Protein-primed replication of bacteriophage phi 29 DNA.
The replication of phi 29 DNA-protein p3 represents a simple model system to study the protein-priming mechanism of initiation of replication. The phi 29 DNA polymerase involved both in the initiation and elongation steps of phi 29 DNA-protein p3 replication, is a very processive enzyme and it is able to produce strand-displacement in the absence of other proteins. To correlate functional and structural domains in the phi 29 DNA polymerase point mutants in the most carboxyl region of amino-acid homology with other DNA polymerases have been constructed. Most of the mutations had a decreased initiation and elongation activity, but normal 3'----5' exonuclease activity, suggesting that this region contributes to the active domain for initiation and elongation. Point and deletion mutants in the terminal protein have allowed the mapping of one DNA-binding region and two DNA-polymerase-binding regions. The viral protein p6, which stimulates the initiation of replication, binds to a set of specific signals present at both phi 29 DNA ends. A good correlation of binding and stimulation of replication has been obtained by using fragments containing phi 29 DNA-terminal sequences and deletion mutants of protein p6. The viral protein p5 has been shown to bind to single-stranded DNA, to protect the latter against nuclease digetion, and to stimulate phi 29 DNA-protein p3 replication in vitro.
Salas
M
M
Centro de Biología Molecular, (C.S.I.C.-U.A.M.), Universidad Autónoma, Madrid, Spain.
Martín
G
G
Bernad
A
A
Garmendia
C
C
Lázaro
J M
JM
Zaballos
A
A
Serrano
M
M
Otero
M J
MJ
Gutiérrez
J
J
Parés
E
E
D001435:Bacteriophages / Q000235:genetics* / Q000502:physiology; D002384:Catalysis; D004261:DNA Replication; D004279:DNA, Viral / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism; D043211:Exodeoxyribonuclease V; D005090:Exodeoxyribonucleases / Q000378:metabolism; D009154:Mutation; D012689:Sequence Homology, Nucleic Acid; D014764:Viral Proteins / Q000378:metabolism / Q000494:pharmacology*; D014779:Virus Replication
D004279:DNA, Viral; D014764:Viral Proteins; D004259:DNA-Directed DNA Polymerase; D005090:Exodeoxyribonucleases; D043211:Exodeoxyribonuclease V