Functional characterization of the genes coding for the terminal protein and DNA polymerase from bacteriophage GA-1. Evidence for a sliding-back mechanism during protein-primed GA-1 DNA replication.

Abstract:

We have determined the nucleotide sequence of 2676 bp at the left part of the linear genome of Bacillus subtilis bacteriophage GA-1. Computer analysis revealed that this fragment contains two open reading frames (ORFs), ORF1 and ORF2, which contain 265 and 578 codons, respectively. Data base search revealed that ORF1 and ORF2 might encode proteins similar to the terminal protein (TP) and DNA polymerase, respectively, of bacteriophage phi29. By using extracts of B. subtilis infected with GA-1, we demonstrated that GA-1 DNA replication occurs by a protein-priming mechanism in which these two viral proteins are involved. Butylphenyl dGTP (BuPdGTP), a specific inhibitor of eukaryotic-type (family B) DNA polymerases, inhibited both the protein-primed initiation step and DNA polymerization during GA-1 DNA replication. These results suggest the involvement of a eukaryotic-type DNA polymerase, probably the product of the viral ORF2, in both stages of a replication process in which the TP primes replication at both DNA ends (replication origins). Using synthetic oligonucleotides, we carried out a mutational analysis of the GA-1 DNA right end to determine the initiation site for replication. The results indicate that initiation of replication mainly occurs opposite the second nucleotide at the 3' end of the template, although the third nucleotide can be used as an alternative initiation site. As in other TP-containing genomes, a sliding-back mechanism is proposed to account for the maintenance of the DNA length at the GA-1 DNA ends.

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