STUDIES ON THE ENZYMOLOGY OF DNA REPLICATION

Abstract

The studies described in this thesis are concerned with the replication of the small icosahedral phage DNAs. Three aspects of DNA multiplication are presented.;(1) An important problem in DNA replication is the mechanism by which polynucleotide chains are initiated de novo. The dnaG encoded protein (Primase) was shown to catalyze that function. The purification of the enzyme is reported as well as the characterization of the reactions catalyzed by it.;(2) The discovery by Eisenberg et al. (Eisenberg, S., Scott, J. F., and Kornberg, A.(1976) Proc.Natl.Acad.Sci.U.S.A.73,3151) that a combination of the purified E. coli rep, SSb, as well as the DNA polymerase III elongation system and (phi)X174 A proteins catalyzed net synthesis of (+)SS(c) DNA when (phi)X174 RFI DNA was used as a template, led to their proposal of a two-step model of (phi)X174 RF (--->) RF DNA replication.;I report here that cell-free extracts of thermosensitive E. coli mutants in the dnaB, dnaC, and dnaG gene synthesize and accumulate (+)SS(c) DNA under nonpermissive conditions. Addition of the purified dnaB, dnaC, or dnaG gene products to their respective heat-inactivated extract results in the accumulation of RF DNA rather than (+)SS(c) DNA. Electron microscopic analysis of (phi)X174 RFI DNA-dependent replication products synthesized under nonpermissive conditions by cell-free extracts prepared from dnaB, dnaC and dnaG E. coli mutants established that the replicative intermediates were identical with those observed in the (+) strand DNA synthesis reactions catalyzed by purified proteins.;(3) The multiple activities of the (phi)X174 A protein are dependent upon a short segment of the (phi)X174 chromosome, the origin of viral strand DNA synthesis. In order to study the rejoining activity of (phi)X174 A protein I cloned two (phi)X174 progeny RF replication origins into the same pBR322 plasmid DNA. I report here the template activity of these new recombinant DNAs in in vitro replication systems which are specific for (phi)X174 sequences. These results indicate that the (phi)X174 gene A protein functions as a sequence specific breakage and reunion enzyme during DNA replication.