Conversion of ϕX174 Viral DNA to Double-Stranded Form by Purified Escherichia coli Proteins

Abstract

The E. coli proteins that catalyze the conversion of ϕX174 single-stranded DNA to duplex DNA have now been purified extensively. The reaction depends on dnaB, dnaC(D), dnaE, and dnaG gene products, DNA elongation factors I and II, E. coli DNA binding protein, and two additional E. coli proteins, replication factors X and Y. DNA synthesis by these proteins requires ϕX174 viral DNA, dNTPs, Mg⁺², and ATP. The product synthesized is full-length linear ϕX174 DNA. The reaction has been resolved into two steps. The first step involves the interaction of ATP and ϕX174 DNA with dnaB and dnaC(D) gene products, E. coli DNA binding protein, and replication factors X and Y in the absence of dNTPs. Subsequent dNMP incorporation requires the addition of DNA polymerase III, DNA elongation factors I and II, dnaG gene product, and dNTPs.