PHAGE-P4 ALPHA-PROTEIN IS MULTIFUNCTIONAL WITH ORIGIN RECOGNITION, HELICASE AND PRIMASE ACTIVITIES

Alpha protein of satellite phage P4 of Escherichia coli is multifuncti onal in P4 replication with three activities. First, the protein (subu nit M(r) = 84 900) complexes specifically the P4 origin and the cis re plication region required for replication. Alpha protein interacts wit h all six type I repeats (TGTTCACC) present in the origin. Second, ass ociated with the alpha protein is a DNA helicase activity that is fuel ed by hydrolysis of a nucleoside 5' triphosphate. All common NTPs exce pt UTP and dTTP can serve as cofactors. Strand separation of partial d uplexes containing tailed ends that resemble a replication fork is pre ferred, although a preformed fork is not absolutely required for the e nzyme to invade and unwind duplex DNA. Alpha protein catalyzes unwindi ng in the 3'-->5' direction with respect to the strand it has bound. F inally, the primase activity already demonstrated for alpha protein is due to synthesis of RNA primers. In vitro, alpha protein generates di - to pentaribonucleotides on single-stranded phage fd DNA. The predomi nant product is the dimer pppApG, on which most of the longer oligorib o-nucleotides are based. Using DNA oligonucleotides of defined sequenc e as templates, synthesis of pppApG was also detectable. To date, amon g prokaryotic and eukaryotic replication systems, gpalpha is the only protein known that combines three activities on one single polypeptide chain.