PubMed Health⌕ Search

Biomedical subjects

J M Hermoso

Publications and source records attributed to J M Hermoso.

14 recordsLinked to original sources
PubMed1990

Characterization of a DNA binding protein of bacteriophage PRD1 involved in DNA replication.

Escherichia coli phage PRD1 protein P12, involved in PRD1 DNA replication in vivo, has been highly purified from E. coli cells harbouring a gene XII-containing plasmid. Protein P12 binds to single-stranded DNA as shown by gel retardation assays and nuclease protection experiments. Binding of protein P12 to single-stranded DNA increases about 14% the contour length of the DNA as revealed by electron microscopy. Binding to single-stranded DNA seems to be cooperative, and it is not sequence specific. Protein P12 also binds to double-stranded DNA although with an affinity 10 times lower than to single-stranded DNA. Using the in vitro phage phi 29 DNA replication system, it is shown that protein P12 stimulates the overall phi 29 DNA replication.

Coliphages↗
PubMed1990

A novel nucleoprotein complex at a replication origin.

The viral protein p6, required for the protein-primed initiation of replication of Bacillus subtilis phage phi 29, forms a nucleoprotein complex at the viral replication origins that shows novel features. Deoxyribonuclease I and hydroxyl radical footprinting data, as well as the induction of positive supercoiling, support a model in which a DNA right-handed superhelix tightly wraps around a multimeric p6 core. The interaction occurs through the DNA minor groove. The activity of p6 not only requires the formation of the complex but also its correct positioning, indicating that the other proteins involved in the initiation of replication recognize, at a precise position, either the p6 core or the DNA conformational change induced by p6.

Bacillus subtilis↗
PubMed1990

Functional domain for priming activity in the phage phi 29 terminal protein.

By site-directed mutagenesis we have changed into Cys the Ser232 of the phi 29 terminal protein (TP) involved in the covalent linkage to dAMP for the initiation of replication. The mutant TP, highly purified, had about 0.7% of the priming activity of the wild-type (wt) protein p3. The linkage between the mutant protein p3 and dAMP was more labile to piperidine treatment than the serine-dAMP linkage in the wt protein p3, suggesting the presence of a different kind of linkage, Cys-dAMP. In the other three mutant TPs, residues Leu220, Ser223 and Ser226 were independently changed into Pro; the purified TP mutants had about 3%, 140% and 1% of the priming activity of the wt p3, respectively. All the mutant TP were able to interact with the phi 29 DNA polymerase and with DNA, suggesting that Leu220 and Ser226, in addition to Ser232, form part of a functional domain involved in the process of initiation of DNA replication.

Amino Acid Sequence↗
PubMed1989

Signals at the bacteriophage phi 29 DNA replication origins required for protein p6 binding and activity.

Protein p6 of Bacillus subtilis phage phi 29 binds specifically to the ends of the viral DNA that contain the replication origins, giving rise to a nucleoprotein structure. DNA regions recognized by protein p6 have been mapped by deletion analysis and DNase I footprinting. Main protein p6-recognition signals have been located between nucleotides 62 and 125 at the right phi 29 DNA end and between nucleotides 46 and 68 at the left end. In addition, recognition signals are also present at other sites within 200-300 bp at each phi 29 DNA end. Protein p6 does not seem to recognize a specific sequence in the DNA, but rather a structural feature, which could be bendability. The formation of the protein p6-DNA nucleoprotein complex is likely to be the structural basis for the protein p6 activity in the initiation of replication.

Bacteriophages↗
PubMed1988

Site-directed mutagenesis in the DNA linking site of bacteriophage phi 29 terminal protein: isolation and characterization of a Ser232----Thr mutant.

By site-directed mutagenesis we have changed the serine residue 232 of the phi 29 terminal protein, involved in the covalent linkage to dAMP for the initiation of replication, into a threonine residue. The mutant terminal protein has been purified to homogeneity and shown to be inactive in the formation of the initiation complex; nevertheless, the mutant protein retains its ability to interact with the phi 29 DNA polymerase and with the DNA. The results obtained indicate a high specificity in the linking site of the terminal protein.

Bacteriophages↗
PubMed1988

Interaction of the bacteriophage phi 29 protein p6 with double-stranded DNA.

The Bacillus subtilis bacteriophage phi 29 protein p6 binds to double-stranded DNA, but not to single-stranded DNA, as determined by a gel retardation assay. The nature of the interaction was further studied by DNase I "footprinting" experiments. Protein p6 binds to fragments containing the right or left terminal sequences of phi 29 DNA, producing a characteristic pattern of hypersensitive bands spaced about 24 nucleotides apart along most of the fragment, flanking protected regions. Binding of protein p6 to an internal phi 29 DNA fragment was also observed, but the footprint pattern was more salt sensitive than that obtained with the terminal phi 29 DNA fragments. By electron microscopy, protein p6 was shown to cover the DNA, totally or partially, from one end. In addition, binding of protein p6 to relaxed circular DNA induced positive supercoiling, indicating that a topological change in the DNA occurred.

Bacillus subtilis↗
PubMed1987

Effect of NH4+ ions on phi 29 DNA-protein p3 replication: formation of a complex between the terminal protein and the DNA polymerase.

Ammonium ions stimulated the formation of the phi diameter 29 protein p3-dAMP initiation complex by decreasing the Km value for dATP in a purified system containing the viral terminal protein p3, the viral DNA polymerase p2, and the phi 29 DNA-protein p3 complex as a template. In addition, NH4+ ions stimulated the amount of p3-dAMP complex elongation and increased by about twofold the rate of elongation. The stimulatory effect of NH4+ ions on in vitro phi 29 DNA replication is probably related to the formation of a stable complex between the terminal protein and the DNA polymerase, which was detected only in the presence of NH4+ ions.

Ammonium Sulfate↗
PubMed1986

Signals in the phi 29 DNA-terminal protein template for the initiation of phage phi 29 DNA replication.

The protein-free terminal fragments HindIII B and L, from the left and right ends of phi 29 DNA, respectively, but not internal fragments of similar size, were active as templates in the formation of the p3-dAMP initiation complex in an in vitro system containing purified phi 29 terminal protein p3 and DNA polymerase p2, although the activity was lower than that obtained with the phi 29 DNA-p3 complex. These results indicate the existence of specific sequences at the ends of phi 29 DNA that allow the initiation of phi 29 DNA replication. The template activity of the protein-free terminal fragments was size dependent. The protein-free single strands of the HindIII L fragment were much less active than the corresponding double-stranded fragment. Terminal protein-DNA complexes of phages PZA and phi 15, with a terminal protein closely related to the phi 29 protein p3, were more active as templates in the initiation reaction with the purified phi 29 proteins than the corresponding protein-free DNAs, as it happens in the case of phi 29. However, the terminal protein-DNA complexes of phages Nf, B103, and GA-1, with a terminal protein less related or unrelated to the phi 29 protein p3, were essentially inactive and became active after removal of the parental terminal protein. These results strongly suggest that the parental terminal protein is the major signal in the template for the initiation of phi 29 DNA replication.

Bacillus subtilis↗
PubMed1986

Formation of a covalent complex between the terminal protein of pneumococcal bacteriophage Cp-1 and 5'-dAMP.

Incubation of extracts of Cp-1-infected Streptococcus pneumoniae with [alpha-32P]dATP produced a labeled treatment with micrococcal nuclease and sensitive to treatment with proteinase K. Incubation of the 32P-labeled protein with 5 M piperidine for 4 h at 50 degrees C released 5'-dAMP, indicating that a covalent complex between the terminal protein and 5'-dAMP was formed in vitro. When the four deoxynucleoside triphosphates were included in the reaction mixture, a labeled complex of slower electrophoretic mobility in sodium dodecyl sulfate-polyacrylamide gels than the terminal protein-dAMP complex was also found, indicating that the Cp-1 terminal protein-dAMP complex can be elongated and, therefore, that it is an initiation complex. Treatment of the 32P-labeled terminal protein-dAMP complex with 5.8 M HCl at 110 degrees C for 2 h yielded phosphothreonine. These results, together with the resistance of the terminal protein-DNA linkage to hydroxylamine, suggest that the Cp-1 terminal protein is covalently linked to the DNA through a phosphoester bond between L-threonine and 5'-dAMP, namely, a O-5'-deoxyadenylyl-L-threonine bond.

Bacteriophages↗
PubMed1977

Nucleic acid helix-unwinding properties of ribosomal protein S1 and the role of S1 in mRNA binding to ribosomes.

The presence of ribosomal protein S1 in 30S ribosomes is indispensable for the formation of 30S initiation complexes with natural mRNA. The 30S subunits lacking S1 retain activity with AUG as mRNA and are also active in poly(rU)-directed binding of Phe-tRNA. Isolated protein S1 stoichiometrically disrupts the secondary structure of helical and stacked single-stranded polynucleotides and converts them into their fully or partially denatured forms. A mono-N-ethylmaleimide derivatives of S1 is nearly devoid of any RNA helix-unwinding properties but is readily incorporated into 30S subunits deficient in S1. The resulting N-ethylmaleimide-S1-containing 30S subunits are completely inactive in the binding of MS2 [3H]RNA and in the formation of an initiation complex with MS2 RNA as mRNA. They retain activity in the binding of the initiator fMet-tRNA in response to the trinucleotide AUG and in the binding of Phe-tRNA in response to poly(U). They also retain the capacity to bind 50S subunits and to form 70S couples. These results suggest that a correlation exists between the RNA helix-unwinding capacity of isolated S1 and the function of S1 in the ribosomal binding of natural mRNA when the protein becomes part of the 30S subunit.

Binding Sites↗
PubMed1975

Ribosomal protein S1 and polypeptide chain initiation in bacteria.

Among several subspecies of 30S subunits of Escherichia coli observed by polyacrylamide-agarose gel electrophoresis, only the slow-moving, protein S1-containing subspecies participates in the formation of the 30S initiation complex with coliphage MS2 RNA as mRNA; the other subspecies retain activity with AUG as mRNA; they are also active in the poly(U)-directed binding of Phe-tRNA. Protein S1 from Caulobacter crescentus substitutes for E. coli S1 despite the fact that C. crescentus ribosomes do not bind MS2 RNA. Under appropriate conditions, the entire population of E. coli 30S subunits can be isolated as the S1-containing subspecies. Protein S1 is lost by salt treatment of ribosomes.

Bacteria↗