Structure of the Escherichia coli primase/single-strand DNA-binding protein/phage G4 ori c complex required for primer RNA synthesis

Abstract

Escherichia coli primase/SSB/single-stranded phage G4 ori c is a simple system to study how primase interacts with DNA template to synthesize primer RNA for initiation of DNA replication. By a strategy of deletion analysis and antisense oligonucleotide protection on small single-stranded G4 ori c fragments, we have identified the DNA sequences required for binding primase and the critical location of single-strand DNA-binding (SSB) protein. Together with the previous data, we have defined the structure of the primase/SSB/G4 ori c priming complex. Two SSB tetramers bind to the G4 ori c secondary structure, which dictates the spacing of 3′ and 5′ bound adjacent SSB tetramers and leaves SSB-free regions on both sides of the stem-loop structure. Two primase molecules then bind separately to specific DNA sequences in the 3′ and 5′ SSB-free G4 ori c regions. Binding of the 3′ SSB tetramer, upstream of the primer RNA initiation site, is also necessary for priming. The generation of a primase-recognition target by SSB phasing at DNA hairpin structures may be applicable to the binding of initiator proteins in other single-stranded DNA priming systems. Novel techniques used in this study include antisense oligonucleotide protection and RNA synthesis on an SSB-melted, double-stranded DNA template.