In vitro protein-primed initiation of pneumococcal phage Cp-1 DNA replication occurs at the third 3' nucleotide of the linear template: a stepwise sliding-back mechanism.
Phage Cp-1 from Streptoccocus pneumoniae makes use of a protein-priming mechanism to start replication of its linear DNA: the first reaction consists of the addition of 5' dAMP to a molecule of the primer protein, an initiation event occurring at both DNA ends. After elongation of the initiation complex, the primer protein remains linked to the 5' end of the nascent DNA chain, and is subsequently referred to as terminal protein (TP). In this paper, using DNA-free extracts from Cp-1-infected S. pneumoniae, we provide evidence that the formation of the covalent complex TP-dAMP is a template-instructed reaction and that ssDNA molecules can serve as templates for TP-primed replication. A mutational analysis of the 3' terminal nucleotides of Cp-1 DNA reveals that a precise DNA sequence is required for efficient template recognition, and that in vitro initiation of Cp-1 DNA replication is directed by the third nucleotide of the template. However, the two terminal nucleotides are recovered during the first steps of elongation. A new variant of the sliding-back mechanism for protein-primed initiation, firstly described for Bacillus subtilis phage phi29, is proposed to account for the maintenance of Cp-1 DNA ends. The results presented here reinforce the hypothesis that sliding-back must be a common feature in all genomes that use protein-priming to initiate replication.
Martín
A C
AC
Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas (CSIC), Madrid, Spain.
Blanco
L
L
García
P
P
Salas
M
M
Méndez
J
J
D017103:Bacillus Phages / Q000235:genetics; D001483:Base Sequence; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000378:metabolism; D004279:DNA, Viral / Q000096:biosynthesis* / Q000235:genetics; D003838:Deoxyadenine Nucleotides / Q000378:metabolism; D008969:Molecular Sequence Data; D010442:Peptide Chain Initiation, Translational; D018741:Replication Origin; D017108:Streptococcus Phages / Q000235:genetics* / Q000378:metabolism; D013296:Streptococcus pneumoniae / Q000821:virology; D013698:Templates, Genetic
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D003838:Deoxyadenine Nucleotides