The mechanism of replication of ΦX174. XVIII. Gene A and A∗ proteins of ΦX174 bind tightly to ΦX174 replicative form DNA

Abstract

Evidence is presented that the gene A and A∗ proteins of bacteriophage ΦX174 form covalent associations with the 5′ ends of the DNA molecules when superhelical ΦX replicative from DNA is nicked by a combination of these proteins in vitro. This evidence is: 1, The 5′ ends of the DNA molecules nicked by the gene A protein and reacted with bacterial alkaline phosphatase were protected against subsequent phosphorylation by polynucleotide kinase even after treatment of the nicked DNA with SDS and pronase followed by centrifugation on a high-salt neutral sucrose gradient. 2, Iodinated pronase-sensitive material remained attached to the nicked replicative from DNA and could not be removed by exposure to SDS or 2 M NaCl, either by sedimentation through high-salt neutral sucrose gradients, or by CsCl equilibrium centrifugation. 3, Iodinated pronase-sensitive material was detected on DNA that had been nicked during the reaction, but not on unreacted DNA. 4, Electrophoresis of the iodinated pronase-sensitive, DNA-bound material in SDS-polyacrylamide gels after DNAase digestion revealed that it was composed almost entirely of polypeptides with electrophoretic mobilities similar to those of the gene A and A ∗ proteins. We speculate that the gene ∗ protein may be essential for normal progeny single-stranded DNA synthesis in vivo.