Docking of the p68 subunit of DNA polymerase ¦á-primase on the SV40 helicase is required for the viral primosome activity

Abstract

DNA polymerase ¦Á-primase (pol-prim) plays a central role in eukaryotic DNA replication, initiating synthesis on both the leading and lagging strand DNA templates. Pol-prim consists of a primase heterodimer that synthesizes RNA primers, a DNA polymerase that extends them, and a fourth subunit, p68, that is thought to regulate the complex. In the Simian Virus 40 (SV40) replication model, the p68 subunit is required for primosome activity and interacts directly with the viral helicase T antigen (Tag), suggesting a functional link between Tag-p68 interaction and primosome activity. To explore this link, I first mapped the interacting regions of the two proteins and discovered a previously unrecognized N-terminal globular domain of p68 (p68N) that physically interacts with the Tag helicase domain. The solution structure of p68N was determined by NMR spectroscopy in the Chazin lab. The putative Tag-interacting surface on p68N was mapped to a hydrophobic patch surrounded by negative charges. Structure-guided mutagenesis of p68 residues in the interface diminished Tag-p68 interaction and primosome activity of the SV40 replisome. The p68N-docking site on Tag was identified using structure-guided mutagenesis of the Tag helicase surface. A charge reversal substitution in Tag specifically disrupted p68N binding, and hence the SV40 primosome activity. These results collectively suggested that the Tag-p68 interaction is vital for SV40 primosome function. A model is presented for how this interaction regulates primosome activity of the pol-prim complex, providing significant insights into the mechanism of eukaryotic DNA replication initiation.