Minute virus of mice initiator protein NS1 and a host KDWK family transcription factor must form a precise ternary complex with origin DNA for nicking to occur
J. Christensen et al., Minute virus of mice initiator protein NS1 and a host KDWK family transcription factor must form a precise ternary complex with origin DNA for nicking to occur, J VIROLOGY, 75(15), 2001, pp. 7009-7017
Parvoviral rolling hairpin replication generates palindromic genomic concat
emers whose junctions are resolved to give unit-length genomes by a process
involving DNA replication initiated at origins derived from each viral tel
omere, The left-end origin of minute virus of mice (MVM), oriL, contains bi
nding sites for the viral initiator nickase, NS1, and parvovirus initiation
factor (PIF), a member of the emerging KDWK family of transcription factor
s, oriL is generated as an active form, oriL(TC), and as an inactive form,
oriL(GAA), which contains a single additional nucleotide inserted between t
he NS1 and PIF sites. Here we examined the interactions on oriL(TC), which
lead to activation of NS1 by PIF, The two subunits of PIF, p79 and p96, coo
peratively bind two ACGT half-sites, which can be flexibly spaced. When coe
xpressed from recombinant baculoviruses, the PIF subunits preferentially fo
rm heterodimers which, in the presence of ATP, show cooperative binding wit
h NS1 on oriL, but this interaction is preferentially enhanced on oriL(TC),
compared to oriL(GAA). Without ATP, NS1 is unable to bind stably to its co
gnate site, but PIF facilitates this interaction, rendering the NSI binding
site, but not the nick site, resistant to DNase I. Varying the spacing of
the PIF half-sites shows that the distance between the NS1 binding site and
the NS1-proximal half-site is critical for nickase activation, whereas the
position of the distal half-site is unimportant. When expressed separately
, both PIF subunits form homodimers that bind site specifically to oriL, bu
t only complexes containing p79 activate the NS1 nickase function.