SEQUENCE MOTIFS IN THE REPLICATOR PROTEIN OF PARVOVIRUS-MVM ESSENTIALFOR NICKING AND COVALENT ATTACHMENT TO THE VIRAL ORIGIN - IDENTIFICATION OF THE LINKING TYROSINE
Jpf. Nuesch et al., SEQUENCE MOTIFS IN THE REPLICATOR PROTEIN OF PARVOVIRUS-MVM ESSENTIALFOR NICKING AND COVALENT ATTACHMENT TO THE VIRAL ORIGIN - IDENTIFICATION OF THE LINKING TYROSINE, Virology, 209(1), 1995, pp. 122-135
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.