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Nucleic Acids Research, 2001, Vol. 29, No. 8 1733-1740
© 2001 Oxford University Press

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

Esther E. Biswas1,2, Robert G. Nagele1 and Subhasis Biswas1,*

1Department of Molecular Biology, School of Osteopathic Medicine and Graduate School of Biomedical Sciences, University of Medicine and Dentistry of New Jersey, Science Center Room 305 A, 2 Medical Center Drive, Stratford, NJ 08084, USA and 2Program in Biotechnology, Department of Laboratory Sciences, Thomas Jefferson University, Philadelphia, PA 19107, USA

We have cloned, expressed and purified a hexameric human DNA helicase (hHcsA) from HeLa cells. Sequence analysis demonstrated that the hHcsA has strong sequence homology with DNA helicase genes from Saccharomyces cerevisiae and Caenorhabditis elegans, indicating that this gene appears to be well conserved from yeast to human. The hHcsA gene was cloned and expressed in Escherichia coli and purified to homogeneity. The expressed protein had a subunit molecular mass of 116 kDa and analysis of its native molecular mass by size exclusion chromatography suggested that hHcsA is a hexameric protein. The hHcsA protein had a strong DNA-dependent ATPase activity that was stimulated >=5-fold by single-stranded DNA (ssDNA). Human hHcsA unwinds duplex DNA and analysis of the polarity of translocation demonstrated 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 expression levels of the hHcsA gene in HeLa cells during various phases of the cell cycle using in situ hybridization analysis. Our results indicated that the expression of the hHcsA gene, as evidenced from the mRNA levels, is cell cycle-dependent. The maximal level of hHcsA expression was observed in late G1/early S phase, suggesting a possible role for this protein during S phase and in DNA synthesis.

* To whom correspondence should be addressed. Tel: +1 856 566 6270; Fax: +1 781 207 8476; Email: biswassb{at}umdnj.edu


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