HUMAN DNA HELICASE-V, A NOVEL DNA UNWINDING ENZYME FROM HELA-CELLS

Using a strand-displacement assay with P-32 labeled oligonucleotide an nealed to M13 ssDNA we have purified to apparent homogeneity and chara cterized 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 helicases. From 300 grams of cultured cells only 0.012 mg of pure protein was iso lated which was free of DNA topoisomerase, ligase, nicking and nucleas e activities. The enzyme also shows ATPase activity dependent on singl e-stranded DNA and has an apparent molecular weight of 92 kDa by SDS-p olyacrylamide gel electrophoresis. Only ATP or dATP hydrolysis support s the unwinding activity. The helicase requires a divalent cation (Mg2 + > Mn2+) at an optimum concentration of 1.0 mM for activity; it unwin ds DNA duplexes less than 25 bp long and having a ssDNA stretch as sho rt as 49 nucleotides. A replication fork-like structure is not require d to perform DNA unwinding. HDH V cannot unwind either blunt-ended dup lex DNA or DNA-RNA hybrids; it unwinds DNA unidirectionally by moving in the 3' to 5' direction along the bound strand, a polarity similar t o the previously described human DNA helicases 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 helicas e IV (Tuteja et al. Nucleic Acid Res. 19, 3613 - 3618, 1991).