Biochemical assays for the characterization of DNA helicases.
Helicases are ubiquitous enzymes that disrupt complementary strands of duplex nucleic acid in a reaction dependent on nucleoside-5'-triphosphate hydrolysis. Helicases are implicated in the metabolism of DNA structures that are generated during replication, recombination, and DNA repair. Furthermore, an increasing number of helicases have been linked to genomic instability and human disease. With the growing interest in helicase mechanism and function, we have set out to describe some basic protocols for biochemical characterization of DNA helicases. Protocols for measuring ATP hydrolysis, DNA binding, and catalytic unwinding activity of DNA helicases are provided. Application of these procedures should enable the researcher to address fundamental questions regarding the biochemical properties of a given helicase, which would serve as a platform for further investigation of its molecular and cellular functions.
Brosh
Robert M
RM
Laboratory of Molecular Gerontology, National Institute on Aging, NIH, Baltimore, MD, USA.
Sharma
Sudha
S