The putative coiled coil domain of the phi 29 terminal protein is a major determinant involved in recognition of the origin of replication

The linear double-stranded genome of phage phi 29 contains a terminal prote in (TP) covalently linked at each 5' DNA end, called parental TP. Initiatio n of phi 29 DNA replication starts with the recognition of the origins of r eplication, constituted by the parental TP-containing DNA ends, by a hetero dimer containing phi 29 DNA polymerase and primer TP. It has been argued th at origin recognition involves protein-protein interactions between parenta l and primer TP. Analysis of the TP sequence revealed that the region betwe en amino acids 84 and 118 has a high probability to form an amphipatic alph a -helix that could be involved in the interaction between parental and pri mer TP. Therefore, this TP region may be important for origin recognition. To test this hypothesis we introduced various mutations in the predicted am phipatic alpha -helix and analyzed the functionality of the corresponding p urified TP mutants. The results obtained show that the identified putative amphipatic alpha -helix of TP is an important determinant involved in origi n recognition.