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A novel human hexameric DNA helicase: expression, purification and characterization.
E E Biswas1, R G Nagele, S Biswas
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.
Nucleic Acids Research
|April 9, 2001
Summary
Researchers cloned and purified human DNA helicase (hHcsA), finding it conserved across species. This hexameric protein unwinds DNA in a 5' to 3' direction and its expression is cell cycle-dependent, peaking in early S phase.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- DNA helicases are crucial enzymes for DNA replication and repair.
- Understanding human DNA helicases is vital for comprehending genome stability.
Purpose of the Study:
- To clone, express, and characterize a novel human DNA helicase, hHcsA.
- To investigate the enzymatic activity and cellular function of hHcsA.
Main Methods:
- Gene cloning and expression in E. coli.
- Protein purification using chromatography.
- DNA-dependent ATPase and helicase assays.
- In situ hybridization for gene expression analysis.
Main Results:
- hHcsA is a hexameric protein with DNA-dependent ATPase and 5' to 3' DNA unwinding activity.
- Helicase activity is stimulated by human and yeast replication protein A.
- hHcsA gene expression is cell cycle-dependent, peaking in late G1/early S phase.
Conclusions:
- hHcsA is a conserved human DNA helicase with essential enzymatic functions.
- The cell cycle-dependent expression suggests a role in DNA replication during S phase.