SEQUENCE MOTIFS IN THE REPLICATOR PROTEIN OF PARVOVIRUS-MVM ESSENTIALFOR NICKING AND COVALENT ATTACHMENT TO THE VIRAL ORIGIN - IDENTIFICATION OF THE LINKING TYROSINE

Parvoviral DNA replication has many features in common with prokaryoti c rolling circle replication (RCR), including the pivotal role of an i nitiator protein which introduces a site-specific, single strand nick into a duplex origin sequence. In this process, the protein becomes co valently attached to the new 5' end of the DNA, while making available a 3' hydroxyl to prime de novo synthesis. Sequence comparisons of pro karyotic RCR initiators has revealed a set of three common motifs, two of which, a putative metal coordination site and a downstream active- site tyrosine motif, could be tentatively identified in parvoviral rep licator proteins. We have introduced mutations into the NS1 gene of th e murine parvovirus minute virus of mice (MVM), in the putative metal coordination site at H129, and into the three candidate tyrosine motif s at Y188, Y197, and Y210. Histidine-tagged mutant proteins were expre ssed in HeLa cells from recombinant vaccinia virus vectors and partial ly purified. None of the mutant proteins were able to initiate replica tion of origin-containing plasmids in vitro, and each showed impaired site-specific binding to the viral origin, with Y188 and Y197 being mo st severely defective. If this deficiency was minimized using low salt conditions, however, Y188 and Y197 mutant proteins were able to nick and become covalently attached to origin DNA, whereas Y210 and H129 mu tant proteins were not, suggesting that the latter residues are part o f the catalytic site of the NS1 nickase. Transfer of [P-32]phosphate f rom substrate DNA to NS1, followed by cyanogen bromide cleavage of the complex, gave the single, labeled peptide consistent with Y210 being the linking tyrosine. (C) 1995 Academic Press, Inc.