Single-strand displacement replication of bacteriophage M2 DNA.

Abstract

Replicative intermediates of M2 DNA, pulse-labeled with [3H]-thymidine, were isolated and the mode of its replication was examined. Compared with mature M2 DNA the replicative intermediates had the following properties: i) higher sedimentation coefficient in sucrose gradient, ii) higher buoyant density in CsC1 equilibrium density gradient, and iii) sensitivity to nuclease S 1. Two types of intermediates were found by electron microscopic observation. One is unit-length duplex molecules with a single-stranded branch of various lengths. The other is unit-length linear molecules consisting of one single-stranded and one doublestranded region of variable proportions. Distribution of the radioactivity in pulse-labeled completed molecules was analyzed with restriction endonucleases. Both ends of the genome had higher radioactivity, showing that the ends are termini of replication. These results indicate that the replication of M2 DNA proceeds from each end by displacing parental single-stranded DNA.

The primer protein of bacteriophage M2 inhibits elongation acivity of its DNA polymerase.

“…Recent homology searches on conserved segments among DNA polymerases and analyses of the mutation sites of aphidicolin-resistant and temperaturesensitive mutants of X29 and M2 suggest that protein priming and polymerase activities are catalyzed by a common functional domain of Pol (1,8,11,12,20,22,24,25). Thus, for catalysis of the apparently different reactions of priming and elongation of M2 Pol, the structure and function of this common domain would be changed in response to the reaction engaged.…”

“…Replication of M2 DNA is initiated at the termini by a protein-priming mechanism followed by displacement of a parental single-strand (20,21), as in the replication in bacteriophages q529 and PRD 1, and in adenovirus (for reviews, see 10,16,27). The replication of M2 DNA requires three viral components: primer protein (PP), DNA polymerase (Pol) and TP-linked DNA (21).…”