RESUMO
Bacteriophage PRD1 is the only protein-primed DNA replication system known to operate in Escherichia coli. The left-genome end of PRD1 contains the early genes for the terminal protein and the DNA polymerase. These genes have been sequenced and the proteins have been produced separately. In this investigation we completed the analysis of the PRD1 early DNA regions by cloning and sequencing the right end genome containing early genes XII and XIX. We compared the structure of the right- and left-terminal regions. The genome organization of both ends was found to be rather uniform. The inverted terminal repeats, the first promoters and the first translation start codons are located almost exactly at the same distance from the genome ends. The PRD1 early gene products, P12 and P19, do not share similarities with proteins found in other protein-primed replication systems.
Assuntos
Bacteriófagos/genética , Escherichia coli/genética , Genes Virais , Proteínas Virais/genética , Proteínas Estruturais Virais/genética , Sequência de Aminoácidos , Sequência de Bases , Replicação do DNA , Dados de Sequência Molecular , Plasmídeos , Homologia de Sequência do Ácido NucleicoRESUMO
The early protein, P19, of bacteriophage PRD1 was purified after overexpression of the cloned gene, XIX, in Escherichia coli DH5 alpha cells. The purified protein binds as multimers to single-stranded DNA (ssDNA), and with a lower affinity to double-stranded DNA (dsDNA), without sequence-specificity. Two distinct P19-ssDNA complexes were discovered in gel- mobility-shift assays at different protein:DNA ratios. P19 was capable of fully protecting ssDNA against nuclease P1. Electron microscopy of protein P19-ssDNA complexes showed DNA molecules which were extensively coated with protein and whose contour length was clearly reduced by P19 binding. The results suggest that P19 binds to ssDNA with moderate cooperativity and are consistent with the DNA being wrapped around the P19 multimers.
Assuntos
Bacteriófagos/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Virais/metabolismo , Clonagem Molecular , DNA de Cadeia Simples/metabolismo , DNA Viral/metabolismo , DNA Viral/ultraestrutura , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Proteínas de Ligação a DNA/ultraestrutura , Desoxirribonucleases/metabolismo , Escherichia coli , Microscopia Eletrônica , Proteínas Virais/genética , Proteínas Virais/isolamento & purificação , Proteínas Virais/ultraestruturaRESUMO
The gene for the lytic enzyme of the lipid-containing, broad-host-range bacteriophage PRD1 codes for a protein of 149 amino acids (17271 Da). The sequence of the protein is unique when compared to other lytic enzymes sequenced. However, three regions of weak similarity with other phage lytic enzymes were observed. The C-terminal region shared seven amino acids in common with phage P22 lysozyme at a site which is conserved in phage-type lysozymes.
Assuntos
Bacteriófagos/enzimologia , Parede Celular/enzimologia , Muramidase/análise , Peptídeo Hidrolases/análise , Peptidoglicano/análise , Sequência de Aminoácidos , Aminoácidos/análise , Sequência de Bases , Parede Celular/análise , Dados de Sequência Molecular , Muramidase/genética , Peptídeo Hidrolases/genéticaRESUMO
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.
Assuntos
Colífagos/metabolismo , Replicação do DNA/efeitos dos fármacos , DNA Viral/biossíntese , Proteínas de Ligação a DNA/genética , Proteínas Virais/farmacologia , Colífagos/efeitos dos fármacos , DNA/metabolismo , DNA de Cadeia Simples/metabolismo , Conformação de Ácido Nucleico , Conformação ProteicaRESUMO
We have determined the nucleotide sequence of the late region (11 kbp) of the lipid-containing bacteriophage PRD1. Gene localization was carried out by complementing nonsense phage mutants with genomic clones containing specific reading frames. The localization was confirmed by sequencing the N-termini of isolated gene products as well as sequencing the N-termini of tryptic fragments of the phage membrane-associated proteins. This, with the previously obtained sequence of the early regions, allowed us to organize most of the phage genes in the phage genome.