Purification andproperties ofhumanDNAhelicase VI

Anovel ATP-dependent DNAunwinding enzyme, called humanDNA helicase VI(HDHVI), waspurified to apparent homogeneity fromHeLacells andcharacterized. From327gofcultured cells, 0.44mg ofpure enzymewasrecovered, free ofDNApolymerase, ligase, topoisomerase, nicking andnuclease activities. The enzymebehaves asamonomerhaving anMrof128 kDa,whether determined withSDS-PAGE, orinnative conditions. Photoaffinity labelling with[ox-32P]ATP labelled the128kDaprotein. OnlyATPordATP hydrolysis supports theunwinding activity forwhicha divalent cation (Mg2+ > Mn2+) isrequired. HDH VI unwinds exclusively DNAduplexes withanannealed portion <32bpandprefers a replication fork-like structure ofthesubstrate. Itcannotunwindblunt-end duplexes andisinactive alsoonDNA-RNAorRNARNAhybrids. HDHVIunwinds DNAunidirectionally by moving inthe3'to5'direction along theboundstrand.