Human DNA helicase V, a novel DNA unwinding enzyme from HeLa cells

Abstract

Using a strand-displacement assay with ³²P labeled ollgonucleotide annealed to M13 ssDNA we have purified to apparent homogeneity and characterized a novel DNA unwinding enzyme from HeLa cell nuclei, human DNA helicase V (HDH V). This Is present In extremely low abundance In the cells and has the highest turnover rate among other human hellcases. From 300 grams of cultured cells only 0.012 mg of pure protein was Isolated which was free of DNA topolsomerase, llgase, nicking and nuclease activities. The enzyme also shows ATPase activity dependent on single-stranded DNA and has an apparent molecular weight of 92 kDa by SDS-polyacrylamide gel electrophoresls. Only ATP or dATP hydrolysis supports the unwinding activity. The helicase requires a divalent cation (Mg²⁺ > Mn²⁺) at an optimum concentration of 1.0 mM for activity; It unwinds DNA duplexes less than 25 bp long and having a ssDNA stretch as short as 49 nucleotides. A replication fork-like structure is not required to perform DNA unwinding. HDH V cannot unwind either blunt-ended duplex DNA or DNA-RNA hybrids; It unwinds DNA unldlrectionally by moving in the 3' to 5' direction along the bound strand, a polarity similar to the previously described human DNA hellcases I and III (Tuteja et al. Nucleic Acids Res. 18, 6785-6792, 1990; Tuteja et al. Nucleic Acid Res. 20, 5329-5337,1992) and opposite to that of human DNA helicase IV (Tuteja et al. Nucleic Acid Res. 19, 3613-3618, 1991).