Involvement of the Yeast DNA Polymerase δ in DNA Repair in Vivo

The POL3 encoded catalytic subunit of DNA polymerase δ possesses a highly conserved C-terminal cysteine-rich domain in Saccharomyces cerevisiae. Mutations in some of its cysteine codons display a lethal phenotype, which demonstrates an essential function of this domain. The thermosensitive mutant pol3-13, in which a serine replaces a cysteine of this domain, exhibits a range of defects in DNA repair, such as hypersensitivity to different DNA-damaging agents and deficiency for induced mutagenesis and for recombination. These phenotypes are observed at 24°, a temperature at which DNA replication is almost normal; this differentiates the functions of POL3 in DNA repair and DNA replication. Since spontaneous mutagenesis and spontaneous recombination are efficient in pol3-13, we propose that POL3 plays an important role in DNA repair after irradiation, particularly in the error-prone and recombinational pathways. Extragenic suppressors of pol3-13 are allelic to sdp5-1, previously identified as an extragenic suppressor of pol3-11. SDP5, which is identical to HYS2, encodes a protein homologous to the p50 subunit of bovine and human DNA polymerase δ. SDP5 is most probably the p55 subunit of Polδ of S. cerevisiae and seems to be associated with the catalytic subunit for both DNA replication and DNA repair.     

Advances in the study of protein–DNA interaction

Protein-DNA interaction plays an important role in many biological processes. The classical methods and the novel technologies advanced have been developed for the interaction of protein-DNA. Recent developments of these methods and research achievements have been reviewed in this paper.     

Conformation of the peptide antibiotic — histatin 8 in aqueous and non aqueous media

Histatin 8 (Lys¹-Phe-His-Glu-Lys⁵-His-His-Ser-His-Arg¹⁰-Gly-Tyr¹²)belongs to a group of related neutral and basic histidine richpeptides present in human salivary secretions that possess fungicidal and bactericidal activities. The conformation of thispeptide has been examined by ¹H and ¹³C 2D-NMR in DMSO-d₆, water (pH 4.0) and 40% HFA solutions. MD simulations incorporating NMR data was used to generate the solution conformations. The structures were refined by MARDIGRAS employing the RANDMARDI approach. In both DMSO-d₆ and water, the peptide is seen to adopt a -pleated sheet, while HFA induces an -helix structure. The role of these structures in its mechanism of action has been explained.     

"Resonances" in the dielectric absorption of DNA?

An attempt was made to confirm previous reports of resonant-like dielectric absorption of plasmid DNA in aqueous solutions at 1-10 GHz. The dielectric properties of the sample were measured using an automatic network analyzer with two different techniques. One technique used an open-ended coaxial probe immersed in the sample; the other employed a coaxial transmission line. No resonances were observed that could be attributed to the sample; however, resonance-type artifacts were prominent in the probe measurements. The coaxial line technique appears to be less susceptible to such artifacts. We note two important sources of error in the calibration of the automatic network analyzer using the probe technique.     

Involvement of the TPR2 subdomain movement in the activities of ?29 DNA polymerase

The polymerization domain of ϕ29 DNA polymerase acquires a toroidal shape by means of an arch-like structure formed by the specific insertion TPR2 (Terminal Protein Region 2) and the thumb subdomain. TPR2 is connected to the fingers and palm subdomains through flexible regions, suggesting that it can undergo conformational changes. To examine whether such changes take place, we have constructed a ϕ29 DNA polymerase mutant able to form a disulfide bond between the apexes of TPR2 and thumb to limit the mobility of TPR2. Biochemical analysis of the mutant led us to conclude that TPR2 moves away from the thumb to allow the DNA polymerase to replicate circular ssDNA. Despite the fact that no TPR2 motion is needed to allow the polymerase to use the terminal protein (TP) as primer during the initiation of ϕ29 TP–DNA replication, the disulfide bond prevents the DNA polymerase from entering the elongation phase, suggesting that TPR2 movements are necessary to allow the TP priming domain to move out from the polymerase during transition from initiation to elongation. Furthermore, the TPR2-thumb bond does not affect the equilibrium between the polymerization and exonuclease activities, leading us to propose a primer-terminus transference model between both active sites.     

Conditional Mutations in the Yeast DNA Primase Genes Affect Different Aspects of DNA Metabolism and Interactions in the DNA Polymerase α-Primase Complex

Different pri1 and pri2 conditional mutants of Saccharomyces cerevisiae altered, respectively, in the small (p48) and large (p58) subunits of DNA primase, show an enhanced rate of both mitotic intrachromosomal recombination and spontaneous mutation, to an extent which is correlated with the severity of their defects in cell growth and DNA synthesis. These effects might be attributable to the formation of nicked and gapped DNA molecules that are substrates for recombination and error-prone repair, due to defective DNA replication in the primase mutants. Furthermore, pri1 and pri2 mutations inhibit sporulation and affect spore viability, with the unsporulated mutant cells arresting with a single nucleus, suggesting that DNA primase plays a critical role during meiosis. The observation that all possible pairwise combinations of two pri1 and two pri2 alleles are lethal provides further evidence for direct interaction of the primase subunits in vivo. Immunopurification and immunoprecipitation studies on wild-type and mutant strains suggest that the small subunit has a major role in determining primase activity, whereas the large subunit directly interacts with DNA polymerase α, and either mediates or stabilizes association of the p48 polypeptide in the DNA polymerase α-primase complex.     

Are there insertions in the ribosomal DNA of vertebrates?

We have analysed the Eco RI restriction pattern of rDNA of the newt Triturus vulgaris and of some other amphibian species by Southern blotting and hybridization with nick-translated Xenopus rDNA prepared from the recombinant plasmids pXlr11 and pXlr12 (21). After hybridization with r11, the 28S coding fragments become visible in two bands, a prominent one of 5.3 kb and a weak band of 5.9 kb representing about 8% of the 28S genes. The evidence obtained so far by additional digestions with Bam HI and Bgl II indicates that in this species and in Triturus helveticus the coding regions of the 5.9 kb fragments are interrupted by an insertion 0.6 kb in length located in a 1.6 kb Bgl II fragment at the 3' end of the Eco RI fragment, which we believe to be the first described in a vertebrate.     

Alanine dehydrogenase of the β-lactam antibiotic producer Streptomyces clavuligerus

l-Alanine dehydrogenase was found in extracts of the antibiotic producer Streptomyces clavuligerus. The enzyme was induced by ammonia, and the level of induction was dependend on the extracellular concentration. l-Alanine was the only amino acid able to induce alanine dehydrogenase. The enzyme was characterized from a 38-fold purified preparation. Pyruvate (K _m =1.1 mM), ammonia (K _m =20 mM) and NADH (K _m =0.14 mM) were required for the reductive amination, and l-alanine (K _m =9.1 mM) and NAD (K _m =0.5 mM) for the oxidative deaminating reaction. The aminating reaction was inhibited by alanine, serine and NADPH. Alanine inhibited uncompetitively with respect to NADH (K _i =1.6 mM) and noncompetitively with respect to ammonia (K _i =2.0 mM) and pyruvate (K _i =3.0 mM). In the aminating reaction 3-hydroxypyruvate, glyoxylate and 2-oxobutyrate could partially (6–7%) substitute pyruvate. Alanine dehydrogenase from S. clavuligerus differed with respect to its molecular weight (92000) and its kinetic properties from those described for other microorganisms.Abbreviation Alanine-DH l-alanine:NAD oxidoreductase     

Defining the role of Emi1 in the DNA replication–segregation cycle

Ordered progression through the cell cycle is essential to maintain genomic stability, and fundamental to this is ubiquitin-mediated proteolysis. In particular, the anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase destabilises specific regulators at defined times in the cycle to ensure that each round of DNA replication is followed by cell division. Thus, the proper regulation of the APC/C is crucial in each cell cycle. There are several APC/C regulators that restrict its activity to specific cell cycle phases, and amongst these the early mitotic inhibitor 1 (Emi1) protein has recently come to prominence. Emi1 has been proposed to control APC/C in early mitosis; however, recent evidence questions this role. In this review we discuss new evidence that indicates that Emi1 is essential to restrict APC/C activity in interphase and, by doing so, ensure the proper coordination between DNA replication and mitosis.     

Is the nuclear matrix the site of DNA replication in eukaryotic cells?

Four types of experiment were carried out to test the recently proposed model of matrix-bound replication in eukaryotic cells. In experiments with pulse-labelling we found preferential association of newly replicated DNA with the matrix only when the procedure for isolation includes first high-salt treatment of isolated nuclei and then digestion with nucleases, or when prior to digestion the nuclei have been stored for a prolonged time. In both cases, however, evidence was found that this preferential association is due to a secondary, artifactual binding of the newly replicated chromatin region to the matrix elements. Pulse-chase experiments and experiments with continuous labelling were carried out to answer the question whether during replication the DNA is reeled through the replication complexes, i.e., whether newly replicated DNA is temporarily or permanently associated with the matrix. The results showed that at that time the matrix DNA does not move from its site of attachment. Since, according to the model of matrix-bound replication, the forks are assumed to be firmly anchored to high-salt resistant proteinaceous matrix structures, the chromatin fragments isolated with endonuclease not recognizing newly replicated DNA and purified by sucrose gradient centrifugation should be free of replication intermediates. The electronmicroscopic analysis of such fragments revealed the existence of intact replication micro-bubbles. Moreover, the fragments with replication configurations appeared as smooth chromatin fibres not attached to elements characteristic for the matrix. All these experiments suggest that the nuclear skeleton is not a native site of DNA replication in eukaryotic cells.     

DNA polymorphisms in the 5′-flanking region of the HLA-DQA1 gene

The HLA-DQA1 gene exhibits haplotype-specific restriction fragment polymorphisms due to DNA rearrangements. We found that some of these polymorphisms extend into the 5 flanking region of the gene and are distinct from other HLA-DQA1 related DNA polymorphisms so far reported. Sequencing of genomic DNA subclones derived from the 5 flanking region of HLA-DQA1 showed the presence, in a DR4 haplotype, of two repetitive elements of the Alu family, oriented in opposite directions and bracketing an approximately 3 kilobase region immediately adjacent to the promoter of the gene. When DNAs extracted from several cell lines were analyzed by genomic hybridization using single-copy probes relative to these intervening sequences, polymorphisms were observed. No structural alterations of the gene immediately outside the DNA portion delimited by the two Alu elements were observed, thus suggesting that polymorphisms of the 5 end of HLA-DQA1 may be limited to the intervening region between the two Alu repeats. The latter includes upstream regulatory elements controlling the expression of the genes. The possibility that the structure of the DNA in this region may influence the regulation of HLA-DQA1 gene expression in different haplotypes is discussed.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession number M72411. Address correspondence and offprint requests to: J. Guardiola.     

Rational antibiotic design: in silico structural comparison of the functional cavities of penicillin-binding proteins and ß-lactamases

The class of ß-lactam antibiotics has proven highly efficient in targeting bacterial penicillin-binding proteins (PBP) leading to the blocking of the bacterial cell wall synthesis. However, the benefit of these drugs is limited because of bacterial resistance mechanisms; the most widespread resistance involves ß-lactamase enzymes (ßLACT) that inactivate ß-lactam-based molecules. We focused on PBPs and ßLACTs from enterobacteria, and performed a detailed in silico study of PBPs whose inactivation is lethal for the bacteria and of ßLACTs that have a PBP-type catalytic mechanism. The comparison of the sequences and structures of PBPs and ßLACTs shows an almost perfect conservation of the catalytic site, and a high spatial resemblance of the whole functional cavity despite a very low overall sequence identity. Some notable differences in the functional cavity were observed in the vicinity of the catalytic site: four tyrosines are well conserved in the PBPs, whereas the residues occurring at equivalent positions in the ßLACT families present other physicochemical properties. These tyrosines are thus good candidates to be targeted in designing new antibiotic molecules with increased affinity and specificity for PBPs, with the goal of overcoming drug resistance. Our analysis also identified residues that have similar characteristics in most ßLACT families and different properties in PBPs; these are interesting targets for new ligands that specifically inhibit ßLACT proteins. The in silico approach presented here can be extended to other protein systems in view of guiding and improving rational drug design.     

WRNIP1 functions upstream of DNA polymerase η in the UV-induced DNA damage response

WRNIP1 (WRN-interacting protein 1) was first identified as a factor that interacts with WRN, the protein that is defective in Werner syndrome (WS). WRNIP1 associates with DNA polymerase η (Polη), but the biological significance of this interaction remains unknown. In this study, we analyzed the functional interaction between WRNIP1 and Polη by generating knockouts of both genes in DT40 chicken cells. Disruption of WRNIP1 in Polη-disrupted (POLH^−/−) cells suppressed the phenotypes associated with the loss of Polη: sensitivity to ultraviolet light (UV), delayed repair of cyclobutane pyrimidine dimers (CPD), elevated frequency of mutation, elevated levels of UV-induced sister chromatid exchange (SCE), and reduced rate of fork progression after UV irradiation. These results suggest that WRNIP1 functions upstream of Polη in the response to UV irradiation.     

Can landscape ecology untangle the complexity of antibiotic resistance?

Bacterial resistance to antibiotics continues to pose a serious threat to human and animal health. Given the considerable spatial and temporal heterogeneity in the distribution of resistance and the factors that affect its evolution, dissemination and persistence, we argue that antibiotic resistance must be viewed as an ecological problem. A fundamental difficulty in assessing the causal relationship between antibiotic use and resistance is the confounding influence of geography: the co-localization of resistant bacterial species with antibiotic use does not necessarily imply causation and could represent the presence of environmental conditions and factors that have independently contributed to the occurrence of resistance. Here, we show how landscape ecology, which links the biotic and abiotic factors of an ecosystem, might help to untangle the complexity of antibiotic resistance and improve the interpretation of ecological studies.     

Ordered distribution of α-putrescinylthymine in the DNA of bacteriophage φW-14

In the DNA of bacteriophage W-14, half the thymine is replaced by a -putrescinylthymine (putThy). Analysis of monopyrimidine tracts shows that putThy and thymine are distributed nonrandomly in W-14 DNA: The sequence purine-putThy-purine occurs more than twice as frequently as the sequence purine-thymine-purine, which means that the post-replicative modification of W-14 DNA is sequence-specific.     

Molecular characterization of co-transcribed genes from Streptomyces tendae Tü901 involved in the biosynthesis of the peptidyl moiety of the peptidyl nucleoside antibiotic nikkomycin

Six genes (nikA, nikB, nikD, nikE, nikF, and nikG) from Streptomyces tendae Tü901 were identified by sequencing the region surrounding the nikC gene, which encodes L-lysine 2-aminotransferase, previously shown to catalyze the initial reaction in the biosynthesis of hydroxypyridylhomothreonine, the peptidyl moiety of the peptidyl nucleoside antibiotic nikkomycin. These genes, together with the nikC gene, span a DNA region of 7.87 kb and are transcribed as a polycistronic mRNA in a growth-phase–dependent manner. The sequences of the deduced proteins NikA and NikB exhibit significant similarity to those of acetaldehyde dehydrogenases and 4-hydroxy-2-oxovalerate aldolases, respectively, which are involved in meta-cleavage degradation of aromatic hydrocarbons. The predicted NikD gene product shows sequence similarity to monomeric sarcosine oxidases, and the deduced NikE protein belongs to the superfamily of adenylate-forming enzymes. The nikF gene and the nikG gene encode a cytochrome P450 monooxygenase and a ferredoxin, respectively. Disruption of any of the genes nikA, nikB, nikD, nikE and nikF by insertion of a kanamycin resistance cassette abolished formation of the biologically active nikkomycins I, J, X, and Z. The nikA, nikB, nikD, and nikE mutants accumulated the nucleoside moieties nikkomycins C_x and C_z. In the nikD and nikE mutants nikkomycin production (nikkomycins I, J, X, Z) could be restored by feeding with picolinic acid and hydroxypyridylhomothreonine, respectively. The nikF mutant exclusively produced novel derivatives, nikkomycins L_x and L_z, which contain pyridylhomothreonine as the peptidyl moiety. Our results indicate that the nikA, nikB, nikD, nikE, nikF, and nikG genes, in addition to nikC, function in the biosynthetic pathway leading to hydroxypyridylhomothreonine; the putative activities of each of their products are discussed. Received: 1 February 1999 / Accepted: 29 April 1999