Protein-primed DNA replication: a transition between two modes of priming by a unique DNA polymerase.
Phage phi29 from Bacillus subtilis is a paradigm of the protein-primed replication mechanism, in which a single-subunit DNA polymerase is involved in both the specific protein-primed initiation step and normal DNA elongation. To start phi29 DNA replication, the viral DNA polymerase must interact with a free molecule of the viral terminal protein (TP), to prime DNA synthesis once at each phi29 DNA end. The results shown in this paper demonstrate that the DNA polymerase-primer TP heterodimer is not dissociated immediately after initiation. On the contrary, there is a transition stage in which the DNA polymerase synthesizes a five nucleotide-long DNA molecule while complexed with the primer TP, undergoes some structural change during replication of nucleotides 6-9, and finally dissociates from the primer protein when nucleotide 10 is inserted onto the nascent DNA chain. This behaviour probably reflects the polymerase requirement for a DNA primer of a minimum length to efficiently catalyze DNA elongation. The significance of such a limiting transition stage is supported by the finding of abortive replication products consisting of the primer TP linked up to eight nucleotides, detected during in vitro replication of phi29 TP-DNA particularly under conditions that decrease the strand-displacement capacity of phi29 DNA polymerase.
Mendez
J
J
Centro de Biologia Molecular Severo Ochoa (C.S.I.C.-U.A.M.), Universidad Autonoma, Canto Blanco, Madrid, Spain.
Blanco
L
L
Salas
M
M
D017103:Bacillus Phages / Q000235:genetics*; D002384:Catalysis; D004252:DNA Mutational Analysis; D017931:DNA Primers / Q000378:metabolism; D004261:DNA Replication; D004279:DNA, Viral / Q000096:biosynthesis* / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D019281:Dimerization; D008957:Models, Genetic; D013698:Templates, Genetic; D014764:Viral Proteins / Q000378:metabolism*
D017931:DNA Primers; D004279:DNA, Viral; D014764:Viral Proteins; C040784:terminal protein, Bacillus phage phi29; D004259:DNA-Directed DNA Polymerase