ϕX174 cistron A protein is a multifunctional enzyme in DNA replication

Abstract

The cistron A protein induced by phase .vphi.X174 nicks (produces a single-strand break in) the viral strand of the superhelical .vphi.X duplex DNA, thereby forming a complex with the DNA. The protein, seen bound to the DNA by EM, was located in the restriction endonuclease fragment between nucleotides 4290 and 4330 on the .vphi.X map. Replication also was initiated at this point, thus identifying the site of cistron A protein nicking and binding as the origin of replication. The cisA-DNA complex (separated from free cistron A protein), upon the addition of Escherichia coli rep protein, ATP and DNA binding protein, is unwound to generate a single-stranded linear [presumably the nicked (+) strand] and a circular [presumably the (-) strand] molecule. The cisA-DNA complex, upon the further addition of DNA polymerase III holoenzyme and deoxynucleoside triphosphates, supports replication to generate viral, single-stranded circles, as many as 15 circles/cisA-DNA complex. The replicating intermediates seen by EM are a novel form of rolling circle. The 5'' end (presumably with the cistron A protein bound to it) is locked in the replication fork and loops back to accompany the strand-separation and replication fork around the template [(-) strand] circle. Thus, the multiple functions of cistron A protein include: nicking the viral strand at the origin of replication to initiate a round of replication, participating in a complex which supports fork movement in strand separation and replication, nicking again at the regenerated origin to produce a unit-length DNA and ligating the newly generated 3''-OH end to the 5''-phosphate-complexed end to form a circular viral molecule.