The functional asymmetry of cosN, the nicking site for bacteriophage lambda DNA packaging, is dependent on the terminase binding site, cosB.

cosN is the site at which terminase, the DNA packaging enzyme of phage lambda, introduces staggered nicks into viral concatemeric DNA to initiate genome packaging. Although the nick positions and many of the base pairs of cosN show 2-fold rotational symmetry, cosN is functionally asymmetric. That is, the cosN G2C mutation in the left half-site (cosNL) causes a strong virus growth defect whereas the symmetrically disposed cosN C11G mutation in the right half-site (cosNR) does not affect virus growth. The experiments reported here test the proposal that the genetic asymmetry of cosN results from terminase interactions with cosB, a binding site to the right of cosN. In the presence of cosB, the left half-site mutation, cosN G2C, strongly affected the cos cleavage reaction, while the symmetric right half-site mutation, cosN C11G, had little effect. In the absence of cosB, the two mutations moderately reduced the rate of cos cleavage by the same amount. The results indicated that the functional asymmetry of cosNdepends on the presence of cosB. A model is discussed in which terminase-cosN interactions in the nicking complex are assisted by anchoring of terminase to cosB.     

Processive action of terminase during sequential packaging of bacteriophage lambda chromosomes

Bacteriophage lambda chromosomes are packaged in a polarized, sequential fashion from a multimeric 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 cosN. 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 cosN, 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 lambda 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 terminase is brought to the central cos by packaging; following cleavage of the central cos, the terminase remains bound to the distal chromosome; and 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 initiate 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 lambda chromosomes.     

Bacteriophage lambda DNA packaging: DNA site requirements for termination and processivity

Bacteriophage lambda chromosomes are processively packaged into preformed shells, using end-to-end multimers of intracellular viral DNA as the packaging substate. A 200 bp long DNA segment, cos, contains all the sequences needed for DNA packaging. The work reported here shows that efficient DNA packaging termination requires cos's I2 segment, in addition to the required termination subsite, cosQ, and the nicking site, cosN. Efficient processivity requires cosB, in addition to cosQ and cosN. An initiation-defective mutant form of cosB sponsored efficient processivity, indicating that the terminase-cosB interactions required for termination are less stringent than those required at initiation. The finding that an initiation-defective form of cosB is functional for processivity allows a re-interpretation of a similar finding, obtained previously, that the initiation-defective cosB of phage 21 is functional for processivity by the lambda packaging machinery. The cosBphi21 result can now be interpreted as indicating that interactions between cosBphi21 and lambda terminase, while insufficient for initiation, function for processivity.     

The lambda terminase enzyme measures the point of its endonucleolytic attack 47 +/- 2 bp away from its site of specific DNA binding, the R site.

lambda terminase is an ATP-interactive, site-specific endonuclease comprising the products of lambda genes Nu1 and A. Terminase binds to cos, at the junction of two chromosomes in a concatemer, catalyzes cos cleavage and initiates the packaging of lambda DNA into proheads. cos consists of a nicking domain, cosN, where terminase cleaves to regenerate the 12 nucleotide cohesive ends of mature lambda chromosomes and a binding domain, cosB, where terminase binds to 16 bp repeat sequences called R3, R2 and R1. Evidence is presented that terminase is a single-strand endonuclease that can nick DNA by one of two mechanisms, both of which require ATP. (i) When bound to any R site, terminase nicks the strand which, within that R site, is purine-rich; the position of this nick is 47 +/- 2 nucleotides away from the mid-point of that R site, measured in the 3' direction; (ii) enzymes that are not bound to R sites nick DNA within certain specific sequences that resemble cosN half sites. These two modes of action are nicely combined for the R3-bound protomer that nicks the bottom strand at position N1 in cosN since the interval between N1 and the R3 midpoint is 47 nucleotides. Within cosN, the bottom and top strand nicks are generated by a rigid protein couple with a 2-fold rotational symmetry. The location of both of these nicks, however, is gauged asymmetrically from R3, 47 nucleotides away. Again, R1 and R2 are separated by 47 bp and orient bound protomers towards each other but, unless the DNA between these R sites is lengthened, the enzymes do not nick, indicating an inhibitory gpA-gpNu1 apposition.     

Mutations in Nu1, the gene encoding the small subunit of bacteriophage lambda terminase, suppress the postcleavage DNA packaging defect of cosB mutations.

The linear double-stranded DNA molecules in lambda virions are generated by nicking of concatemeric intracellular DNA by terminase, the lambda DNA packaging enzyme. Staggered nicks are introduced at cosN to generate the cohesive ends of virion DNA. After nicking, the cohesive ends are separated by terminase; terminase bound to the left end of the DNA to be packaged then binds the empty protein shell, i.e., the prohead, and translocation of DNA into the prohead occurs. cosB, a site adjacent to cosN, is a terminase binding site. cosB facilitates the rate and fidelity of the cosN cleavage reaction by serving as an anchoring point for gpNu1, the small subunit of terminase. cosB is also crucial for the formation of a stable terminase-DNA complex, called complex I, formed after cosN cleavage. The role of complex I is to bind the prohead. Mutations in cosB affect both cosB functions, causing mild defects in cosN cleavage and severe packaging defects. The lethal cosB R3- R2- R1- mutation contains a transition mutation in each of the three gpNu1 binding sites of cosB. Pseudorevertants of lambda cosB R3- R2- R1- DNA contain suppressor mutations affecting gpNu1. Results of experiments that show that two such suppressors, Nu1ms1 and Nu1ms3, do not suppress the mild cosN cleavage defect caused by the cosB R3- R2- R1- mutation but strongly suppress the DNA packaging defect are presented. It is proposed that the suppressing terminases, unlike the wild-type enzyme, are able to assemble a stable complex I with cosB R3- R2- R1- DNA. Observations on the adenosine triphosphatase activities and protease susceptibilities of gpNu1 of the Nu1ms1 and Nu1ms3 terminases indicate that the conformation of gpNu1 is altered in the suppressing terminases.     

Genetic analysis of cosB, the binding site for terminase, the DNA packaging enzyme of bacteriophage lambda.

cosB, the binding site for terminase, the DNA packaging enzyme of bacteriophage lambda, consists of three binding sites (called R3, R2 and R1) for gpNu1, the small subunit of terminase; and I1, a binding site for integration host factor (IHF), the DNA bending protein of Escherichia coli. cosB is located between cosN, the site where terminase introduces staggered nicks to generate cohesive ends, and the Nu1 gene; the order of sites is: cosN-R3-I1-R2-R1-Nu1. A series of lambda mutants have been constructed that have single base-pair C-to-T transition mutations in R3, R2 and R1. A single base-pair transition mutation within any one of the gpNul binding sites renders lambda dependent upon IHF for plaque formation. lambda phage with mutations in both R2 and R3 are incapable of plaque formation even in the presence of IHF. Phages that carry DNA insertions between R1 and R2, from 7 to 20 base-pairs long, are also IHF-dependent, demonstrating the requirement for a precise spacing of gpNu1 binding sites within cosB. The IHF-dependent phenotype of a lambda mutant carrying a deletion of the R1 sequence indicates that IHF obviates the need for terminase binding to the R1 site. In contrast, a lambda mutant deleted for R2 and R1 fails to form plaques on either IHF+ or IHF- cells, indicating terminase binding of R2 is involved in suppression of R mutants by IHF. A fourth R sequence, R4, is situated on the left side of cosN; a phage with a mutant R4 sequence shows a reduced burst size on both an IHF+ and an IHF- host. The inability of the R4- mutant to be suppressed by IHF, plus the fact that R4 does not bind gpNu1, suggests R4 is not part of cosB and may play a role in DNA packaging that is distinct from that of cosB.     

Structure of the bacteriophage lambda cohesive end site. Genetic analysis of the site (cosN) at which nicks are introduced by terminase.

A collection of mutations affecting the site (cosN) at which the bacteriophage lambda DNA packaging enzyme, terminase, introduces nicks to generate mature lambda chromosomes has been studied. A good correlation was found for mutational effects on burst size, accumulation of unused proheads, packaging of DNA into heads and cos cutting by terminase in vitro, indicating that defective cosN cleavage by terminase is the molecular explanation for the phenotypic effects of the mutations. Although the base-pairs of cosN display partial twofold rotational symmetry, cosN was found to be asymmetric functionally. Certain mutations to the left side of the center of rotational symmetry have more pronounced phenotypic effects than rotationally symmetric mutations to the right. The cosN11G mutation has no phenotypic effects when present as a single mutation, but does affect DNA packaging and cosN cutting in the presence of the symmetrically disposed cosN2C mutation. Mutations that decrease cosN cleavage result in the accumulation of unexpanded proheads, indicating that prohead expansion depends on cosN cutting.     

Defining cosQ, the site required for termination of bacteriophage lambda DNA packaging

Bacteriophage lambda is a double-stranded DNA virus that processes concatemeric DNA into virion chromosomes by cutting at specific recognition sites termed cos. A cos is composed of three subsites: cosN, the nicking site; cosB, required for packaging initiation; and cosQ, required for termination of chromosome packaging. During packaging termination, nicking of the bottom strand of cosN depends on cosQ, suggesting that cosQ is needed to deliver terminase to the bottom strand of cosN to carry out nicking. In the present work, saturation mutagenesis showed that a 7-bp segment comprises cosQ. A proposal that cosQ function requires an optimal sequence match between cosQ and cosNR, the right cosN half-site, was tested by constructing double cosQ mutants; the behavior of the double mutants was inconsistent with the proposal. Substitutions in the 17-bp region between cosQ and cosN resulted in no major defects in chromosome packaging. Insertional mutagenesis indicated that proper spacing between cosQ and cosN is required. The lethality of integral helical insertions eliminated a model in which DNA looping enables cosQ to deliver a gpA protomer for nicking at cosN. The 7 bp of cosQ coincide exactly with the recognition sequence for the Escherichia coli restriction endonuclease, EcoO109I.     

Chromosome end formation in phage lambda, catalyzed by terminase, is controlled by two DNA elements of cos, cosN and R3, and by ATP.

The terminase enzyme of phage lambda is a site-specific endonuclease that nicks DNA concatemers to regenerate the 12 nucleotide cohesive ends of the mature chromosome. The enzyme's DNA target, cos, consists of a nicking domain, cosN, and a binding domain, cosB. cosB, situated to the right of cosN, comprises three 16 bp repeat sequences, R1, R2 and R3. A similar sequence, R4, is present to the left of cosN. It is shown here that terminase has an intrinsic specificity for cosN which is independent of the R sites. The interaction with cosN is mediated by binding to target sites that include 12 bp on the 5', and 2-7 bp on the 3' side of the nick. Of the four R sites, only R3 is required for the proper formation of ends. When R3 is present, an ATP-charged terminase system correctly catalyzes the production of staggered nicks in cosN, at sites N1 and N2 on the bottom and top strands, respectively. When ATP is omitted, the bottom strand is nicked incorrectly, at the site Nx, 8 bp to the left of N1. If R3 is removed or disabled by a point mutation, nicking in cosN becomes dependent upon ATP but, even in the presence of ATP, bottom strand nicking is divided between sites N1, the correct site, and Nx, the incorrect one. Thus, R3 is an important regulatory element and must reside in cis in respect to cosN. Furthermore, cosN substrates bearing point mutations at N1 and N2 are nicked at sites Nx and Ny, 8 bp to the left of N1 and N2, respectively. When R3 is present and ATP is added, nicking is redirected to the N1 and N2 positions despite the mutations present. Thus, terminase binding to R3, on one side of cosN, regulates the rotationally symmetric nicking reactions on the bottom and top strands within cosN.     

Integration host factor (IHF) stimulates binding of the gpNu1 subunit of lambda terminase to cos DNA.

The lambda terminase enzyme binds to the cohesive end sites (cos) of multimeric replicating lambda DNA and introduces staggered nicks to regenerate the 12 bp single-stranded cohesive ends of the mature phage genome. In vitro this endonucleolytic cleavage requires spermidine, magnesium ions, ATP and a host factor. One of the E. coli proteins which can fulfill this latter requirement is Integration Host Factor (IHF). IHF and the gpNu1 subunit of terminase can bind simultaneously to their own specific binding sites at cos. DNase I footprinting experiments suggest that IHF may promote gpNu1 binding. Although no specific gpNu1 binding to the left side of cos can be detected, this DNA segment does play a specific role since a cos fragment that does not include the left side or whose left side is replaced by non-cos sequences, is unable to bind gpNu1 unless either spermidine or IHF is present. Binding studies on the right side of cos using individual or combinations of gpNu1 binding sites I, II and III indicate that binding at sites I and II is not optimal unless site III is present.     

Molecular analysis of the cos region of the Lactobacillus casei bacteriophage A2. Gene product 3, gp3, specifically binds to its downstream cos region

The terminal nucleotide sequence of the Lactobacillus casei bacteriophage A2 DNA revealed a single-stranded extension 13 bases in length (5'-AACGGTCGGCCTC-3') at its 3' termini that defines the packaging initiation nicking site ( cosN ). The cosN sequence is bisected by an axis of hyphenated twofold rotational symmetry. Directly and inverted repeated sequences located to the left ( cosL ) and the right ( cosR ) of the cosN site were observed. Analysis of the 3.4 kb Eco RI DNA sequence surrounding the cos region revealed four complete and one incomplete open reading frames ( orf s). Northern blots indicated that all were cotranscribed in a single mRNA molecule in excess of 10 kb that appeared late during infection. Minicell studies indicated that the four orf s were translated into protein. From the ORF3 amino acid sequence DNA-binding and NTP-binding domains can be predicted. The purified ORF3 (predicted molecular mass 16.8 kDa) shows specific binding to the A2 cos region, so it was renamed gp3. Gp3 forms a specific complex with a 369 bp cos DNA segment in the presence of ATP. Gp3 interaction with the intrinsically bent cos DNA segment induces intramolecular ligation in the presence of T4 DNA ligase. The data presented here suggest that gp3 is the small subunit of the terminase enzyme.     

The last duplex base-pair of the phage lambda chromosome. Involvement in packaging, ejection and routing of lambda DNA.

cosN is the site at which the bacteriophage lambda DNA packaging enzyme, terminase, introduces staggered nicks to generate the cohesive ends of mature lambda chromosomes. Genetic and molecular studies show that cosN is recognized specifically by terminase and that effects of cosN mutations on lambda DNA packaging and cosN cleavage are well correlated. Mutations affecting a particular base-pair of cosN are unusual in being lethal in spite of causing only a moderate defect in cosN cleavage and DNA packaging. The particular base-pair is the rightmost duplex base-pair in mature chromosomes, at position 48,502 in the numbering system of Daniels et al; herein called position - 1. A G.C to T.A transversion mutation at position - 1, called cosN - 1T, reduces the particle yield of lambda fivefold, and the particles formed are not infectious. lambda cosN - 1T particles have wild-type morphology, and contain chromosomes that have normal cohesive ends. The chromosomes of lambda cosN - 1T particles, like the chromosomes of lambda + particles, are associated with the tail. lambda cosN - 1T particles, in spite of being normal structurally, are defective in injection of DNA into a host cell. Only approximately 25% of lambda cosN - 1T particles are able to eject DNA from the capsid in contrast to 100% for lambda +. Furthermore, for the 25% that do eject, there is a further injection defect because the ejected lambda cosN - 1T chromosomes fail to cyclize, in contrast to the efficient cyclization found for wild-type chromosomes following injection. The cosN - 1T mutation has no effect on Ca2+ mediated transformation by lambda DNA, indicating that the effect of the mutation on DNA fate is specific to the process of DNA injection. Models in which specific DNA : protein interactions necessary for DNA injection, and involving the rightmost base-pair of the lambda chromosome, are considered.     

Genetic Evidence That Recognition of Cosq, the Signal for Termination of Phage λ DNA Packaging, Depends on the Extent of Head Filling

Packaging a phage λ chromosome involves cutting the chromosome from a concatemer and translocating the DNA into a prohead. The cutting site, cos, consists of three subsites: cosN, the nicking site; cosB, a site required for packaging initiation; and cosQ, a site required for termination of packaging. cosB contains three binding sites (R sequences) for gpNu1, the small subunit of terminase. Because cosQ has sequence identity to the R sequences, it has been proposed that cosQ is also recognized by gpNu1. Suppressors of cosB mutations were unable to suppress a cosQ point mutation. Suppressors of a cosQ mutation (cosQ1) were isolated and found to be of three sorts, the first affecting a base pair in cosQ. The second type of cosQ suppression involved increasing the length of the phage chromosome to a length near to the maximum capacity of the head shell. A third class of suppressors were missense mutations in gene B, which encodes the portal protein of the virion. It is speculated that increasing DNA length and altering the portal protein may reduce the rate of translocation, thereby increasing the efficiency of recognition of the mutant cosQ. None of the cosQ suppressors was able to suppress cosB mutations. Because cosQ and cosB mutations are suppressed by very different types of suppressors, it is concluded that cosQ and the R sequences of cosB are recognized by different DNA-binding determinants.     

Genetic analysis of mutations affecting terminase, the bacteriophage lambda DNA packaging enzyme, that suppress mutations in cosB, the terminase binding site.

Terminase, the DNA packaging enzyme of phage lambda, binds to lambda DNA at a site called cosB, and introduces staggered nicks at an adjacent site, cosN, to generate the cohesive ends of virion lambda DNA molecules. Terminase also is involved in separation of the cohesive ends and in binding the prohead, the empty protein shell into which lambda DNA is packaged. Terminase is a DNA-dependent ATPase, and both subunits, gpNu1 and gpA, have ATPase activity. cosB contains a series of gpNu1 binding sites, R3, R2 and R1; between R3 and R2 is a binding site, I1, for integration host factor (IHF), the Escherichia coli DNA bending protein. In this work, a series of mutations in Nu1 have been isolated as suppressors of cosB mutations. One of the Nu1 mutations is identical to the previously described Nu1ms1/ohm1 mutation predicted to cause the change L40F in the 181 amino acid-long gpNu1. Three other Nu1 missense mutations, the Nu1ms2 (L40I), ms3 (Q97K) and ms4 (A92G) mutations, have been isolated; the relative strengths of suppression of cosB mutations by the Nu1ms mutations are: ms1 > ms2 > ms3 > ms4. The Nu1 missense mutations all affect amino acid residues that lie outside of the putative helix-turn-helix DNA binding motif of gpNu1. The Nu1ms1 and Nu1ms2 mutations alter an amino acid residue (L40) that lies directly between two segments of gpNu1 proposed to be involved in ATP binding and hydrolysis; thus these mutations are likely to alter the gpNu1 ATP-binding site. The Nu1ms3 and Nu1ms4 mutations both affect amino acid residues in the central region of gpNu1 that is predicted to form a hydrophilic alpha-helix. To explain how the Nu1ms mutations suppress cosB defects, models involving alterations of the DNA binding and/or catalytic properties of terminase are considered. The results also indicate that terminase occupancy of a single gpNu1 binding site (R3) is necessary and sufficient for the efficient initiation of DNA packaging; the Nu1ms1, ms2 and ms3 mutations permit IHF-independent plaque formation by a phage lacking R2 and R1.     

Analysis of a mutation affecting the specificity domain for prohead binding of the bacteriophage lambda terminase.

Genetic studies have identified a specificity domain for prohead binding in the C-terminal 32 amino acids of gpA, the large subunit of bacteriophage lambda terminase (S. Frackman, D. A. Siegele, and M. Feiss, J. Mol. Biol. 180:283-300, 1984). In the present work, an amber mutation, Aam42, in the fifth-to-last codon of the A gene was found to be lethal in nonsuppressing hosts. The mutation, expected to generate gpA lacking the last five amino acids, caused the production of a terminase that cut cos efficiently both in vivo and in vitro but was defective in DNA packaging. lambda Aam42 lysates contained unused proheads, consistent with a defect in prohead binding. Aam42 terminase was more strongly dependent than wild-type terminase on gpFI, the catalyst of prohead binding. Like wild-type terminase, Aam42 terminase did not cut cos in vivo when prohead assembly was blocked by a mutation in one of the genes encoding the prohead.     

Delimitation of cohesive ends site (cos) of Streptomyces temperate bacteriophage R4.

The cohesive ends site (cos) of actinophage R4 was delimitated by assaying the in vivo packaging activity of cosmid derivatives in Streptomyces lividans. A region of 66 bp from -30 to +36 from the center of cohesive ends was required for the basal level of packaging activity. Two additional regions outside the basal sequences from -39 to -31 and from +37 to +97 were necessary for the high level of activity, defined as the accessory sequences. Direct- or inverted-repeat sequences were found within the delimitated region, which might be involved in the recognition by the terminase of actinophage R4.     

The interaction of E. coli integration host factor and lambda cos DNA: multiple complex formation and protein-induced bending.

The interaction of E. coli's integration Host Factor (IHF) with fragments of lambda DNA containing the cos site has been studied by gel-mobility retardation and electron microscopy. The cos fragment used in the mobility assays is 398 bp and spans a region from 48,298 to 194 on the lambda chromosome. Several different complexes of IHF with this fragment can be distinguished by their differential mobility on polyacrylamide gels. Relative band intensities indicate that the formation of a complex between IHF and this DNA fragment has an equilibrium binding constant of the same magnitude as DNA fragments containing lambda's attP site. Gel-mobility retardation and electron microscopy have been employed to show that IHF sharply bends DNA near cos and to map the bending site. The protein-induced bend is near an intrinsic bend due to DNA sequence. The position of the bend suggests that IHF's role in lambda DNA packaging may be the enhancement of terminase binding/cos cutting by manipulating DNA structure.     

The Nu1 subunit of bacteriophage lambda terminase

The maturation and packaging of bacteriophage lambda DNA are catalyzed by the phage terminase enzyme. Terminase is composed of two protein subunits, gpNu1 and gpA. The holoenzyme is multifunctional in vitro; it binds to and cleaves lambda DNA at the cos site (where cos represents cohesive-end site), packages DNA into lambda proheads, and is also a DNA-dependent ATPase. The genes of the two subunits have been cloned separately into powerful expression vectors which allow for very high levels of protein overproduction. The gpNu1 protein has been purified to homogeneity and has a monomeric molecular weight of 21,200, in close agreement with the Mr of 20,444 expected from its amino acid sequence. Both gel filtration and sedimentation velocity centrifugation indicate that the native gpNu1 protein exists as a Mr greater than 500,000 aggregate. The sequence of the first 20 amino acids and the overall composition both match those predicted by the nucleotide sequence of the Nu1 gene. Purified gpNu1 is able to complement gpA-containing extracts in both lambda DNA packaging and cos cleavage assays. The Nu1 gene amino acid sequence predicts DNA binding by the protein, and gpNu1 does show specific binding to lambda DNA by filter binding assays. Also, as predicted from its sequence, gpNu1 exhibits ATPase activity; but in contrast to the holoenzyme, this activity is DNA-independent.     

Self-association properties of the bacteriophage lambda terminase holoenzyme: implications for the DNA packaging motor

Terminases are enzymes common to complex double-stranded DNA viruses and are required for packaging of viral DNA into a protective capsid. Bacteriophage lambda terminase holoenzyme is a hetero-oligomer composed of the A and Nu1 lambda gene products; however, the self-association properties of the holoenzyme have not been investigated systematically. Here, we report the results of sedimentation velocity, sedimentation equilibrium, and gel-filtration experiments studying the self-association properties of the holoenzyme. We find that purified, recombinant lambda terminase forms a homogeneous, heterotrimeric structure, consisting of one gpA molecule associated with two gpNu1 molecules (114.2 kDa). We further show that lambda terminase adopts a heterogeneous mixture of higher-order structures, with an average molecular mass of 528(+/-34) kDa. Both the heterotrimer and the higher-order species possess site-specific cos cleavage activity, as well as DNA packaging activity; however, the heterotrimer is dependent upon Escherichia coli integration host factor (IHF) for these activities. Furthermore, the ATPase activity of the higher-order species is approximately 1000-fold greater than that of the heterotrimer. These data suggest that IHF bending of the duplex at the cos site in viral DNA promotes the assembly of the heterotrimer into a biologically active, higher-order packaging motor. We propose that a single, higher-order hetero-oligomer of gpA and gpNu1 functions throughout lambda development.     

Genetics of cosQ, the DNA-packaging termination site of phage lambda: local suppressors and methylation effects

The cos site of the bacteriophage lambda chromosome contains the sites required for DNA processing and packaging during virion assembly. cos is composed of three subsites, cosQ, cosN, and cosB. cosQ is required for the termination of chromosome packaging. Previous studies have shown cosQ mutations to be suppressed in three ways: by a local suppressor within cosQ; by an increase in the length of the lambda chromosome; and by missense mutations affecting the prohead's portal protein, gpB. In the first study reported here, revertants of a set of cosQ mutants were screened for suppressors, and cis-acting suppressors of cosQ mutations were studied; these included second-site cosQ point mutations, base-pair insertions within cosQ, and an additional genome-lengthening suppressor. The 7-bp-long cosQ, with the sequence 5'-GGGTCCT-3', coincides exactly with the recognition site for the EcoO109I restriction/methylation system, which has the consensus sequence 5'-PuGGNCCPy-3'. In a second study, EcoO109I methylation was found to strongly interfere with the residual cosQ function of leaky cosQ mutants. cis-acting suppressors that overcome methylation-associated defects, including a methylation-dependent suppressor, were also isolated. Models of cosQ suppression are presented.