Sf. Cotmore et P. Tattersall, HIGH-MOBILITY-GROUP-1 2 PROTEINS ARE ESSENTIAL FOR INITIATING ROLLING-CIRCLE-TYPE DNA-REPLICATION AT A PARVOVIRUS HAIRPIN ORIGIN/, Journal of virology (Print), 72(11), 1998, pp. 8477-8484
Rolling-circle replication is initiated by a replicon-encoded endonucl
ease which introduces a single-strand nick into specific origin sequen
ces, becoming covalently attached to the 5' end of the DNA at the nick
and providing a 3' hydroxyl to prime unidirectional, leading-strand s
ynthesis. Parvoviruses. such as minute virus of mice (MVM), have adapt
ed this mechanism to amplify their linear single-stranded genomes by u
sing hairpin telomeres which sequentially unfold and refold to shuttle
the replication fork back and forth along the genome, creating a cont
inuous, multimeric DNA strand. The viral initiator protein, NS1, then
excises individual genomes from this continuum by nicking and reinitia
ting synthesis at specific origins present within the hairpin sequence
s. Using in vitro assays tea study ATP-dependent initiation within the
right-hand (5') MVM hairpin, we have characterized a HeLa cell factor
which is absolutely required to allow NS1 to nick this origin. Unlike
parvovirus initiation factor (PIF), the cellular complex which activa
tes NS1 endonuclease activity at the left-hand (3') viral origin, the
host factor which activates the right-hand hairpin elutes from phospho
cellulose in high salt, has a molecular mass of around 25 kDa, and app
ears to bind preferentially to structured DNA, suggesting that it migh
t be a member of the high-mobility group 1/2 (HMG1/2) protein family.
This prediction was confirmed by showing that purified calf thymus HMG
1 and recombinant human HMG1 or murine HMG2 could each substitute for
the HeLa factor, activating the NS1 endonuclease in an origin-specific
nicking reaction.