Two widely spaced initiator binding sites create an HMG1-dependent parvovirus rolling-hairpin replication origin

Minute virus of mite (MVM) replicates via a linearized form of rolling-circ le replication in which the viral nickase, NS1, initiates DNA, synthesis by introducing a site-specific nick into either of ho distinct origin sequenc es. In vitro nicking and replication assays with substrates that had deleti ons or mutations were used to explore the sequences and structural elements essential for activity of one of these origins, located in the right-end ( 5') viral telomere. This structure contains 248 nucleotides, most-favorably arranged as a simple hairpin with six unpaired bases. However, a pair of o pposing NS1 binding sites, located near its outboard end, create a 33-bp pa lindrome that could potentially assume an alternate cruciform configuration and hence directly bind HMG1, the essential cofactor for this origin. The palindromic nature of this sequence, and thus its ability to fold into a cr uciform, was dispensable for origin function, as was the NS1 binding site o ccupying the inboard arm of the palindrome, In contrast, the NS1 site in th e outboard arm was essential for initiation, even though positioned 120 bp from the nick site. The specific sequence of the nick site and an additiona l NS1 binding site which directly orients NS1 over the initiation site were also essential and delimited the inboard border of the minimal right-end o rigin. DNase I and hydroxyl radical footprints defined sequences protected by NS1 and suggest that HMG1 allows the NS1 molecules positioned at each en d of the origin to interact, creating a distortion characteristic of a doub le helical loop.