HIGH-MOBILITY-GROUP-1 2 PROTEINS ARE ESSENTIAL FOR INITIATING ROLLING-CIRCLE-TYPE DNA-REPLICATION AT A PARVOVIRUS HAIRPIN ORIGIN/

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.