A single-molecule approach to visualize the unwinding activity of DNA helicases

N Fili, CP Toseland, MS Dillingham, MR Webb, JE Molloy

Research output: Contribution to journalArticle (Academic Journal)peer-review

6 Citations (Scopus)

Abstract

Almost all aspects of DNA metabolism involve separation of double-stranded DNA catalyzed by helicases. Observation and measurement of the dynamics of these events at the single-molecule level provide important mechanistic details of helicase activity and give the opportunity to probe aspects that are not revealed in bulk solution measurements. The assay, presented here, provides information about helicase unwinding rates and processivity. Visualization is achieved by using a fluorescent single-stranded DNA-binding protein (SSB), which allows the time course of individual DNA unwinding events to be observed using total internal reflection fluorescence microscopy. Observation of a prototypical helicase, Bacillus subtilis AddAB, shows that the unwinding process consists of bursts of unwinding activity, interspersed with periods of pausing.
Translated title of the contributionA single-molecule approach to visualize the unwinding activity of DNA helicases
Original languageEnglish
Pages (from-to)193-214
Number of pages22
JournalMethods in Molecular Biology
Volume778
DOIs
Publication statusPublished - 2011

Bibliographical note

Other: Chapter 13

Fingerprint Dive into the research topics of 'A single-molecule approach to visualize the unwinding activity of DNA helicases'. Together they form a unique fingerprint.

Cite this