Analysis of the DNA unwinding activity of RecQ family helicases.
The RecQ family of DNA helicases is highly conserved in evolution from bacteria to mammals. There are five human RecQ family members (RECQ1, BLM, WRN, RECQ4 and RECQ5), defects, three of which give rise to inherited human disorders. Mutations of BLM have been identified in patients with Bloom's syndrome, WRN has been shown to be mutated in Werner's syndrome, while mutations of RECQ4 have been associated with at least a subset of cases of both Rothmund-Thomson syndrome and RAPADILINO. The most characteristic features of these diseases are a predisposition to the development of malignancies of different types (particularly in Bloom's syndrome), some aspects of premature aging (particularly in Werner's syndrome), and on the cellular level, genome instability. In order to gain understanding of the molecular defects underlying these diseases, many laboratories have focused their research on a study of the biochemical properties of human RecQ helicases, particularly those associated with disease, and of RecQ proteins from other organisms (e.g., Sgs1p of budding yeast, Rqh1p of fission yeast, and RecQ of E.coli). In this chapter, we summarize the assay systems that we employ to analyze the catalytic properties of the BLM helicase. We have successfully used these methods for the study of other RecQ and non-RecQ helicases, indicating that they are likely to be applicable to all helicases.
Bachrati CZ, Hickson ID