Effects of altered RGD (ARG-GLY-ASP) sequences in the primer protein on the protein primed DNA replication of bacteriophage M2 in vitro (original) (raw)
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Protein-primed replication of bacteriophage Φ29 DNA
Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression, 1988
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Characterization of the Origins of Replication of Bacteriophage ø29 DNA
Nucleic acids research, 1988
The origins of replication of p29 DNA have been studied by analyzing the activity as templates in the 029 in vitro replication system of E. coli recombinant plasmids and M13 derivatives containing 029 DNA terminal sequences. Plasmid pITR, containing the 6 bp long inverted terminal repeat of 929 DNA, was shown to be essentially inactive. The analysis of a series of deletion derivatives of plasmid pID13, that contains the 73 and 269 bp from the left and right 029 DNA ends, respectively, indicated that the minimal origins of replication are comprised within the terminal 12 bp at each DNA end. Point site-directed and random mutagenesis at these sequences was carried out. Changes of the second or third A into a C completely abolished the template activity. In the case of changes at position from 4 to 12, only 3 out of 14 mutations reduced the template activity; these 3 mutations were double changes and 2 of them affected the inverted terminal repeat. The results suggest that the sequence requirement at the end-proximal region of the origin of replication is more strict than that at the distal region.
Nucleic Acids Research, 1992
Bacteriophage PRD1 replicates its DNA by means of a protein-primed replication mechanism. Compared to Mg2 +, the use of Mn2+ as the metal activator of the phage DNA polymerase results in a great stimulation of the initiation reaction. The molecular basis of the observed stimulatory effect is an increase in the velocity of the reaction. The phage DNA polymerase is also able to catalyze the formation of the initiation complex in the absence of DNA template. Although the presence of Mn2 + does not affect either the polymerization activity or the processivity of the DNA polymerase, this metal is unable to activate the overall replication of the phage genome. This can be explained by a deletorious effect of Mn2+ on the 3'-5'-exonucleolytic and/or the strand-displacement activity, the latter being an intrinsic function of the viral DNA polymerase required for protein-primed DNA replication.
An inhibitory effect of RGD peptide on protein, priming reaction of bacteriophages ?29 and M2
Molecular and General Genetics - MOL GEN GENET, 1989
The amino acid sequence, arginine-glycine-aspartic acid (RGD), found in some cell adhesive proteins, is a recognition signal for the receptor protein. It is interesting that we have found the RGD sequence in terminal protein (TP) of bacteriophages ~b29 and M2 near an amino acid, the serine residue at 232, covalently linked to the terminal nucleotide of their DNAs. At the initiation of proteinprimed DNA replication, TP is essential for the recognition of replication machinery containing DNA polymerase and primer protein (PP; PP becomes TP upon linking the first nucleotide, and hence the primary structure of TP is the same as that of PP). Synthetic peptide RGD specifically inhibited transfection of ~b29 and M2. The target of the RGD peptide is shown to be TP by marker rescue experiments, suggesting that a receptor for the RGD sequence exists in TP. Furthermore, the peptide inhibited the in vitro protein-priming reaction of DNA replication. We propose that the RGD sequence of PP and a putative receptor on TP is utilized for the molecular recognition initiating DNA replication.
Proceedings of the National Academy of Sciences, 1980
In the presence of single-stranded DNA, the bacteriophage T4 gene 41 and gene 61 proteins catalyze the synthesis of a group of pentaribonucleotides which are homogeneous in chain length but heterogeneous in nucleotide sequence. When single-stranded T4 DNA is used as template, a unique dinucleoside sequence, pppApC, is found at the 5' end of these pentaribonucleotides with the general sequence pppApCpNpNpN. In the presence of the remaining five T4 replication proteins, the pentaribonucleotides can be utilized with high efficiency to prime de novo DNA chain starts; as a result, the vast majority of them can be detected at the 5' end of newly made DNA molecules in an unaltered form. There are multiple, but specific, sites at which new DNA chains are primed in this way on a natural single-stranded DNA. Because identical RNA primers have been isolated from the 5' end of the Okazaki fragments made in T4-infected cells, we suggest that the T4 gene 41 and gene 61 proteins also m...
The functional origin of bacteriophage f1 DNA replication
Journal of Molecular Biology, 1984
The origin of DNA replication of bacteriophage fl functions as a signal, not only for initiation of viral strand synthesis, but also for its termination. Viral (plus) strand synthesis initiates and terminates at a specific site (plus origin) that is recognized and nicked by the viral gene II protein. Mutational analysis of the 5' side (upstream) of the origin of plus strand replication of phage fl led us to postulate tile existence of a set of overlapping functional domains. These included ones for strand nicking, and initiation and termination of DNA synthesis. Mutational analysis of the 3' side (downstream) of the origin has verified the existence of these domains and determined their extent. The results indicate that the fl "functional origin" can be divided into two domains: (1) a "core region", about 40 nucleotides long, that is absolutely required for plus strand synthesis and contains three distinct but partially overlappingsignais, (a)the gene II protein recognition sequence, which is necessary both for plus strand initiation and termination, (b)the termination signal, which extends for eight more nucleotides on the 5' side of the gene II protein recognition sequence, (c) the initiation signal that extends for about ten more nucleotides on the 3' side of the gene II protein recognition sequence; (2) a "secondary region", 10O nucleotides long, required exclusively for plus strand initiation. Disruption of the secondary region .does not completely abolish the functionality of the fl origin but does drastically reduce it (1% residual biological activity). We discuss a possible explanation of the fact that this region can be interrupted (e.g. fl, M]3 cloning vectors) by large insertions of foreign DNA without significantly affecting replication.
Studies of bacteriophage P2 DNA replication: localization of the cleavage site of the A protein
Nucleic Acids Research, 1994
Bacteriophage P2 replicates via a modified rolling circle-type of mechanism, where the P2 A protein acts as an Initiator of the replication by inducing a singlestranded cut at the origin of replication (ori). The exact location of the cut Induced by the A protein In vivo is determined In this report by: (i) restriction analysis; (ii) DNA sequence analysis; and (Ml) primer extensions. It Is located 89.2% from the left end of the P2 genome, which is within the coding part of the A gene, In a region devoid of secondary structures. The A gene has been cloned Into an expression vector, and the A protein has been purified. The purified A protein does not bind to double-stranded ori containing DNA, but It cleaves single-stranded ori containing DNA, which indicates that a special DNA structure and/or protein is required to make the ori accessible for the A protein.
Journal of Virology, 1993
Escherichia coli bacteriophage PRD1 and its relatives contain linear double-stranded DNA genomes, the replication of which proceeds via a protein-primed mechanism. Characteristically, these molecules contain 5'-covalently bound terminal proteins and inverted terminal nucleotide sequences (inverted terminal repeats [ITRs]). The ITRs of each PRD1 phage species have evolved in parallel, suggesting communication between the molecule ends during the life cycle of these viruses. This process was studied by constructing chimeric PRD1 phage DNA molecules with dissimilar end sequences. These molecules were created by combining two closely related phage genomes (i) in vivo by homologous recombination and (ii) in vitro by ligation of appropriate DNA restriction fragments. The fate of the ITRs after propagation of single genomes was monitored by DNA sequence analysis. Recombinants created in vivo showed that phages with nonidentical genome termini are viable and relatively stable, and hybri...