PARVOVIRUS INITIATION-FACTOR PIF - A NOVEL HUMAN DNA-BINDING FACTOR WHICH COORDINATELY RECOGNIZES 2 ACGT MOTIFS

A novel human site-specific DNA-binding factor has been partially puri fied from extracts of HeLa S3 cells. This factor, designated PIF, for parvovirus initiation factor, binds to the minimal origin of DNA repli cation at the 3' end of the minute virus of mice (MVM) genome and func tions as an essential cofactor in the replication initiation process. Here we show that PIF is required for the viral replicator protein NS1 to nick and become covalently attached to a specific site in the orig in sequence in a reaction which requires ATP hydrolysis, DNase I and c opper ortho-phenanthroline degradation of the PIF-DNA complexes showed that PIF protects a stretch of some 20 nucleotides, covering the enti re region in the minimal left-end origin not already known to be occup ied by NS1. Methylation and carboxy-ethylation interference analysis i dentified two ACGT motifs, spaced by five nucleotides, as the sequence s responsible for this binding. A series of mutant oligonucleotides wa s then used as competitive inhibitors in gel mobility shift assays to confirm that PIF recognizes both of these ACGT sequences and to demons trate that the two motifs comprise a single binding site rather than t wo separate sites, Competitive inhibition of the origin nicking assay, using the same group of oligonucleotides, confirmed that the same cel lular factor is responsible for both mobility shift and nicking activi ties. UV cross-linking and relative mobility assays suggest that PIF b inds DNA as a heterodimer or higher-order multimer with subunits in th e 80- to 100-kDa range.