EFFECTS OF ALTERED RGD (ARG-GLY-ASP) SEQUENCES IN THE PRIMER PROTEIN ON THE PROTEIN PRIMED DNA REPLICATION OF BACTERIOPHAGE M2 IN VITRO

Abstract

To elucidate the detailed role of the RGD (Arg-Gly-Asp) sequence of the primer protein (PP) on the protein primed DNA replication of phage M2, we introduced the site-specific mutations in the nucleotide sequence for the RGD sequence of PP. Using an in vitro DNA replication system which was composed of three indispensable components, PP, Pol (phage M2 coded DNA polymerase) and double-stranded template DNA of phage M2 with a covalently bound terminal protein (TP) at both 5′ termini (TP-DNA), the priming activity of modified PPs was tested. The modified PPs decreased in priming activity by various levels, indicating an essential role of the RGD sequence to initiate DNA replication. Then, the modified PPs were tested for their binding ability to the other components, Pol and TP. Though all the modified PPs formed heterodimers with Pol, a significant reduction of the binding ability to TP was evident. The degrees of the reduction corresponded to the decrease of the ability of each modified PP to prime DNA replication. The results strongly suggest that the RGD motif of phage M2 primer protein is a key sequence for binding TP to initiate the protein primed DNA replication.