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M Feiss

Publications and source records attributed to M Feiss.

At least 37 records · Page 2Linked to original sources
PubMed1993

The role of cosB, the binding site for terminase, the DNA packaging enzyme of bacteriophage lambda, in the nicking reaction.

cosB is the binding site for terminase, the DNA packaging enzyme of ai-12581mbda, and cosN is the adjacent site at which terminase gm-07228es staggered nicks to generate mature lambda DNA molecules. There are three binding sites (R3, R2 and R1) within cosB for gpNu1, the small subunit of terminase. A particular transition mutation of R1, known to weaken binding of gpNu1 to R1, has been introduced into the other R sites, and in the present work the effects of R site mutations on nicking of cosN have been examined. Nicking experiments performed in the presence of ATP suggest that the most profound cosB mutation tested (the R3-R2-R1- mutation) would, at most, reduce cos nicking to congruent to 30% of the level observed for the wild-type substrate. In the presence of ATP, the R3-R2-R1- mutation had no significant effect on terminase nicking of the 1 strand and reduced r-strand nicking to 35% of the wild-type level. The other cosB mutations had no effect on the nicking of either DNA strand when nucleotide was added, but in the absence of ATP, most of the cos mutations resulted in some form of cosN nicking defect; the nicking defects, however, are milder than the in vivo packaging defects that result from the mutations. Quantitatively, only the effect of the R3-R2-R1- mutation on in vitro cosN nicking is reflective of the growth defect exhibited by a R3-R2-R1- phage but the nicking defect is only observed when ATP is omitted from the reaction. The proposal that the cosB mutations primarily affect DNA packaging rather than cosN nicking is discussed. All of the cosB mutations affect r-strand nicking to a greater extent than 1-strand nicking, implying that the interaction of terminase with the left half of cosN occurs via the direct recognition of cosNL by terminase. The level of DNA substrate required for half-maximal cos nicking is approximately equivalent for reactions performed in the presence or absence of ATP, indicating that ATP does not increase the affinity of terminase for cosB. ATP does accelerate the rate of cos nicking, suggesting that the role of ATP in promoting nicking of the cosB- DNAs is primarily to increase the rate of conversion of a cosN-terminase complex into product. A possible fourth R site, R4, is located on the other side of cosN from cosB.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphate↗
PubMed1993

A site required for termination of packaging of the phage lambda chromosome.

Lambda chromosomes are cut and packaged from concatemeric DNA by phage enzyme terminase. Terminase initiates DNA packaging by binding at a site called cosB and introducing staggered nicks at an adjacent site, cosN, to generate the left cohesive end of the DNA molecule to be packaged. After DNA packaging terminase recognizes and cuts the terminal cosN, an event that does not require a wild-type cosB. In this work a site, called cosQ, has been identified that is required for termination of DNA packaging. cosQ, defined by mutations in a sequence called R4, is located approximately 30 bp upstream from cosN. The order of sites is cosQ-cosN-cosB. Helper packaging of repressed, tandem prophage chromosomes demonstrated that a cosQ point mutation affects DNA packaging only when placed at the terminal cos site, whereas cosB mutations only affect packaging initiation. In vitro packaging studies confirmed that cosQ mutations do not affect packaging initiation. In vivo studies indicated that cosQ mutations do not affect cutting of initial cos sites but do cause a defect in packaging termination. cosQ mutants accumulated expanded phage heads, indicating that cosQ mutations affect a step that occurs after packaging of a substantial length of phage DNA. These results show that cosQ mutations define a site required for use of cos sites present at the ends of lambda chromosomes undergoing packaging. Available evidence suggests that other viruses, including phages T3 and T7 and the herpesviruses, may ultimately prove to use cosQ-like sites for packaging termination.

Bacteriophage lambda↗
PubMed1993

Sequence analysis of the phage 21 genes for prohead assembly and head completion.

Phage 21 is a temperate lambdoid coliphage, and its head-encoding genes, as well as those of phage lambda, are descended from a common ancestral phage. The head protein-encoding genes of phage 21 have been sequenced, confirming earlier genetic studies indicating that the head-encoding genes of 21 and lambda are analogous in location, size, and function. The phage 21 head-encoding genes identified (and their lambda analogues) include: 3(W), 4(B), 5(C), 6(Nu3), shp (D), 7(E), and 8(FII), respectively. An open reading frame, orf1, is analogous in position and shares some sequence identity with FI, a phage lambda gene involved in DNA packaging. The phage 21 major head protein, gp7, is predicted to have strong sequence identity (65%) with the lambda major capsid protein, gpE, including amino acids known to be important for capsid form determination. The nested genes 5/6 of phage 21 and C/Nu3 of lambda differ by several rearrangements including deletions and a triplication. The possibility that lambda genes C/Nu3 evolved from ancestal nested genes containing a triplication is discussed.

Amino Acid Sequence↗
PubMed1993

Function of IHF in lambda DNA packaging. I. Identification of the strong binding site for integration host factor and the locus for intrinsic bending in cosB.

Integration host factor (IHF) plays an accessory role in lambda DNA packaging. IHF affects the interaction of the lambda DNA packaging protein, terminase, with cos, the site on lambda DNA at which terminase binds and introduces staggered nicks to generate cohesive ends of mature lambda chromosomes. cos includes cosB, the terminase binding site and cosN, the adjacent nicking site. cosB includes multiple binding sites for gpNu1, the small subunit of terminase, and an IHF binding site, I1. I1 contains two overlapping sequences, called I1A and I1B, that closely match the consensus sequence for IHF binding sites. The I1A sequence was determined to be the site of IHF binding by hydroxyl radical footprinting experiments. Comparison of the pattern of IHF-induced enhancements and diminishments at I1 with published patterns for IHF binding sites at the lambda attachment site identifies I1A as the IHF binding site at I1. The conclusion that I1A is the IHF binding site was confirmed by studies with DNA mutant in I1A. The I1A- mutation, consisting of three adjacent base-pair changes in I1A, abolished IHF binding. In contrast to the I1A- mutation, a mutation in I1B, also consisting of three adjacent base-pair changes, caused a reduction in the affinity of IHF for I1A, and caused a reduction in the magnitude of the net intrinsic bending of cos lambda.

Bacterial Proteins↗
PubMed1993

Function of IHF in lambda DNA packaging. II. Effects of mutations altering the IHF binding site and the intrinsic bend in cosB on lambda development.

cosB is the binding site on lambda DNA for terminase, the phage DNA packaging protein. cosB contains three binding sites for gpNu1, the small subunit of terminase, and a site for integration host factor (IHF). IHF plays an accessory role in lambda DNA packaging, and IHF stimulates the burst size of lambda several-fold, presumably by assisting the interaction of terminase with cosB. The present work includes a study of the effect on lambda development of a mutation, called I1A-, which consists of three adjacent base-pair changes in the IHF binding site. The I1A- mutation was found to abolish IHF stimulation of the lambda burst size, indicating that IHF is unable to bind to the mutant I1A site. A second mutation, called I1B- and also consisting of three adjacent base-pair changes, is a mutation that reduces an intrinsic bend found in cosB. lambda I1B- was more dependent on IHF than lambda+, raising the possibility that the intrinsic bend in cosB plays a role in cos function for lambda+ under the IHF- conditions. In vitro DNA packaging experiments established that the I1 mutations affect DNA packaging per se. A series of Nu1 mutations that create terminases able to suppress a variety of cosB defects were found to suppress the defects of the I1A- and I1B- mutations under IHF- conditions.

Bacterial Proteins↗
PubMed1993

Sites and gene products involved in lambdoid phage DNA packaging.

21 is a temperate lambdoid coliphage, and the genes that encode the head proteins of lambda and 21 are descended from a common ancestral bacteriophage. The sequencing of terminase genes 1 and 2 of 21 was completed, along with that of a segment at the right end of 21 DNA that includes the R4 sequence. The R4 sequence, a site that is likely involved in termination of DNA packaging, was found to be very similar to the R4 sequences of lambda and phi 80, suggesting that R4 is a recognition site that is not phage specific. DNA packaging by 21 is dependent on a host protein, integration host factor. A series of mutations in gene 1 (her mutations), which allow integration host factor-independent DNA packaging by 21, were found to be missense changes that affect predicted alpha-helixes in gp1. gp2, the large terminase subunit, is predicted to contain an ATP-binding domain and, perhaps, a second domain important for the cos-cutting activity of terminase. orf1, an open reading frame analogous in position to FI, a lambda gene involved in DNA packaging, shares some sequence identity with FI. orf1 was inactivated with nonsense and insertion mutations; these mutations were found not to affect phage growth. 21 was also not able to complement a lambda FI mutant.

Amino Acid Sequence↗
PubMed1992

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.

Bacteriophage lambda↗
PubMed1992

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.

Amino Acid Sequence↗
PubMed1992

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.

Bacteriophage lambda↗
PubMed1991

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.

Bacteriophage lambda↗
PubMed1991

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.

Bacteriophage lambda↗
PubMed1991

Interference with phage lambda development by the small subunit of the phage 21 terminase, gp1.

Bacteriophage lambda development is blocked in cells carrying a plasmid that expresses the terminase genes of phage 21. The interference is caused by the small subunit of phage 21 terminase, gp1. Mutants of lambda able to form plaques in the presence of gp1 include sti mutants. One such mutation, sti30, is an A. T-to-G.C transition mutation at base pair 184 on the lambda chromosome. The sti30 mutation extends the length of the ribosome-binding sequence of the Nul gene that is complementary to the 3' end of the 16S rRNA from GGA to GGAG. The sti30 mutation causes a approximately 50-fold increase in the level of expression of a Nul-lacZ reporter gene, indicating that the sti30 mutation overcomes the gp1 inhibition by increasing the level of expression of gpNul. Although the Nul and A genes of lambda overlap, the sti30 mutation has little effect on the level of gpA expression, indicating that translational coupling does not occur.

Bacteriophage lambda↗
PubMed1990

Structure of the bacteriophage lambda cohesive end site: bent DNA on both sides of the site, cosN, at which terminase introduces nicks during chromosome maturation.

Packaging of lambda DNA is mediated by the phage-encoded enzyme, terminase, which acts at a site termed cos. cos consists of cosB, the site where terminase binds lambda DNA, and cosN, the site where nicks are introduced to generate the cohesive ends of virion DNA. cos contains multiple binding sites for gpNu1, the small subunit of terminase, and integration host factor (IHF), an Escherichia coli DNA binding protein. Polyacrylamide gel electrophoresis of circularly permuted segments of cos DNA has been used to locate major bend loci in cos. Two major bends have been located; one bend is ca 146 bp to the left of cosN while the second major bend is located ca 92 bp to the right of cosN. The major bend at 92 coincides roughly with I1, the strongest IHF binding site in cos. The possible roles of static bending in DNA packaging are discussed.

Bacteriophage lambda↗
PubMed1988

The interaction of Escherichia coli integration host factor with the cohesive end sites of phages lambda and 21.

The interaction of E. coli integration host factor (IHF) with the cohesive end sites (cos's) of phages lambda and 21 has been studied by the DNAase I footprinting technique. Six potential sites in cos lambda differ from the consensus IHF binding sequence by 1 to 3 base pairs. Of the six, one site, I1, binds IHF strongly. The I1 segment protected by IHF contains two sequences that closely match the IHF consensus binding sequence. Another site, I2, binds IHF moderately well, and three sites: 10', 13 and 14 bind IHF very weakly. The 10 site does not bind IHF under the conditions used here. In phage 21 the DNA segment extending to the right from the cohesive ends, which contains three potential IHF binding sites, was examined. Two sites bind IHF well; I1, the 21 analogue of one of the lambda I1 sites, and I0, a site not analogous to a lambda site. The third 21 site, I2, binds IHF moderately well, as does the analogous I2 site in lambda. The significance of the results for lambda DNA packaging is discussed.

Bacterial Proteins↗
PubMed1988

Bacteriophage lambda DNA packaging: a mutant terminase that is independent of integration host factor.

Lambda+ is able to grow in Escherichia coli cells lacking integration host factor (IHF), producing a burst of approximately 25% that produced in IHF+ cells. In vitro, however, we find that the lambda DNA packaging enzyme terminase is strongly dependent on IHF in both cos cleavage reactions and DNA packaging reactions. The cos59 mutation renders lambda dependent on IHF in vivo. The cos59 mutation is a deletion of 3 base pairs at the XmnI site in the cohesive end site (cos) of lambda. Variants of lambda cos59 that were able to grow in the absence of IHF were isolated and found to carry a mutation, called ms1, in the Nu1 gene, which codes for the small subunit of terminase. The Nu1ms1 mutation results in a change of the 40th amino acid of the Nu1 gene product from leucine to phenylalanine. The Nu1ms1 terminase was independent of IHF in packaging reactions in vitro. The results indicate that the mutation either renders terminase: (1) able to utilize some host protein other than IHF, or (2) totally independent of host factors.

Bacterial Proteins↗
PubMed1988

The bacteriophage lambda cohesive end site: isolation of spacing/substitution mutations that result in dependence on Escherichia coli integration host factor.

Substitution, insertion and deletion mutations have been constructed at the XmnI restriction site in cos lambda. The XmnI site is located between cosB, the site where terminase binds lambda DNA; and cosN, the site where terminase introduces staggered nicks to generate cohesive ends. Substitution mutations and deletion of a base pair (a -1 change) do not obviously affect lambda growth and DNA packaging. Changes of -2, +2 and -3 render lambda unable to grow on host cells lacking integration host factor (IHF). The -3 mutant has a reduced burst size in IHF+ cells, due to a defect in the initiation of packaging. A -7 deletion mutation is lethal. Models for the basis of these mutational effects are discussed.

Bacterial Proteins↗
PubMed1988

Partial FI gene-independence of lambda-21 hybrid phages specifying chimeric terminases.

The role of the FI gene in the life cycles of a series of lambda-21 hybrid phages that produce chimeric lambda-21 terminases has been examined. An isogenic series of FI+ and FI- derivatives of the hybrids was constructed, and the growth properties of the phages were examined. It was found that three of the four hybrids (hybrids 51, 67, and 33) are able to form plaques and produce a small burst in the absence of the FI gene product (gpFI), but each of the three phages is much healthier in the presence of gpFI. It is concluded that each of the three chimeric terminases is dependent on gpFI. The ability of the FI- hybrids to grow better than lambda FI- is postulated to be due to a minor qualitative or quantitative difference between the chimeric terminases and lambda terminase. The fourth hybrid (54), known from earlier work to produce an infirm terminase, is more dependent on gpFI than the other hybrids and lambda itself.

Bacteriophage lambda↗