DNA Synthesis at the Site of a Red-Mediated Exchange in Phage λ

In phage lambda, progeny particles bearing unreplicated chromosomes are recombinant by action of lambda's Red system only near the right end of the chromosome. These recombinants are frequently heterozygous (heteroduplex) for markers located there. In replication-blocked crosses involving two heavy-labeled parents we find that particles in the solitary peak, containing progeny with fully conserved DNA, vary in density. Those on the heavy side of this peak are more apt to be heterozygous than are those on the light side. The data fit a model in which a double chain cut at cos, lambda's packaging origin, is followed by partial exonucleolytic degradation of lambda's r chain from the right end leftward. The exposed l chain, which thereby constitutes a 3' overhang, invades an intact, circular homologue after itself suffering some degradation. Completion of the recombinant chromosome sometimes involves DNA synthesis primed by the invading chain.     

Endopeptidase Activity of Phage λ-Endolysin

JACOB and Fuerst^1,2 demonstrated the presence of a bacteriolytic enzyme (λ-endolysin) in the induced cultures of lysogenic Escherichia coli K12 (λ). The enzyme was later identified as the product of gene R; of phage λ³ which is involved in bacterial lysis at the end of a latent period. The enzyme is apt to form spheroplast-like structures in E. coli² and one would therefore expect its substrate to be murein.     

Gene Regulation in N Mutants of Bacteriophage λ

Mutants (N(-)nin) of bacteriophage lambda in which the N gene product is not required for growth on wild-type Escherichia coli do not plate on recA bacterial mutants. Secondary mutants, selected for growth on recA, lie within the immunity region to the right of gene cI and appear identical to the cro mutants of Eisen et al. In an N(+) phage, a cro mutation causes enhanced and prolonged production of lambda exonuclease. N(-)cro phages make no detectable exonuclease, but show an increased rate of specific excision from lysogens and are excluded by P2 prophage. These properties, together with the ability to plate on recA, suggest that N(-)cro phages express genes to the left of N at a rate that is very low but higher than that for N(-)cro(+) phages. N(-)nin phages can integrate at the normal site on the bacterial chromosome, but specific excision from lysogens is immeasurably low.     

Directionality and Nonreciprocality of Chi-Stimulated Recombination in Phage λ

Chi's are genetic elements that stimulate generalized recombination in their locale in phage λ. All Chi's, wherever located on λ's chromosome, act asymmetrically in crosses blocked in DNA replication: (1) They stimulate exchange primarily to their left on the conventional λ map, and (2) the stimulated exchange is frequently nonreciprocal, the recombinant carrying the Chi element being produced less often than the complementary product.     

Molecular Analysis of λbio Transducing Phage Produced by Oxolinic Acid-Induced Illegitimate Recombination in Vivo

To study the mechanism of DNA gyrase-mediated illegitimate recombination in Escherichia coli, we examined the formation of λ Spi(-) phage during prophage induction. The frequency of Spi(-) phage was two to three orders of magnitude higher in the presence of oxolinic acid, an inhibitor of DNA gyrase A subunit, than in the absence of the drug, while it was very low in nalA(r) bacteria with the drug. RecA function is not required for the formation of these phages, indicating that this enhancement is not caused by the expression of SOS-controlled genes. Analyses of att region and recombination junctions of Spi(-) phages revealed that they have essentially the same structures as λbio transducing phages but are classified into two groups with respect to recombination sites. In the majority class of the transducing phages, there were not more than 3-bp homologies bewteen the parental E. coli bio and λ recombination sites. In the minority class of the transducing phages, on the other hand, 9-10-bp homologies were found between the parental recombination sites. These results suggested that oxolinic acid-induced illegitimate recombination takes place by two variants of a DNA gyrase-dependent mechanism.     

Ubiquitylation of DNA polymerase λ

DNA polymerase (pol) λ, one of the 15 cellular pols, belongs to the X family. It is a small 575 amino-acid protein containing a polymerase, a dRP-lyase, a proline/serine rich and a BRCT domain. Pol λ shows various enzymatic activities including DNA polymerization, terminal transferase and dRP-lyase. It has been implicated to play a role in several DNA repair pathways, particularly base excision repair (BER), non-homologous end-joining (NHEJ) and translesion DNA synthesis (TLS). Similarly to other DNA repair enzymes, pol λ undergoes posttranslational modifications during the cell cycle that regulate its stability and possibly its subcellular localization. Here we describe our knowledge about ubiquitylation of pol λ and the impact of this modification on its regulation.     

“Maturation” of DNA duplexes

Reassociation of typical single-copy DNAs, like E. coli DNA, even when performed at relatively low temperatures, results in the formation of perfect duplexes with thermal stability very close to that of the native DNA. In contrast, duplexes of mouse repeated DNA as well as duplexes of Streptomyces DNA prepared under the same conditions, show a low thermal stability and undergo post-reassociation changes upon prolonged incubation. These changes, called maturation of the DNA duplexes, result in increasing of their thermal stability. Some of the factors affecting the rate of maturation are studied. The implication of the maturation process in reassociation analysis and in characterization of the heterogeneity of DNA is discussed.     

A Stereoselective Synthesis of (±)-endo-Brevicomin,a Pheromone Inhibitor Produced by Dendroctonus Bark Beetles

Prediction of elution curves in gel chromatography was attempted on the basis of a mass balance model which gave consideration to gel phase diffusion and longitudinal dispersion in a column. The basic differential equations for the model were solved by means of Laplace transformation, and then the solution in Laplace domain was inverted into time domain numerically. The calculated elution curves were in good agreement with the experimental ones of NaCl and myoglobin with various Sephadex gel columns. This indicated the validity of the calculation method and the model employed in this study.

Furthermore, the elution curves were calculated tentatively for various combinations of the parameters appearing in the mass balance model. Then, the magnitude of peak asymmetry, the shift of peak position and the maximum peak height of the elution curves were correlated with various parameters and operational variables. These correlations might permit prediction of suitable operational conditions for gel chromatography, especially for molecular weight determination.     

On the Clustered Exchanges of the Recbcd Pathway Operating on Phage λ

Lytic cycle crosses of Red(-) Gam(-) phage λ were conducted in rec(+) Escherichia coli carrying one or another plasmid with homology to λ. λ X λ recombinants and λ X plasmid recombinants were formed by RecBCD-mediated recombination. We showed previously that the act of recombining with a plasmid alters the disposition of selected λ X λ exchanges. This work reports that the relationships between the λ X plasmid and the λ X λ exchanges is unaltered by the removal from one λ parent of the homology shared with the plasmid. This result supports our view that a reciprocal exchange, allowing for cointegrate formation, is associated with but mechanistically separable from a (presumably) nonreciprocal λ X λ exchange. The nature of this relationship is independent of λ's Rap function, which is shown to alter the ratio of cointegrate formation (splices) to marker pick-up (patches) in λ X plasmid recombination mediated by the RecBCD pathway.     

Synthesis of oligosaccharides by reversal of a fungal β-glucanase

Summary A crude commercial preparation of -glucanase fromPenicillium emersonii was used to synthesise glucose-containing oligosaccharides by condensation reactions in high concentrations of glucose at elevated temperature. Gentiobiose, laminaribiose, cellobiose, isomaltose and trehalose were identified as products. Heterooligosaccharides were produced by enzyme in some mixtures of glucose and an acceptor sugar. High performance ion-exchange chromatography was used to analyse synthetic products.     

Direct cloning of cDNA inserts from λgt11 phage DNA into a plasmid vector by a novel and simple method

Bacteriophage λgt11 has been used quite extensively for producing cDNA libraries. The cDNA inserts are usually subcloned into a plasmid vector for large scale production and analysis. However, isolating the recombinant DNA of interest from the phage clones can be a tedious task. Since the E. coli strain Y1088 used for λgt11 phage infection carries a pBR322-derived plasmid endogenously, we reasoned that this endogenous plasmid could be used directly for cloning the cDNA phage insert. In this report, we describe a method in which cDNA inserts from λgt11 phage were cloned directly into the pBR322 plasmid vector, by-passing the time-consuming procedures of preparing plasmid DNA as a subcloning vector. This method is likely to be extended to the cloning of DNA inserts derived from other phage λ vectors when bacteria containing endogenous pBR322 are used as host cells.     

Homology Requirements for Double-Strand Break-Mediated Recombination in a Phage λ-Td Intron Model System

Many group I introns encode endonucleases that promote intron homing by initiating a double-strand break-mediated homologous recombination event. A td intron-phage λ model system was developed to analyze exon homology effects on intron homing and determine the role of the λ 5'-3' exonuclease complex (Redαβ) in the repair event. Efficient intron homing depended on exon lengths in the 35- to 50-bp range, although homing levels remained significantly elevated above nonbreak-mediated recombination with as little as 10 bp of flanking homology. Although precise intron insertion was demonstrated with extremely limiting exon homology, the complete absence of one exon produced illegitimate events on the side of heterology. Interestingly, intron inheritance was unaffected by the presence of extensive heterology at the double-strand break in wild-type λ, provided that sufficient homology between donor and recipient was present distal to the heterologous sequences. However, these events involving heterologous ends were absolutely dependent on an intact Red exonuclease system. Together these results indicate that heterologous sequences can participate in double-strand break-mediated repair and imply that intron transposition to heteroallelic sites might occur at break sites within regions of limited or no homology.     

Translesion Synthesis of Abasic Sites by Yeast DNA Polymerase ?

Studies of replicative DNA polymerases have led to the generalization that abasic sites are strong blocks to DNA replication. Here we show that yeast replicative DNA polymerase ϵ bypasses a model abasic site with comparable efficiency to Pol η and Dpo4, two translesion polymerases. DNA polymerase ϵ also exhibited high bypass efficiency with a natural abasic site on the template. Translesion synthesis primarily resulted in deletions. In cases where only a single nucleotide was inserted, dATP was the preferred nucleotide opposite the natural abasic site. In contrast to translesion polymerases, DNA polymerase ϵ with 3′–5′ proofreading exonuclease activity bypasses only the model abasic site during processive synthesis and cannot reinitiate DNA synthesis. This characteristic may allow other pathways to rescue leading strand synthesis when stalled at an abasic site.     

Interaction between the Sbcc Gene of Escherichia Coli and the Gam Gene of Phage λ

gam mutants of phage lambda carrying long palindromes fail to form plaques on wild-type Escherichia coli but do grow on strains that are mutant in the sbcC gene. gam + lambda carrying the same palindrome grow on both hosts and on a host deleted for the recB, C and D genes. These results suggest that the Gam protein of lambda, known to interact also with E. coli's recBCD protein, can interact with the product of the sbcC gene.     

Temperature-Sensitive DNA Synthesis Mutants of BACILLUS SUBTILIS—APPENDIX: Theory of Density Transfer for Symmetric Chromosome Replication

A simple method for characterizing temperature-sensitive DNA synthesis mutants is described. The method uses density transfer and transformation techniques and is based on expected theoretical behavior of the chromosome population. A direct proof of inhibition of initiation of DNA replication is provided. The mutant dna-1, showing quick inhibition of initiation, is further characterized and mapped. An independent method for mapping genetic markers close to the origin, based on their transfer behavior after inhibition of initiation, is presented.     

DNA Packaging by λ-Like Bacteriophages: Mutations Broadening the Packaging Specificity of Terminase,the λ-Packaging Enzyme

The DNA-packaging specificities of phages λ and 21 depend on the specific DNA interactions of the small terminase subunits, which have support helix-turn-recognition helix-wing DNA-binding motifs. λ-Terminase with the recognition helix of 21 preferentially packages 21 DNA. This chimeric terminase''s ability to package λDNA is reduced ∼20-fold. Phage λ with the chimeric terminase is unable to form plaques, but pseudorevertants are readily obtained. Some pseudorevertants have trans-acting suppressors that change codons of the recognition helix. Some of these codons appear to remove an unfavorable base-pair contact; others appear to create a novel nonspecific DNA contact. Helper-packaging experiments show that these mutant terminases have lost the ability to discriminate between λ and 21 during DNA packaging. Two cis-acting suppressors affect cosB, the small subunit''s DNA-binding site. Each changes a cosB^λ-specific base pair to a cosB²¹-specific base pair. These cosB suppressors cause enhanced DNA packaging by 21-specific terminase and reduce packaging by λ-terminase. Both the cognate support helix and turn are required for strong packaging discrimination. The wing does not contribute to cosB specificity. Evolution of packaging specificity is discussed, including a model in which λ- and 21-packaging specificities diverged from a common ancestor phage with broad packaging specificity.VIRUSES must package viral chromosomes from nucleic acid pools that include host-cell nucleic acids, so specific recognition of the viral nucleic acid is essential during virion assembly. For large DNA viruses, including the tailed double-strand DNA (dsDNA) bacteriophages, the herpesviruses, and the adenoviruses, DNA-packaging proteins recognize specific sequences on the viral chromosomes (reviewed in Baines and Weller 2005 and Ostapchuk and Hearing 2005, respectively). For the dsDNA viruses that produce virion chromosomes by processing concatemeric DNA, a viral terminase enzyme functions in the recognition and cutting of concatemeric DNA and subsequently sponsors DNA translocation. λ-Terminase is a heterooligomer of large and small subunits, gpA and gpNu1, respectively. Cutting of concatemeric DNA is carried out by gpA''s endonuclease activity (Becker and Gold 1978; Davidson and Gold 1992; Hwang and Feiss 1996). Three DNA subsites, cosQ, cosN, and cosB, are contained in the ∼200-bp-long cos site and orchestrate DNA packaging through interactions with terminase (Figure 1A; reviewed in Feiss and Catalano 2005). gpA introduces staggered nicks in cosN to generate the 12-bp cohesive ends of mature λDNA molecules. Efficient and accurate nicking of cosN requires anchoring of gpA by gpNu1, which binds to the adjacent cosB subsite (Higgins and Becker 1994b; Hang et al. 2001).Open in a separate windowFigure 1.—The cos and terminase region of the λ-chromosome. (A) (Top) Map of cos and the terminase-encoding Nu1 and A genes. The black bar indicates the location of the winged helix-turn-helix DNA-binding motifs in the N-terminal domain of gpNu1. (Bottom) cos subsites: cosQ is required for termination of DNA packaging; cosN is the site where the large terminase subunit, gpA, introduces staggered nicks to generate the cohesive ends of virion DNA molecules; and cosB contains the gpNu1-binding sites R1, R2, and R3 along with the IHF-binding site I1. (B) (Top) Schematic of gpNu1 residues 1–42, including the support (blue) and recognition (red) α-helixes and the wing loop (magenta). β1 and β2 are short β-strands flanking the DNA-binding elements. (Bottom) Sequences are a comparison of residues of λ''s gpNu1 and phage 21''s gp1, with conserved resides indicated by vertical lines. Note that the recognition helixes of gpNu1 and gp1 differ by four residues, all likely solvent-exposed (Becker and Murialdo 1990; de Beer et al. 2002). (C) Three-dimensional structure of the winged helix-turn-helix-containing, N-terminal domain of gpNu1 (residues 1–68) (de Beer et al. 2002). Side groups of solvent-exposed residues of the recognition helix are displayed. Color coded as in B.λ''s cosB (cosB^λ) is a complex subsite containing three copies of a gpNu1-binding sequence, the R sequence, plus a site, I1, for the integration host factor (IHF), the Escherichia coli DNA-bending protein. The order of sites is cosN–R3–I1–R2–R1. The amino-terminal half of gpNu1 contains a winged helix-turn-helix DNA-binding motif (Figure 1, B and C; Gajiwala and Burley 2000) that interacts with the R sequences. Further, the amino-terminal domain of gpNu1 is a tight dimer (Figure 1C, de Beer et al. 2002). The IHF-induced bend at I1 creates a DNA hairpin in cosB that positions the major grooves of R3 and R2 to face inward, so that the helix-turn-helix motifs of dimeric gpNu1 can be docked into them. The wing loops are positioned to make minor groove contacts with R3 and R2. Thus it is proposed that gpA is positioned to nick cosN by assembly of a bent structure with dimeric gpNu1 bound to R3 and R2 (Becker and Murialdo 1990; de Beer et al. 2002). A variety of studies indicate that the positioning of gpNu1 at R3 is crucial and that the other interactions function to create and/or stabilize the R3–gpNu1 interaction (Cue and Feiss 1993a; Higgins and Becker 1994a; Hang et al. 2001).DNA packaging initiates when terminase binds and nicks a cos. Following cosN nicking and separation of the cohesive ends, terminase remains bound to the cosB-containing chromosome end (Becker et al. 1977; Yang et al. 1997). The DNA-bound terminase docks on the portal vertex of a prohead, the empty, immature virion head shell. Assembly of the ternary prohead–terminase–DNA complex activates gpA''s potent translocation ATPase, and the viral DNA is translocated into the prohead (Yang and Catalano 2003; Dhar and Feiss 2005). Translocation brings the next cos along the concatemer to the portal-docked terminase (Feiss and Widner 1982). The downstream cos is cleaved by terminase, completing packaging of the chromosome. Recognition of the downstream cos requires cosQ and cosN (Cue and Feiss 2001). Following DNA packaging, terminase undocks from the filled head. Attachment of a tail to the DNA-filled head completes virion assembly. The undocked terminase remains bound to and sponsors the packaging of the next chromosome along the concatemer.The interactions between the recognition helix of gpNu1 and an R sequence are typical for helix-turn-helix proteins, as shown by genetic studies of chimeras between λ and its relative, phage 21, as follows: λ and 21 have similarly organized cos sites; the cosB of 21 also has the R3–I1–R2–R1 structure. Nevertheless, the two phages have distinct packaging specificities. Base-pair differences in the R sequences account for packaging specificity (Becker and Murialdo 1990; Smith and Feiss 1993). cosN and cosQ are interchangeable between λ and 21 (Feiss et al. 1981). The consensus R sequences are 5′-CGTTTCCtTTCT-3′ for cosB^λ and 5′-CaTGTCGGncCT-3′ for cosB²¹, where capitalized residues are conserved in all three R sequences of both phages; underlined and capitalized are two residues conserved in all three R sequences of both phages, but which differ between cosB^λ and cosB²¹ (Becker and Murialdo 1990). These two conserved but phage-specific base pairs are likely to be of major importance for specificity. Similarly, the recognition helixes of the helix-turn-helix motifs of the small subunits of λ (gpNu1) and 21 (gp1) terminases differ in four amino acid residues that account for packaging specificity (Figure 1; Becker and Murialdo 1990).In earlier work (de Beer et al. 2002), we showed that modifying λ-terminase by replacing the gpNu1 recognition helix with that of 21''s gp1 created a terminase (gpNu1^hy1 terminase) that was specific for the cosB of phage 21 (designated cosB²¹). That is, λ cosB²¹ Nu1^hy1 was viable, but λ cosB^λ Nu1^hy1 was inviable due to the specificity mismatch between cosB^λ and the cosB²¹-specific recognition helix of the chimeric small terminase subunit, gpNu1^hy1. The Nu1^hy1 terminase packages cosB²¹ chromosomes ∼10-fold more efficiently than it does cosB^λ chromosomes. This 10-fold discrimination between cosB²¹ and cosB^λ chromosomes is much weaker than the >10⁴-fold discrimination shown by wild-type λ and 21 terminases (de Beer et al. 2002). Because of the modest discrimination of Nu1^hy1 terminase, the yield of λ cosB^λ Nu1^hy1 is only slightly below the yield required for plaque formation. Lysates of λ cosB^λ Nu1^hy1 contain plaque-forming pseudorevertants at a level expected for single mutations. A number of these pseudorevertants were sequenced and found to contain mutations in cosB^λ or in the Nu1^hy1 gene. Here we report on in vivo packaging studies on the effects of these Nu1^hy1 and cosB^λ suppressor mutations on packaging specificity.     

Second-Site Suppressors of a Cold-Sensitive Prohead Accessory Protein of Bacteriophage φX174

This study describes the isolation of second-site suppressors which correct for the defects associated with cold-sensitive (cs) prohead accessory proteins of bacteriophage phi X174. Five phenotypically different suppressors were isolated. Three of these suppressors confer novel temperature-sensitive (ts) phenotypes. They were unable to complement a ts mutation in gene F which encodes the major coat protein of the phage. All five suppressor mutations confer nucleotide changes in the gene F DNA sequence. These changes define four amino acid sites in the gene F protein. Three suppressor mutations placed into an otherwise wild-type background display a cold resistant phenotype in liquid culture infections when compared to a wild-type phi X174 control.