Suchergebnisse

DNA replication of ϕ29 and related phages takes place via a strand displacement mechanism, a process that generates large amounts of single-stranded DNA (ssDNA). Consequently, phage-encoded ssDNA-binding proteins (SSBs) are essential proteins during phage ϕ29-like DNA replication. In the present work we analyze the helix-destabilizing activity of the SSBs of ϕ29 and the related phages Nf and GA-1, their ability to eliminate non-productive binding of ϕ29 DNA polymerase to ssDNA and their stimulatory effect on replication by ϕ29 DNA polymerase in primed M13 ssDNA replication, a situation that resembles type II replicative intermediates that occur during ϕ29-like DNA replication. Significant differences have been appreciated in the functional behavior of the three SSBs. First, the GA-1 SSB is able to display helix-destabilizing activity and to stimulate dNTP incorporation by ϕ29 DNA polymerase in the M13 DNA replication assay, even at SSB concentrations at which the ϕ29 and Nf SSBs do not show any effect. On the other hand, the ϕ29 SSB is the only one of the three SSBs able to increase the replication rate of ϕ29 DNA polymerase in primed M13 ssDNA replication. From the fact that the ϕ29 SSB, but not the Nf SSB, stimulates the replication rate of Nf DNA polymerase we conclude that the different behaviors of the SSBs on stimulation of the replication rate of ϕ29 and Nf DNA polymerases is most likely due to formation of different nucleoprotein complexes of the SSBs with the ssDNA rather than to a specific interaction between the SSB and the corresponding DNA polymerase. A model that correlates the thermodynamic parameters that define SSB–ssDNA nucleoprotein complex formation with the functional stimulatory effect of the SSB on ϕ29-like DNA replication has been proposed. ; This investigation was aided by research grant 2RO1 GM27242-20 from the National Institutes of Health, by grant PB93-0173 from the Dirección General de Investigación Científica y Técnica, by grant ERBFMRX CT970115 from the European Union and by an institutional grant from the Fundación Ramón Areces. I.G. was holder of a pre-doctoral fellowship from the Fundación Ramón Areces. ; Peer reviewed

Phage phi 29 encodes a DNA-dependent DNA polymerase belonging to the eukaryotic-type (family B) subgroup of DNA polymerases that use a protein as the primer for initiation of DNA synthesis. In one of the most important motifs present in the 3'5' exonucleolytic domain of proofreading DNA polymerases, the ExoII motif, phi 29 DNA polymerase contains three amino acid residues, Y59, H61 and F69, which are highly conserved among most proofreading DNA polymerases. These residues have recently been shown to be involved in proper stabilization of the primer terminus at the 3'5' exonuclease active site. Here we investigate by means of site-directed mutagenesis the role of these three residues in reactions that are specific for DNA polymerases utilizing a protein-primed DNA replication mechanism. Mutations introduced at residues Y59, H61 and F69 severely affected the protein-primed replication capacity of phi 29 DNA polymerase. For four of the mutants, namely Y59L, H61L, H61R and F69S, interaction with the terminal protein was affected, leading to few initiation and transition products. These findings, together with the specific conservation of Y59, H61 and F69 among DNA polymerases belonging to the protein-primed subgroup, strongly suggest a functional role of these amino acid residues in the DNA polymerase–terminal protein interaction ; This investigation was aided by research grant 5R01 GM27242-22 from the National Institutes of Health, by grant PB98-0645 from the Dirección General de Investigacíon Científica y Técnica, by grant ERBFMX CT97 0125 from the European Union and by an institutional grant from Fundación Ramón Areces. R.E. was a post-doctoral fellow of the European Union ; Peer reviewed

Alignment of the protein sequence of DNA-dependent DNA polymerases has allowed the definition of a new motif, lying adjacent to motif B in the direction of the N-terminus and therefore named pre-motif B. Both motifs are located in the fingers subdomain, shown to rotate towards the active site to form a dNTP-binding pocket in several DNA polymerases in which a closed ternary complex pol:DNA:dNTP has been solved. The functional significance of pre-motif B has been studied by site-directed mutagenesis of phi 29 DNA polymerase. The affinity for nucleotides of phi 29 DNA polymerase mutant residues Ile364 and Lys371 was strongly affected in DNA- and terminal protein-primed reactions. Additionally, mutations in Ile364 affected the DNA-binding capacity of phi 29 DNA polymerase. The results suggest that Lys371 of phi 29 DNA polymerase, highly conserved among families A and B, interacts with the phosphate groups of the incoming nucleotide. On the other hand, the role of residue Ile364 seems to be structural, being important for both DNA and dNTP binding. Pre-motif B must therefore play an important role in binding the incoming nucleotide. Interestingly, the roles of Lys371 and Ile364 were also shown to be important in reactions without template, suggesting that phi 29 DNA polymerase can achieve the closed conformation in the absence of a DNA template ; This investigation has been aided by research grant 2R01 GM27242-22 from the National Institutes of Health, by grant PB98-0645 from the Dirección General de Investigación Científica y Técnica, by grant ERBFMRX CT97 0125 from the European Union and by an institutional grant from Fundación Ramón Areces. V.T. was a post-doctoral fellow of the Comunidad Autónoma de Madrid. ; Peer reviewed