The left packageable prophage chromosome will be called chromosome 1 and phages carrying this chromosome, 41 particles. The right or downstream chromosome will be called chromosome 2 and phages carrying chromosome 2 will be called ~$2 particles. In a second experiment we have asked whether terminase that has scanned for and cleaved a cosN’ cosB- terminal cos can both (1) initiate packaging of, and then (2) scan. the downstream chromosome. This experiment requires the con- struction of a multilysogen (tri or higher order) with the cos sites arranged cosA-+ (cos$21), as diagrammed in Figure 1 (e).

2. Materials and Methods

The strains used are listed in Table 1.

(b) Marker tests

The following is a list of the tests used t’o score phage markers.

(1) imm. The immunity of a phage was scored by spotting or printing a resuspended plaque onto lawns of Escherichia COli C600(1+), C600(iim~m21) and C6OO(limm434).

(2) biol. Phages carrying the biol substitution were identified as spi-; that is, able to grow on C6OO(P2) (Zissler et a,/.. 1971b).

(3) red+. red+ phages were scored as able to grow on the polA- strain *MF894.

(4) red3. Phages carrying the red3 mutation were those scoring as spi’ but unable to grow on pol.V bacteria (Zissler et al., 1971a).

(5) Pam902. The Pam902 mutation was scored as enabling a phage to grow on the supE’ yrol’A15 strain, MF208 (Georgopoulos & Herskowitz. 1971).

(6) guZ8. The marker was scored by spotting phages on a lawn of AB1157 (a supE gaZK2 strain) on an EMB- galactose plate: phages carrying gal8 generate pink Gal’ transductants.

(7) DNA packaging specificity. The packaging specificity of helper-packaged chromosomes was determined by identification of the first trrminase gene (Nuz or I). The specificity was determined by complementation tests with lNul- and 1-21 hybrid 33 I-. A phage with 1 specificity complements INul- but not 1-21 hybrid 33 l-, and a phage with 21 specificity can complement 1-21 hybrid 33 I- but not LVul- (Feiss et al., 1979; Siegele et al., 1983). A second packaging specificity test employed was the ability to grow on a himA - host. K5282. Phages with 21 packaging specificit? are unable to grow. but phages with I packaging specScIty can grow (Feiss et al.. 1985).

(8) Aam32, Aaml21 and 2am149. These markers were scored by genetic crosses of each phage under study with Mam32. &lam121 and A-21 hybrid 19 %am14!).

(9) am+/am-. Phages unable to grow on sup+ (non- suppressing) cells (e.g. 594) were scored as am-.

(10) b+ bill 1. b+ phages are inactivated to a survival level of <O.lO;,, bv incubation in 0.01 M-EDT.\ for 10 min at 42°C. and b511 phages show > IO”,, survival (Parkinson & Huskey, 1971).

762 M. E’eiss, J. Sippy and C?. Jfilk~

Table 1 Strains

Strain Remarks Sourre~reference ------_-..

A. Escherichia coli K12 derivatiws

HS331 Aamlll, thy- derivative of It901 H. Sh~/.u\-ai(:arnI,bell et trl. ( l!IciQ) KL-16QQ rrcA I Hfr, t.ransfers rcc,-l 2 early Low ( 1 Qb%) MF1343 Dilysogen This work MF1362 Trilysogen: cosl+cos@Zl +cosi This work MFl363 Trilysogen: cos~-~os~-+~os~ This work MFl774 Multilysogen : co8A+ (co.xj%l ). This work ( :(ioo WApE Campbell (1961) MFX94 supE, p&591, gal- Our collrc%ion C”600( 1’2) .ayE, P2 lysogen Our collec~tion (‘600 (&mm434 fh82) s1tpE Our collection R594 SllJf (‘ampbell (1961) M F208 gyoPAl5 derivative of (I600 Georgopoulos B Hrrskowitz ( IQ7 1) MF327 s11pF Our collection KS282 .wpF himdA82 H. 1 Millel MF713 b511 recA1 (gal-IS) deletion derivative Shapiro & Atihya (lQ6Q):

of AM3100 (A-1857) Friw B Siepele (1979) -.- __--

13. Bnetwio~hages

i.g& hiol iwm21 Pam902 Used for construction of MF1343 ()ur collrct~on icI26 b53X rrd3 Helper phage, used to analyze MF1343 Our collection i-21 hybrid 33 2am 149 ind- [ised for construction of MF1362 ()ur collection iAam32 in& ITsed for construction of MPl363 Our collection AC126 ti21 red270 Phage for selecting lysogens Our collect ion i.-21 hybrid 19 b53X 21 -specific helper phage This work

red3 imm434 CI p4 ib53H red3 imm434 cl p-l i.-specific helper phage Thih work IAaml21 gal8 41 This work lAam32 biol imm21 Pam902 ~$2 of MF1363 This work E.-P1 hybrid 33 Zaml49

hi0 1 “imm2 1 42 of MF1362 This work

Pam902 1-21 hybrid 33 Iam red3 This work

imm434 cI 1Aam32, L4am121: ( ‘ampbell (IQ61 ) A-21 hvbrid 19 Zam149 Siegelr tt nl. (1983) A-21 hirbrid 51 Iam r&3 ind- Used for construction of MFl774 Frackman rf (I/. (1985)

Tryptone broth (TB). TB soft agar. T13 agar (ail supplemented with 0.01 3f-MgSO,). and 1, broth were prepared as described by Campbell (1961). Eosin-1’. methylene blue plus galactose (EMHG) plates were made as outlined by Campbell et al. (1961). Minimal media plates were made according to Davis & Mingioli (1950). and appropriately supplemented.

(d) Trnnductims and mntin,qs

The transductions and matings u-err done according to Ueary $ Campbell (1972) and Miller (1972). respectively.

(e) Phayrs c~~~~s~s nn,d (:omplrmmtutior2s

Cells were grown in TB, 0.1”,, (w/v) maltose to 108/ml. resuspended in 0.01 M-MgSO, and infe&d with thr 2 appropriate phages at a mult,iplicity of fi phagesjcell for each. After 15 min adsorpt,ion at, room temperature. free phages were inactivated b? incubation with anti-i serum (K = 6) for 7 min. The Infected cells were diluted to 104/ml in TR and incubat,ed for 70 min in a 37°C wat.t-t bath without shaking. After t)reatment with (‘HU,. the Iysate was titered for phage yield. For (Brasses. thr anti-R step was omitted.

Cells were grown t,o 10s]ml in LB. O.%“, maltose. 1 pg desthiobiotin. resuspended in an equal volume of 0.01 M-

MgS0,. and infected at a multiplicity of d helper phi~grs, cxell. After 15 min adsorption at roon1 temperat ur(a. the, cells were diluted to < lOhim in LB anti incubate(l fcjl 90 min in a 37°C water bat,h without shaking. The l\.satt*s were treat,ed with (‘H(‘I,. spun in a11 TE(’ c>linicA czrntrifuge. and plated

For packaging t rilysogerm ib5SX 1~13 i~~~w43~ ~1 p-C was the helper phagr with i, packaging sl)et’itic-it,>-: it will br called the i.-spcitic~ helper. i.-21 hybrid 19 h,?:iri ~d:i imm.434 CI p4 was the helper phagr with 21 parkaging specificity. or the 21 -specific helper. i-21 hytwitl 19 is a i derivative in which the left Aromosome end. inc~lutling all the head genes. ha,s been repla,cetl by 21 11X.4 (Fries I’/ nl.. 1979). Both helpers are defective in the phaye g!cllc~ral (R.ed) and sit,tb-specitic recombination systems (the lattet deleted by h53X) to IJrevrnt, crossing-orrr with ln~~pb;lgc~. Both helpers are 114. ;I substitution that caonfers :I latc, gene expression specificity that, is diRerent. from t,hat of i (Strathern & Herskowitz. 1975). Hence t,he prophagr Iate genes are not transac%ivated in helper packqiny experiments.

MF1343 is a dilysogrn. in the sense t,hat it contains one packageablr cahromosome bounded by 2 cod sit,es. MF1343 was used to prepare the trilysogens as described below-. To ronstrurt MF1343. we start,ed with HS331. which is a drrirat,ivr of t,hr st,rain R901. Figure 2 shows t,hr normal

Processive Action of I Terminase 763

prophage structure of K12(/1+) strains. R901 (Campbell et al.. 1969) is a derivative of K12(n’) deleted for A5061 and A904. shown in Figure 2. The 1 DNA in R901 consists only of the segment extending from a site within Q through cos to a site between the A and B genes. HS331 is simply a derivative of R901 that has a thyA - mutation and the j. Aaml21 mutation. We lysogenized HS331 with iyal8 hi01 imm21 Pam902. The biol substitution contains the bioB gew and confers on Abio strains such as HS331 the ability to grow on glucose minimal medium containing 1 pg desthiobiotin/ml. Accordingly, the lysogens were selected as transductants able to grow on medium containing desthiobiotin. Four transductants were grown up and crossed with KL1699, an Hfr that is thy + recrl 1 cstrS and which transfers recA1 early. Recombinants that were thy+ and sir’ were selected and screened for rrcA 1. The recA1 recombinants were identified as rxtremely sensitive to ultraviolet light. Loopfulls of overnight cultures were spotted on TB plates. given a u.v. dose of lOergs/mm* and incubated. The recA1 strains were completely killed and ret+ strains were not. The wcA1 thy+ derivatives of the 4 putative HS331(jLgal8 biol imm21 Pam902) isolates were used in a helper packaging experiment. Only one of the 4, called MFl343. contained a prophage chromosome t,hat’ was packageable by the helper phage, 1~126 b.538 red3. The yield of phages bearing prophage chromosomes was 0.32 phagelinfected cell. This yield is a typical positive helper packaging result. In addition, the yield reflects fairly efficient packaging of the limited number of prophages per cell. Four phages carrying prophage chromosomes were purified and tested for markers. Each was found to be iAaml21 gal8 bioll imm21 Pam902. From these markers the structure of MF1343 was deduced t,o be as shown in Figure 2. The crossover by which iLga/8 biol imm21 Pam902 integrated into HS331 must have occurred to the right of the Aaml21 marker of HS331. so the Barn121 marker is on the packageable chromosome. MF1343 is useful for the construction of trilysogens of defined structure because it has a single attachment, site (attB), into which an incoming chromosome can insert.

(ii) Thr trilyxogen MF1363, with cos sites arranged: cod -+ cd + cod (Fig. 3)

MF1343 was infected with 1Aam32 imml ind-, and lysogens were selected by spreading the infected cells on a TB plate with 10’ 1~126 b221 red270. Trilysogen candidates were purified and studied by helper packaging. As can be seen in Figure 3? infection of MF1363 with the i-specific helper. 13538 red3 imm434 CT p-l. is rxpect.ed to yield lysates containing the helper phage. particles carrving the left chromosome from the prophage structure. XAaml21 gaZ8 imml, and particles carrying the right chromosome. kAam32 biol imm21 Pam902. Phages bearing the left chromosome from a trilysogen prophage structure, as a result of helper packaging, will be referred to as 41, and phages bearing the right chromosome will be referred to as 42. The helper phages are in the vast majority in the lysate, and so platings were employed to select phages bearing prophage chromosomes. 41 is red+, and is able to grow on polA - (MF894) cells (with an e.o.p. (efficiency of plating) relative to (‘600. of 16). whereas the helper and 42 are rrd - and unable to grow on polA- cells. $2 is biol, a substitution that, confers insensitivity to P2 inhibition. On C660(1’2). neither the helper nor 41 can grow, but 42 plates with an e.o.1). of 0.5 to 0.6.

Of 4 potential trilysogens that were helper-packaged.

two (isolates 1 and S), yielded phages able to grow on MF894 or CSOO(P2). See Results, section (a) (ii) for yields. Ten plaques on MF894 from each trilysogen candidate were single-plaque-isolated and tested for markers: all 10 were immi gal8 and am-, m addition to being red+. Two of the isolates (from each trilysogen candidate) were subjected to crosses t,hat showed each was ilam121. /I:r concluded that the red+ phapes have the genot,ype expected for 41.

Similarly, the lysates from trilysogen candidates I and 8 were plated on CSOO(P2) and from each 10 phages were purified and tested for markers. Earh was found t,o he 1:mmBl and Pam902. in addition to being hi01 Each was non-gal transducing and had a very low revertant frequency, consist,ent with the presence of an amber mutation in addit,ion to the Pam902 marker. Two from each trilysogen candidate were subjected to crosses and found to be dam32 as expected. We concluded t,hat

these biol phages had t,he genotype expected for &!, and that t’he 2 candidates had the st,ructure of MFl363 as drawn in Figure 3.

(iii) The trilysogen MF1362, with ws sitvs nrrung~d: cosi-+cos~21+cosi (Fig. 4)

To make t,his trilvsogen we used i-21 hybrid 33 to provide cos#Bl. Hybrid 33 is an am+ recombinant from a cross of iAam1 lam32 and pBW3. a plasmid containing the left end of the 21 chromosome (from cos to a point beyond the terminase genes: Frackman et., 1984). Hybrid 33 has been found to have 21 packaging specificity (cos$Zl). the 2 gene of 21. and a hybrid %/A gene (hereafter referred to as the 2 gene). The carboxyl 38 amino acids of the hybrid 2/A gene product are from 1: and they confer i prohead specific&v on the terminase. We infected MF1343 with 1-21 hybrid 33 %am149 imml ind-, and lysogens were selected and purified as described. The trilysogen candidates were helper- packaged with /I- and 21-specific helpers. i\s described for MF1363, phages bearing the left and right chromosomes (41 and 42, respectively) can be selected by plating on polA - (MF894) and P2-lysogenic (CSOO(P2)) bacteria, respectively. From the expected structure of MF1362. shown in Figure 4, packaging by the I-specific helper should yield 41. and packaging by the Pl-specific helper should yield 42. Packaging of the right chromosome to give 42 by the i-specific helper constitutes the experimental question: this is discussed in Results section (a) (iii). Of 4 potential trilysogens. 2 (isolates 4 and 5) gave the expected packaging. The 2 lysates produced by infection of isolates 4 and 5 by the A-specific helper were pla.ted on MF894; 10 red+ phages from each lysate were isolated, grown up and t.est,ed for markers. Each of the red’ phages was imml, gal8 and am-. Two from each lysate were shown to be Aarnl21 by phage crosses. We conclude that the red+ phages have t,he genotype expected for $1. The 2 tril?sogen candidates were helper-packaged with the 21.specific helper and t,he lysates plated on CSOO(P2) to select biol phages. Ten biol phages from each trilysogen candidate were isolated. grown up, and tested for markers. Each of the biol phages was found to be imm21. Pam902. non-gal transducing and had a very low revertant frequency, t)he latter indicating the presence of a second am mutation. Two biol phages from each of t’he candidates were shown to carry the Zam149 marker by phage (rosses. We concluded that these biol phages had the genot,ype expect.ed for (52: A-21 hybrid 33 Zam 149 biol imm21 Pam902. and that isolates 4 and 5 had the structure shown for MF1362 in Figure 4.

A5061 Agod

5061 904

lm OS-0

HS331 K-T- R A- A

A gol bio imm21 P-

A’

c

P-

tmm21

bio PO/

764 M. E’eiss. J. Sippy and 0. Miller

K12(k) 901 chl 0ttff b,o chl or*

KTE D N P SR Nul A E J A A

MFl343

5061 904

90’ bio (mm21 _ lm

R A- ott6 P- R A+

A-gal bm imm21 P- I +

Figure 2. Construction of the dilysogen, MFf343. The top line shows the structure of the i. prophape (straight line) nncf adjacent bacterial genes (wavy lines) in a normal K12(L) lysogen. Filled cG%=s indicat.e ws sites. Thv hatcahed segments represent the extents of 2 deletions of HS331. 65061 and A904 (indicated b\- arrows in the HS331 strucaturr). These deletions removed most of the prophage DNA except for a segment including WA. HS331 also carries an dam1 “1 mutation. HS331 was lysogenized by igal Oiol imm21 Pam902 and made ~.-d I to generate MF13-G. MB1343 is a dilysogen in the sense that the prophage structure cont,ains a chromosome t’hat fan be packaged by a L-sprcifk hrlf>rl (arrow). MF1343 contains a single att site. facilitating the const.rurt.ion of trif,vsogens of defined st,ruc?urr.

(iv) J1F17iP. a vnultilysogen with, thr cm sites arranged

rosl-+ (cos~21), (Fig. 5)

The construction started with MF713. a derivative of K f2Q’) deleted for A509. A509 removes a DNA segment that includes the bacterial gal genes, the left attachment site and most of the early genes of the prophage (Shapiro & Adhya. 1969). The prophage DNA not deleted by A509 extends from a site in gene S through cos and the late genes to the right attachment site. The MF713 prophage is marked with the 0511 deletion. MF713 is recA1. MF713 was infected wit,h 1-21 hy 51 Iam red3 ind- (hereaft.er called i-21 hy 51 I-) and lysogens were sefect,ed as described above. L-21 hy 51 is a recombinant between 1 and 21 in which the left 490 base-pairs. which include the amino-terminal half of the 1 gene, are of 21 origin and the remainder is I DNA (Frackman ut al.. 1985). A-21 hy 51 has ~0~~21. We expected that most lysogens would contain a single 1-21 hy 51 I- prophage, but that a few would contain multiple 1-21 hy 51 I- prophages inserted in the MF7 I3 att sit,e. Single and multiple lysogens of L-21 hy 51 I- were identified by helper packaging with the PI - specific, helper. Single lysogens contain only one prophage cahromosome that can be packaged by a helper. This fjrophagr chromosome is Ah511 and it is therefore onI> f)a(-kaged by t,he Z-specific helper, not by the 21-specific helper. On the other hand. MF713 fysogens carrying muftifjle A-21 hy 51 l- prophages (as shown in Fig. 5) do contain one or more prophages that can be packaged by the %I-specific helper: these are 1-21 hy 51 I- rshromosomes.

Of 25 lysogens helper-packaged by the 21 -specitic hrlf)er. 2 (MFf774 isolates I7 and 18) yielded phages with

fjackagrd prophape chromosomes that t’ormed turbid plaques on C16OO. The turbid plaques were all (24 from isolate I7 and 10 from isolate 18) an- LS expected fat 1-21 hy .il lam.5 phages. Ten from each isolate were also trsted for I)+ or h5f I and each was b’. also as rxprctrd. 12’~ concluded t,hat MFfii4 isolates Ii and 1 X \\csrc’ MF7 I3 derivatives carrying 2 or more i-21 hy .‘,I I-- f)rophages. \Vr assump tha,t one or I)oth isolatc*a 01 MFfi74 arc trilysogrns with two i-21 hy Sl I fnyhagrs. but the number of prophages has not hryn drtrrminetf. Tt is not caritical for thr experimental quc+tion that thr number of pruphages be known. Results of packaging with thr i-specific helper agree with the prof)osed structure and are given in Itrsufts. section (I)).

Processive Action of i Term.inase 765

gal ott bio MFl343 ‘rrrrrrm

immel

A- X

P-

immi h A-immk

MFl363

P- A+

I k A-gal immk k A- bio imm21 P-

b

Figure 3. Construction of the cosi-+co~l-+cos~ trilysogen. MF1363. MF1343 was lysogenized with id imn/i to yield MF1363. whicah has the prophage structure shown (straight lines, I DKA: wary lines. bacterial DXA: filled c-ircles, ~0s sites). A i.-specific helper packages the left chromosome, which is IA - gal imml (left) arrow): and the right) chromosome. which is Ail - bio imm21 P- (right arrow). All the cos sites are of i origin and hence cos,V’ cosB+. The right chromosome can be packaged by initiation of packaging at the central cos or by sequential packaging of the right c*hromosomr. following packaging of the left chromosome. Phape part,icles containing the left and right rhromosomes are ca.lled 41 and $2.‘&pect~~e~~. as described in the text. ” -

negligiblr. Each lysate was plated independently in quadruplicatr on C600 (hum434 Rts2). Lrge plat)rs (1-H) mm dia.) were used and suitable dilutions were used to give approx. 250 plaques/plate. All plaques separated 1~~ 5 mm from the edge and from other plaques were plcked onto (!6OO(P2). on which 41 could not, prow and t.he biol 42 phages could grow.

Statist,ical analysis of the ~$1 : $12 proportions from the MF1363 helper packagings was carried out using a (i-test

(Sokal 8r Rohlf. 1969). The data were first tested for heterogeneity between different platings of the same

lysat’e. When these were shown not t,o be signiticantl\ heterogeneous (a > @I), the results of different plating of each lysate were pooled and the pooled totals for the 2 lysates from each isolat,r were tested for heterogeneity. Again. when t,he resu1t.s from the 2 different lysates were shown not to be significaantly heterogeneous (n > 0.1). the results for the lysates were pooled and the pooled tot,als for the 2 MN363 isolates were tested for heterogeneity. The results for the 2 independent isolates were not significant by t,hr C&test, (n > 0.1); so the results were pooled and used t’o establish confidence limit,s (~1 = 0%) for the proportions of $1 and $2.

Ht,atistical analysis of the outcome of the MF1362 helper packaging experiments was carried out in the same wa>- except’ only one plating was perfortned nit,h each lysate. A test for heterogeneit,y between the platings of the lysates of the isolates was not significant (a > 0.1). so the data were pooled and used t’o establish confidence limit,s at CI = 0.05.

3. Results (a) Terminase is able to read across cos to

initiate pxkayiny of the downstream chromosome

(i) Design of triZysoyen8

Several schemes for generating trilysogens were tried before the following constructions succeeded. Instead of starting with a monolysogen carrying a complet#e wild-type prophage, which would have the K12(A) structure shown in Figure 2, we used a

prophage deleted on both sides of cos (HS331. Fig. 2). HS331 was lysogenized with lgal bio imm21

r-. and a lysogen was identified in which integration had occurred by homologous re- combinat’ion of A sequences as diagrammed in Figure 2. This strain, a dilysogen wit.h respect to cos. was made recA- to prevent prophage excision and named MF1343. The structure of MF1343 was carefully examined, as described in Materials and Methods. Because a gal bio phage was used. MF1343 contains the normal bacterial attachment site, attB. This attB site is the only att site present, since the att sites of the first prophage were delet’ed in HS331. Trilysogens were then constructed by lysogenizing MF1343.

A co& phage, IA - immA, was used to construct. the control trilysogen, as shown in Figure 3. The resulting strain was called MF1363. As shown in the Figure, MF1363 has three cod sites (co&+ cosB+) and hence two chromosomes that may be helper- packaged by L. The various markers allow con- firmation of t,he prophage structure. as described in .Materials and Methods.

For the experimental trilysogen, a ~0~421 phage, A-21 hybrid 33 2- immll, was used (Fig. 4). The experimental trilysogen has three cos sites: the center site is ~0421, so it is cosN+ rosB- for the A packaging system. MFI 362 contains two packageable chromosomes, the left chromosome has 1 packaging specificity and should be packaged by a I helper. The right chromosome has 21 specificity, and should be packaged by a 21 -specific helper. The right chromosome might also be packaged by a 3. helper if scanning during packaging of the left chromosome allows J. terminase to function in reinitiat,ion of packaging of the right chromosome at cos@21. Remembering that I. does not initiate packaging from a COSN’ cosB- site, the experi- mental quest.ion may be rephrased as: does prior

M. Feiss, J. Sippy and 0. Miller

go1 att bi4 irnrn2l

MFl343

,i-21hyb33 i’-immi

2-

cosi pal ott immh cos821 oit b/O COSh MFl362

A- Z-

IL A- 9al immi ,.-21 hyb33 2- bi0 /mm21 P-

I m ä

Figure 4. Construction of the cosl+cos@l +COS~ trilysogen, 1MF1362. MF1343 was lysogenized with E.-Z1 hybrid 33 %- immA to give MFI362, which has the structure shown (straight lines. I DKA; wavy lines. bacterial DN;A: thick lines. 21 DNA; filled circles, cos sites). The central cos has phage 21 specificity. and hence is co~V+ cosBe wit,h respect to the packaging system provided by a I-specific helper. For further disrussion and a different drawing of the MF1362 structure see the Introduction and Fig. l(d), respectively. A i-specific helper packages the left chromosome. which is id gal &ml (left arrow). The right chromosome could only be packaged b?- continuation of packaging from the left- most CWS. since the cosB- defect of the central cos prevents packaging initiation by a i. helper. Phage particles bearing

the left and right chromosomes are called ~$1 and ~$2. respectively. as described in the text.

packaging of a A chromosome potentiate t,he packaging of a 21-specific chromosome by A?

(ii) Packaging of the cos/1-+cosi+cos~ trilysogen

Two independent isolates of MF1363 were grown and infected with helper phages having 1 or 21 packaging specificity. The lysates were plated to determine the yields of the helper phages and particles bearing the left and right prophage chromosomes (called 41 and ~$2, respectively). The result’s are presented in Table 2. As expected, only the helper phage with A specificity packaged the prophage chromosomes. The vield of 42 is somewhat in excess of the yield of 41 (see Discussion). The genotypes of 41 and $2 were exactly as expected from the structure drawn (10 studied for each MFI363 isolate).

(iii) Packaging of the cosd-+cos@l +cwsic trilysogen

Two independent isolates of ,MF1362 were grown and infected with the helper phages. and t,he lysates plated to determine the yields of $1 and ~$2. The results are presented in Table 3. With the 21. specific helper. the right, but, not’ the left. prophage chromosome was packaged, as expected for the MF1362 structure. We checked all the markers in these pubative ~$2 phages and all (10 t,ested from each MF1362 isolate) had the proper genotype fm 42. When the i-specific helper was used, we found that ~$1 was produced. and that each phage studied (10 from each MFl362 isolate) had the proper genotype. Thus, the results so far were completely consistent wit,h the expected structure of MF1362. Therefore, we concluded that the st,ructure of each

Table 2 Helper packaging of cosA-+ co&-+ cosi. trilysogen

lsolatr

Helper packaging specificity I,\-sate?

t For the i-specific helper, 2 independent infections of each isolate were done. giving lysates A and 13. For 1 hc 21 -specific helper. it single infection of each isolate was done.

$ The proportion between phages bearing the left (41) and right (42) prophagr ch~.omosomes (Fig. 3) was determrrwd ah drsr~ribrd ill Materials ;md Methods. section (h). The numbers given for each lysate represent the sum of 4 independent platings of the Iysatrs

4 The yields of 41 and 42 are calculated from the total yield of 41 plus $2. dekwnined on C6OO(limm434 Rts2). and the 41 to $2 proportion

Processive Action of A Terminase

Aso9 S A J b511 bio MF713 >

X Otf

j.-21 hy 51 /-

(circular dimer)

(I)

I-

I-

Oft

MF1774

b511

Lb511 t

1 k-21 hy 51 l-

m H

Figure 5. Construction of MF1774, a multilysogen with cos arrangement of cosd + (eos~21),. The top line shows the prophage structure of MF713 (straight lines, 1 DPU’A; heavy lines, 21 DNA; wavy lines, bacterial DKA; hatched lines deletions: filled circles, cos sites). The 509 deletion removes a DPU’A segment extending from the ba,cterial gal operon through the left attachment site to a site in the 1 S gene. The prophage also is marked by the 6511 deletion at the right at’tachment site. MF1774 is drawn as a trilysogen resulting from integration of a dimeric circle of A-21 hy 51 lam5 into MF713. It is possible that MF1774 isolates are tetra or higher order multilysogens resulting from integration of 3 or more I-21 hy 51 Iam chromosomes.

of the MFI362 isolates was correct as drawn in Figure 4.

The experimental question was asked by determining whether the right chromosome was also packaged by the I-specific helper, and we found that it was. 42 particles were produced by t.he 1 helper in an amount one-quarter that of the yield of $1 (see Discussion). The genotype of these ~$2 phages was determined, and all those tested (10 from each MF1362 isolate) were shown to have the expected genotype. The most critical marker was the hvbrid 33 substitution that confers 21 packaging specificity: each putative 42 was found to complement, A-21 hybrid 33 lam5, indicating each to have 21 packaging specificity. Two 42 phages from each trilysogen isolate were also studied by phage crosses and each was able to give

am+ recombinants with 1-21 hybrid 33 lam5 but not with A-21 hybrid 33 2am149. The level of ~$2 packaging by A is about lOO-fold greater than the level obtained when there is no R-specific prophage chromosome (the left chromosome) preceding the 21 -specific chromosome (the right chromosome) (Feiss et al., 1979). Thus, prior packaging of a 1 chromosome potentiates the packaging by I of a 21. specific chromosome. We conclude that terminase that has scanned the first chromosome is able to nick the central cos and initiate packaging of downstream chromosome.

the

(b) Scanning of the downstream chromosome

In MF1362, the experimental trilysogen, it is not possible to determine whether scanning occurs on

Table 3 Helper packaging of a cod + cos42 1 -+ cos1 trilysogen

Helper Yield (phage/infected cell)$ packaging

Isolate specificity Lysatet 41: 42:: Helper $1 42

MF1362-1 A A 114:32 48.0 0.75 0.21 i s 80.18 39.8 0.46 0.10

21 A .51.6 <O.Ol 1.32

MF136%5 1. A 141:45 24.6 1.19 I).38 /, u 102 : 37 17.9 040 0.3”

21 A 11.0 <O.Ol 0.26

t For 1 he E.-specific helper, 2 independent infections of each isolate were done. giving lysates A and B. For t,he 21 -specific helper, a single infection of each isolate was done.

$ The proportion between phages bearing the left (~$1) and the right (I#J~) prophage chromosomes (Fig. 4) was determined as described in Materials and Methods, section (h). The numbers given for each lysate represent one plating of the lysate.

9 The yields of Cpl and 42 are calculated from the t.otal yield of #II plus 42. determined on C6OO(limm434 Rts2). and the ~$1 to ~$2 proportion.

768 M. F&S, J. Sippy and C. Nill~r

Table 4 Helper packaging of a cos%+ (cos&21), rt~ultilysogPtl

Helper Sield (phapr;infrc~ted cell J$ packaging ______..- - - - ~~.

Isolate specificity 41 C&5”? Hrlp~ (bl @i -----------

Ii i 13 x 72 !bO!J I I4 I 21 0 : 21 3) ( I~Xi

IX i. 17.7 35 lbT4 (b3ll 2 1 0. IO 23 0.41

t The proportion between phages bearing the left (4) and right (42) prophayr t~hrornosomes was detwminrd by woriug 1i1r au1 * or am and h+ or b511 as described in Materials and Methods, section (b). $1 is lb511 and 42 is &?I hy 51 lam5. In this ‘l’ablc~ $2 r&w to particles bearing downstream chromosomes. since the MP1771 strains are tri- or higher order multilysogrns.

$ The yields of ~$1 and 42 are calculated from the total yield of packaged prophape chromosomes. determined OII (‘WO ii,~~r~r-~:i-I lit&). and the 41 to ~$2 proportion.

the second chromosome because the rightmost cos is cosE.. Because cosl is cosR+, terminase can bind to it directly to terminate packaging of the second chromosome. To study scanning of the second cahromosome, multilysogens (tri or higher order) were constructed that had an initial co& and ~08421 in t’he downstream positions (MF1774 isolates 17 and 18; Fig. 5). The ~08421 sites are from 1-21 hy 51 lam5 prophages. The two isolates of MF1774 were grown and infected with the helper phages and plat,ed to determine the yields (Table 4).

With the Sl-specific helper, the downstream chromosome (n-21 hy 51) was packaged. as expected for MF1774 structure (see Materials and Methods. section (g) (iv) for data). With the i,-specific helper, both prophage chromosome types were packaged. 41 particles were identified a,s phages that formed turbid plaques on C600 and that were am+ when tested for growth on 594. We checked these am+ phages for the 6511 marker and each (21 from isolate 17 and 24 from isolate 18) was 6511. Each also grew on hinzA - cells and hence has 1” packaging specificity. These results are completely consistent with the proposed prophage structure for MF1774. Particles carrying A-21 hy 51 cahromosomes were also found in the lysates. These scored as am- and were concluded to be L-21 hy 51 lam5 phages. None of these am- phages was able to grow on the supF himA strain K5282, indicating each has 21 packaging specificity. Ten am- phages from the lysates of each isolate were t’ested and found to be b+, as expected for the MF1774 struct,ure. The yield of L-21 hy 51 lam5 phages was more than lOO-fold greater than the level obtained in the absence of a &rpecifir chromosome preceding the 21 -specific chromosome (Feiss et al.. 1979). The results indicate the terminase that reads across cos can efficiently scan a downstream chromosome.

4. Discussion

We have found that’ the 1 packaging system can package a 21specific chromosome provided t’he 21 chromosome is preceded by a L-specific chromosome on the same DNA molecule. Since ~0~421 is COSN+ cosB- for II terminase; initiation of packaging at

cos~P1 does not occur: the packaging of the L’I specific* chromosome must, be caontinuation of’ a packaging sequence begun upst,ream. The basic conclusions from this result are: (1) that terminase can rea,d across cos, i.e. complete pa,okaging of one

chromosome and initiate packaging of the next: and (2) that cosH is not required for initiat,ion of packaging of that next chromosome. For a multilysogen in which the first cos is cosl and the distal cos sites are ~0~~21, downstream chrome somes are packaged by 2. This result indicates that terminase can not only- initiate packaging of ii downstream chromosome but caan scan for und

cleave the terminal cos of a downst~rram (ahromosome as well.

The read-through frequency c’an be calculat~ed from the proportions of 41 and 42 by making a simple model for packaging. In the ca,se of the 1’o~s~-+co~s;1-+cos~ trilysogens, 42 can be packaged by initiation at the central cos, or b?- continuatjion of packaging initiated at the first cos. Initiation i:: ra,ndom at available cos sites (Feiss 8 Bublitz. 1975). so t,he probability of initiation at the first c’on ([Jo) should be equal to the probability of initiation at the second site (p2). The fraction of 42 (,f2) is:

where r is the frequency of reading across (YIN to init’iate packaging of the next, chromosome. In this equation, because r can vary from 0 to I f; varies only from 0.5 to 0.67, which is a narrow range. Therefore a small change in f2 results in a large difference in t)he calculated value of Y. Thus. /’ cannot be precisely estimated. From all the pooled data. J; is 0.60+0.017 (95?& confidence limits). Therefore. sett,ing pi = p,. r is 0~49+0~11

For t,he cosE~--+cos~21 -+cosi trilysogens (,lilF1362). pz = 0, and equation (1) simplifies to:

In this case. as r varies from 0 tjo I, ,j’* varies from 0

Processive Action of 1 Terminase 769

to 0.5, a broader range. Hence the calculated r values are less inexact. From the pooled data, f2 is 0.23 &O-O34 (95% confidence limits), and r is 0.30 + O-06. Comparison of the control (cod + cod + cod) and experimental (cos~+cos1$2l+cos~) values of r indicate that the frequencies of reading across cos are similar, with the ~0~421 value being somewhat less than the value for cos1. If co& played a crucial role in reading across cos the r values for COSL and ~0~421 would be expected to show a much greater difference than observed. We conclude that cosR function is not necessary for terminase to read across cos.

How do our r values compare with Emmon’s (1974) estimate t.hat the average packaging series length is two to three chromosomes long during lytic I growth? If one assumes that during lytic growth the packaging series length is not limited by concatemer length, then an r value of 0.5 would generate an average packaging series length of two, a value close to that calculated by Emmons. Our r values of 0.5 for COST and O-3 for eos&21 are in good agreement with the value of 0.5 calculated for lytic 1 growth. Many assumptions have been made in estimating the r values so the comparisons must be viewed with caution (see the following paragraphs).

What might account for the difference in our r estimates for cosl (0.5) and ~0~421 (0.3)?

One possibility is that our model does not fully describe the packaging of the tandem prophages in the ~os~-+~os~--+cos~ trilysogen. For example, the model assumes that the left and right chromosomes are packaged independently except when reading across COR occurs. This assumption of independence may be an oversimplification. Packaging of one chromosome may reduce the probability that the other will be packaged; for example, the free cohesive end of the unpackaged chromosome might be subject tJo nuclease attack. If such attack preferentially inactivat,ed the left chromosome, t,hen the apparent value of r would be increased. This particular possibility is somewhat likely because the yield of ~$1 is about the same for MF1363 where packaging can be initiated at the central cos and MF1362 where packaging cannot be initiated at the central cos.

A second possible explanation for the difference in r values for cosi and ~0~421 is that terminase may dissociate from the downstream chromosome (when reading across cos does not occur), and then reassociate with the downstream chromosome if a functional cosB is present, i.e. in MF1363 but not MFl362. Should such dissociation-reassociation take place at the central cod of MF1363, then the apparent value of r would be increased.

A third possible explanation for the difference in r values is t,hat co&21 differs from co& in base- pairs important for reading across cos. We discuss this possibility below.

Finally, t’he absolute plating efficiencies of ~$1 and 42 might. vary, though @I and 42 in each experiment make large, easily scored plaques.

(b) Scanning of downstream chromosome

The downstream chromosome(s) of the cosl+(cos~2l), multilysogen is packaged by a E.-specific helper. The initial cos in this multilysogen is the only cos with a functional binding site for 1 t,erminase. Hence the initial cos is the entry site for terminase that initiates, scans and terminates packaging of the downstream chromosome.

The downstream chromosomes from MF1774 are 0.33 of the helper packaged chromosomes. a value similar to the value of the cosA-+ cos~p21 -+cosL trilysogen. Since the number of prophages in MF1774 is undetermined the value of r cannot be estimated.

(c) A model for tertilinase read-through and sequential packaging

The above results suggest a model for sequential chromosome packaging. The model has several elements: (1) packaging delivers a terminase molecule to the terminal cos; (2) the terminal cosN is directly recognized and cleaved by terminase: (3) the terminase molecule remains bound to the left end of the next chromosome; and (4) the terminase binds a prohead to initiate packaging. Step 1 is then repeated as terminase scans the next chromosome to cleave the next cosN.

The first element, that packaging delivers terminase to the terminal cos during packaging, is supported by several lines of evidence showing that cutting of a terminal cos is packaging-dependent. These are that (I) segregation of cos duplications indicates that cos cleavage is packaging-dependent (Emmons, 1974); (2) t’erminase is required after packaging has occurred (Becker et a,Z., 1977; Kaiser et al., 1975); (3) the efficiency of cleavage of the terminal cos depends on chromosome length (Henderson & Weil, 1977; Feiss & Biegele, 1979). The most likely model is that terminase traverses t’he DNA during packaging.

The second postulate, that the terminal cosN is directly recognized and cleaved, comes from the finding that the terminal cos can be cut. even when it is cosN+ cosR- and unable to bind L terminase (Feiss et al., 1979; Feiss & Widner, 1982) as long as the initial cos is cosN+ co&‘. Terminase can encounter cosN by scanning during packaging or by rosH binding.

The third step, terminase binding to the left end of the next chromosome following cleavage, is proposed to generate a DNA-terminase complex that can bind a prohead to form complex II and begin packaging. There is little evidence directly bearing on t,his point, but alternatives are more complex. For example, one alternative mechanism to initiate packaging of a 21-specific chromosome following packaging of a i-specific chromosome would be that free ,J terminase might’ bind to the left end of the cosB- chromosome following co8 cleavage. This mechanism would require that free A terminase would be able to bind to the left end of a cleaved 21 -specific chromosome. This alternative is

770 M. Feiss, J. Sippy and G. Miller

unlikely because mature 21 DNA is not packaged in vitro by a A-specific system, which indicates that free % terminase does not bind mature 21 DNA. See section (d), below, for further discussion.

Step 4 would simply be prohead binding followed by packaging. The r values for b0t.h the exprri- mental and control trilysogens indicate that reading across cos occurs only some of the time, Two mechanisms by which reading across cos could he interrupted are (1) failure to form the DNA- terminase complex, and (2) failure to bind a prohead by the DNA-terminase complex.

(d) ‘Twminase binding at thr kft chromoaom~ end

If the terminase that is delivered bv packaging to a terminal cosN+ cosB- site can initiate packaging from the site. it must bind the cosB- chromosome. To what part of cos might terminase bind at t,he left cohesive end 2

One possibility is that terminase does interact with COSB when it binds to the left end of t,he downstream chromosome. and that the t’erminasee cosH int’eraction involves COSB base-pairs that are the same in cod and cos~$dl. The specificity difference between co& and co4421 might. involve other base-pairs of cosB that have another function such as the initial binding of terminase. i.e. t’he strep bypassed by scanning.

Another speculative model (suggested by a reviewer) is that cosB is an entry site that is necessarv for t,he formation but, not the maintenance of a terminase complex with the DNA. Formation of this complex at’ t’he initial cos would properly position terminase for cosN cleavage and prohead binding. Once formed, t,he terminase-DNA complex perhaps could move away from cosB (as in scanning) while maintaining proper conformation. That is, once formed at cosB, t’he DNA-terminase complex would be stable to terminase movement and independent of DNA sequence. Scanning would bring the terminal eosN into proper posit.ion for nicking and terminase would remain complexed at the left end of the next chromosome. This binding would be sequence-independent, because the proper DNA-terminase conformat,ion was set up at the initial cosB. Upon binding of a prohead. packaging of thr second chromosome would oc(*ur. What, might be the nat,ure of t,erminase entry at COSB! (!rrtain parallels between cos and the phage attachment site. attP, exist). Integration requires t.hr phage enzyme integrase and the bacterial prot’ein. integrative host’ factor, IHF (reviewed by Nash. 1981; Weisberg & Landy. 1983). The attP site contains multiple, interspersed binding sites for integrase and IHF. Topological changes in L DNA a.cacompanying integration suggest that. the binding of intrgrase and TH% to attP results in wrapping to the DNA to form a nucleosome-like structure (T’ollock $ Nash, 1983). cosB might also be a sit’e at wflich such a structure is formed, expecially as co& is about, 100 base-pairs long and rich m direct repeat,s and palindromes that are pot,ential protein

binding sites (Miwa $ Matsubara, 1983: Feiss ul /xl.. 1983. 198.5: Rear ut al., 1984). The att-cos analogy it; strengthened bv the finding t,hat the phage 21 t)ermina)sr requires IHF as host factor and that there are several potential THF binding sites in cos$21 (Friss et al.. 1985). THF is also implicated in the action of ,? t,erminase (1lea.r of nl.. 1984).

Man!, viruses pckage chromosomes by cAut,ting unit-length molecules from concat,emrrir DNA. For phagr P22, packaging of the first chromosome of a series initiat’es unidirretionally from a site caalletl /XX. The first cut is made near par and t)hr second cut occurs when the head is full with approximately 107’)~ ’ 10 of the unique sequence length of DNA. generat,ing a trrminall~ redundant molPcwle. According to a model for sequential packaging, t,hch site of the second cut generates the start point of bhe next chromosome in the packaging sequence. so that the remaining chromosomes are pa.ckaged without sequence specificity (Ty~b it nl.. 1974). Thr initial cxut, is made near pat in one of six regions (pzd sit’es) witShin a 120 base-pair segment of l)NA4 (Casjens 8 Huang. 19X2). Sequential packaging in /. and P22 may be mechanistically analogous: init,iation of a packaging sequence by t,he binding of trrminwsc~ t 0 a unique site (cosH or pt7C) Ad caontinuatiou of packaging by proc’easive action of trrminase in a co.sR~ or p”c-independent marmel*. The c>ssent,ial tlifferenc>e between i and 1’22 packaging may 1)~ the cleavage st!ep, wflich is sequencbe-specific for I and not sequence-specific tbl P22.

Thanks to Hiroaki Shizuya for HSS3L a.nd to .loseph Hegmann for help wit,h statisticx Max (Mtesman rt~couragetl us to ronstrurt and study t~ht, cvsA ((0~~21 ),, multilq-sogen. His interest in our work is appreciated. ‘lVe apprwiatr cwmments on the manuscript from ,Jran \Valkrr and Marty Stoltzfus. Thanks to Mar& Reeve for preparing ali the vvrsionn. Suppcded by NIH rrsrarch grant AI-1%51, (;.?/I. is the recipient of * Xatiomd Rrsearc,h Srrvicv Award from t,he Xational Jnstitut,rs of Health (GM-05335).

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Edited by M. Gottesman