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.     

Break-Join Recombination in Phage λ

In phage lambda, when DNA replication is blocked, recombination mediated by the Red pathway occurs only near the double-chain break site, cos, that defines the termini of the virion chromosome. The recombinants initiated by cos contain newly synthesized DNA near cos, in amount corresponding to a few percent of the length of lambda. A restriction enzyme cut delivered to one parent far from cos results in elevated recombination near the restriction site. Recombinants induced by this cut have a similarly small amount of DNA synthesis in these replication-blocked crosses. When restriction cuts are introduced in the presence of normal amounts of all of the DNA replication enzymes, many of the resulting recombinants still enjoy, at most, a small amount of DNA synthesis associated with the exchange event. Thus, these experiments fail to support the previously considered possibility that Red-mediated recombination in lambda proceeds largely through a break-copy pathway.     

N-terminal modification of proteins—a review

The first efforts to modify the terminal α-amino groups of proteins without reaction of the ?-amino groups of lysine residues made use of their lower pK values. A pH below 7 favors modification of weaker bases, since the stronger bases, although more reactive, are protected to an even greater extent by protonation. Unfortunately, this approach only favors modification of terminal over side-chain amino groups to a limited extent. N-Terminal serine and threonine residues may be selectively acylated on the amino group by an acyl transfer reaction after a peptide has been selectively acylated on its hydroxyl groups. This approach is severely limited by the need for the peptide to be stable to the acidic and anhydrous conditions necessary for selective O-acylation, and to the alkaline conditions necessary for removing the remaining O-acyl groups. Terminal serine and threonine residues may also be selectively oxidized by periodate, since this reaction is a thousand-fold faster than other oxidations of periodate, e.g., of 1,2-diols or disulfides. Further, it forms glyoxyloyl groups, which may be converted into terminal glycine residues by transamination. The last observation provided the basis for the one general modification of N-terminal residues, namely their conversion into 2-oxoacyl groups by reaction of the α-amino group with glyoxylate, a reaction catalysed by a bivalent cation, e.g., Cu²⁺, and a base, e.g., acetate. Participation of the neighboring peptide bond in the reaction ensures specificity of the reaction for the N-terminus. Scission of the N-terminal residue is possible after such a transamination; hence residues may be removed from the N-terminus under nondenaturing conditions. Other exploitations of transamination may be developed.     

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.     

Sequence preference of α-helix N-terminal tetrapeptide

The α-helix is the most abundant secondary structure in proteins. Due to the specific i, i+4 hydrogen bond pattern, the two termini have unsatisfied hydrogen bonds, and are less constrained; in order to compensate for this, specific residues are preferred for the terminal positions. However, a naive combination of the statistically-preferred residues for each position may not result in a stable N-terminal helical sequence. In order to provide a set of preferable N-terminal peptides for α-helix design, we have studied the N-terminal tetrapeptide sequence motifs that are favorable for helix formation using statistical analysis and atomistic simulations. A set of tetrapeptide sequences including TEEE and TPEE were found to be favorable motifs. In addition to forming more hydrogen bonds in the helical conformation, the favorable motifs also tended to form more capping boxes. To empirically test our predictions, we obtained 10 peptides with different N-terminal motifs and measured their α-helical content by circular dichroism spectroscopy. The experimental results agreed qualitatively with the statistical and simulation results. Furthermore, some of the suggested preferable tetrapeptide sequences have been successfully applied in de novo protein design.     

Preparation of Plasmid λdv from Bacteriophage λ: Role of Promoter-Operator in the Plasmid Replicon

A technique has been described for selection of bacteria carrying plasmid lambdadv. With this technique, the effects of mutations in the promoter-operators were compared on the production and perpetuation of the plasmid. It was found that "left" promoter-operator that controls leftward gene expressions can be deleted from the plasmid genome. Some mutations of "right" promoter-operator (pRoR) that controls expression of genes tof, O, and P affect the stability of the plasmid. However, the plasmid genome accomodates a variety of pRoR mutations within a reasonable but different degree of constitutivity. Some new promoter mutations that allow bypass of the pRoR cannot be carried in the plasmid genome. From these findings it was proposed that the plasmid replicon has one indispensable promoter-operator that controls expression of all the genes related to its own replication, although a variety of constitutive mutations can be accommodated in the pRorR.     

Cloning bacterial genes with plasmid λdv

Summary Fragments ofEscherichia coli DNA carrying genes for -galactosidase, or for biosynthesis of guanine or biotin were recombined in vitro with dv DNA. The cloned recombinant molecules recovered from transformedE. coli cells consisted of a biologically functional bacterial DNA fragment and, except for dv-bio30-7, two dv monomer units: one of the dv units was used as the insertion site for the bacterial DNA, whereas the other was intact, and seemed to be responsible for the replication of the recombinant plasmid. The process which gives rise to these recombinant molecules at high frequency from mixtures of monomeric dv DNA's and bacterial DNA fragments is discussed.     

N-terminal acetylation and other functions of Nα-acetyltransferases

Abstract Protein N-terminal acetylation by Nα-acetyltransferases (NATs) is an omnipresent protein modification that affects a large number of proteins. The exact biological role of N-terminal acetylation has, however, remained enigmatic for the overall majority of affected proteins, and only for a rather small number of proteins, N-terminal acetylation was linked to various protein features including stability, localization, and interactions. This minireview tries to summarize the recent progress made in understanding the functionality of N-terminal protein acetylation and also focuses on noncanonical functions of the NATs subunits.     

Mapability of Very Close Markers of Bacteriophage λ

Recombinant frequency was compared with nucleotide distance in crosses involving markers in either the P_RM or the cy region of phage λ. For each pair of markers, we performed reciprocal four-factor crosses of the following types: (I) A⁺m₁ ⁺m₂^-B^- x A^-m₁ ^-m₂⁺B⁺; and (II) A⁺m₁ ^-m₂⁺B^- x A^-m₁ ⁺m₂^-B⁺. In crosses of type I, the frequency of A⁺m₁ ⁺m₂⁺B⁺ recombinants among total (selected) A⁺B⁺ progeny was directly proportional to nucleotide distance between m₁ and m₂ in the range from 3 to 160 nucleotides. When less than three nucleotides separated m₁ and m₂, the measured yields of m₁⁺m₂⁺ recombinants were significantly depressed.

We also found that the frequency of A⁺m₁ ⁺m₂⁺B⁺ recombinants among total A⁺B⁺ progeny was significantly lower (about 10-fold on the average) in crosses of type II than in the corresponding crosses of type I. Since mismatch correction should yield A⁺m₁ ⁺m₂⁺B⁺ recombinants with approximately equal frequencies in type I and II crosses, we suggest: (1) that most m₁⁺m₂⁺ recombinants produced in type I crosses must arise from the formation of heteroduplex structures with a discontinuity (in the source of genetic information) between sites m₁ and m₂, and (2) that mismatch correction is not a major pathway for production of recombinants for close markers in normal λ infection.

     

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.     

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.     

Chromosomal arrangement of the chicken β-type globin genes

We have isolated the chicken β-type globin genes from a library of chicken DNA-λ Charon 4A recombinant bacteriophage. There are four β-type genes within this segment of the genome; we believe this represents all of the β-type genes of the chicken. The recombinant λCβG1 contains the embryonic ?- and adult β-globin genes. The hatching β^H and embryonic p-globin genes are found in the recombinant λCβG2. Although λCβG1 and λCβG2 do not physically overlap, we present evidence that all four genes are closely linked and transcribed from the same DNA strand. These experiments demonstrate that the chromosomal regions represented by λCβG1 and λCβG2 lie approximately 1.6 kb apart in the chicken genome. A third recombinant λCβG3 extends the genomic locus studied in the vicinity of the β-type globin genes to approximately 39 kb. The physical order of the chicken β-type globin genes within this segment of the chromosome is 5′ … ?-β^H-β-? … 3′. This arrangement is unique among the vertebrate β-type globin gene clusters thus far examined, in that embryonic genes are located at the 5′ and 3′ ends of the cluster while the hatching and adult genes occupy central positions.     

Structure of a dimeric fungal α-type carbonic anhydrase

The crystal structure of Aspergillus oryzae carbonic anhydrase (AoCA) was determined at 2.7 Å resolution and it revealed a dimer, which only has precedents in the α class in two membrane and cancer-associated enzymes. α carbonic anhydrases are underrepresented in fungi compared to the β class, this being the first structural representative. The overall fold and zinc binding site resemble other well studied carbonic anhydrases. A major difference is that the histidine, thought to be the major proton shuttle residue in most mammalian enzymes, is replaced by a phenylalanine in AoCA. This finding poses intriguing questions as to the biological functions of fungal α carbonic anhydrases, which are promising candidates for biotechnological applications.Structured summaryAoCA binds to AoCA by molecular sieving (View interaction)AoCA binds to AoCA X-ray crystallography (View interaction)     

Thermal mobility of β-O-4-type artificial lignin

Several lignin model polymers and their derivatives comprised exclusively of β-O-4 or 8-O-4' interunitary linkages were synthesized to better understand the relation between the thermal mobility of lignin, in particular, thermal fusibility and its chemical structure; an area of critical importance with respect to the biorefining of woody biomass and the future forest products industry. The phenylethane (C6-C2)-type lignin model (polymer 1) exhibited thermal fusibility, transforming into the rubbery/liquid phase upon exposure to increasing temperature, whereas the phenylpropane (C6-C3)-type model (polymer 2) did not, forming a char at higher temperature. However, modifying the Cγ or 9-carbon in polymer 2 to the corresponding ethyl ester or acetate derivative imparted thermal fusibility into this previously infusible polymer. FT-IR analyses confirmed differences in hydrogen bonding between the two model lignins. Both polymers had weak intramolecular hydrogen bonds, but polymer 2 exhibited stronger intermolecular hydrogen bonding involving the Cγ-hydroxyl group. This intermolecular interaction is responsible for suppressing the thermal mobility of the C6-C3-type model, resulting in the observed infusibility and charring at high temperatures. In fact, the Cγ-hydroxyl group and the corresponding intermolecular hydrogen bonding interactions likely play a dominant role in the infusibility of most native lignins.