A novel human hexameric DNA helicase: expression, purification and characterization

We have cloned, expressed and purified a hexameric human DNA helicase (hHcs A) from HeLa cells. Sequence analysis demonstrated that the hHcsA has stron g sequence homology with DNA helicase genes from Saccharomyces cerevisiae a nd Caenorhabditis elegans, indicating that this gene appears to be well con served from yeast to human, The hHcsA gene was cloned and expressed in Esch erichia coil and purified to homogeneity. The expressed protein had a subun it molecular mass of 116 kDa and analysis of its native molecular mass by s ize exclusion chromatography suggested that hHcsA is a hexameric protein, T he hHcsA protein had a strong DNA-dependent ATPase activity that was stimul ated greater than or equal to5-fold by single-stranded DNA (ssDNA). Human h HcsA unwinds duplex DNA and analysis of the polarity of translocation demon strated that the polarity of DNA unwinding was in a 5'-->3' direction. The helicase activity was stimulated by human and yeast replication protein A, but not significantly by E,coli ssDNA-binding protein, We have analyzed exp ression levels of the hHcsA gene in HeLa cells during various phases of the cell cycle using in situ hybridization analysis, Our results indicated tha t the expression of the hHcsA gene, as evidenced from the mRNA levels, is c ell cycle-dependent. The maximal level of hHcsA expression was observed in late G(1)/early S phase, suggesting a possible role for this protein during S phase and in DNA synthesis.