Mutational analysis of Bloom helicase.
DNA helicases are biomolecular motors that convert the chemical energy derived from the hydrolysis of nucleotide triphosphate (usually ATP) into mechanical energy to unwind double-stranded DNA. The unwinding of double-stranded DNA is an essential process for DNA replication, repair, recombination, and transcription. Mutations in human RecQ helicases result in inherent human disease including Bloom's syndrome, Werner's syndrome, and Rothmund-Thomson syndrome. Bloom's syndrome (BS) is a rare human autosomal recessive disorder characterized by a strong predisposition to a wide range of cancers commonly affecting the general population. In order to understand the molecular basis of BS pathology and the mechanism underlying the function of Bloom helicase, we have analyzed BS-causing missense mutations by a combination of structural modeling, site-directed mutagenesis, and biochemical and biophysical approaches. Here, we describe the methods and protocols for measuring ATPase, ATP and DNA binding, DNA strand annealing, and DNA unwinding activities of Bloom protein and its mutant variants. These approaches should be applicable and useful for studying other helicases.
Xi
Xu Guang
XG
Institut CURIE Recherche, Orsay, France.
D000251:Adenosine Triphosphatases / Q000378:metabolism; D016880:Anisotropy; D001816:Bloom Syndrome / Q000201:enzymology / Q000235:genetics; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004252:DNA Mutational Analysis / Q000379:methods*; D006801:Humans; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation
D004247:DNA; D000251:Adenosine Triphosphatases