Fork sensing and strand switching control antagonistic activities of RecQ helicases
Daniel Klaue,Daniela Kobbe,Felix E. Kemmerich,Alicja Kozikowska,Holger Puchta,Ralf Seidel,Ralf Seidel +6 more
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TLDR
This work investigates the DNA unwinding of RecQ helicases from Arabidopsis thaliana, AtRECQ2 and AtRECZ3 at the single-molecule level using magnetic tweezers and provides a simple explanation for how different biological activities can be achieved by rather similar members of the RecQ family.Abstract:
RecQ helicases have essential roles in maintaining genome stability during replication and in controlling double-strand break repair by homologous recombination. Little is known about how the different RecQ helicases found in higher eukaryotes achieve their specialized and partially opposing functions. Here, we investigate the DNA unwinding of RecQ helicases from Arabidopsis thaliana, AtRECQ2 and AtRECQ3 at the single-molecule level using magnetic tweezers. Although AtRECQ2 predominantly unwinds forked DNA substrates in a highly repetitive fashion, AtRECQ3 prefers to rewind, that is, to close preopened DNA forks. For both enzymes, this process is controlled by frequent strand switches and active sensing of the unwinding fork. The relative extent of the strand switches towards unwinding or towards rewinding determines the predominant direction of the enzyme. Our results provide a simple explanation for how different biological activities can be achieved by rather similar members of the RecQ family.read more
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Journal ArticleDOI
Camera-based three-dimensional real-time particle tracking at kHz rates and Ångström accuracy
Alexander Huhle,Daniel Klaue,Hergen Brutzer,Peter Daldrop,Sihwa Joo,Oliver Otto,Ulrich F. Keyser,Ralf Seidel,Ralf Seidel +8 more
TL;DR: This work demonstrates that camera-based imaging can provide a similar performance for all three dimensions of particle tracking with Ångström accuracy as laser detection through photodiodes, and provides a simple and robust way for high-resolution tweezers experiments using multiple particles at a time.
Journal ArticleDOI
Mechanistic insight into the interaction of BLM helicase with intra-strand G-quadruplex structures.
Sujoy Chatterjee,Jennifer Zagelbaum,Pavel Savitsky,Andreas Sturzenegger,Diana Huttner,Pavel Janscak,Ian D. Hickson,Opher Gileadi,Eli Rothenberg +8 more
TL;DR: It is shown that the activity of BLM is substrate dependent, and highly regulated by a short ssDNA segment that separates the G4 motif from dsDNA, and a model is presented that proposes a unique role for G4 structures in modulating theActivity of DNA processing enzymes.
Journal ArticleDOI
Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases
TL;DR: It is shown that the helicase of hDNA2 functionally integrates with BLM or WRN helicases to promote dsDNA degradation by forming a heterodimeric molecular machine, which collectively suggests that the h DNA2 motor promotes the enzyme's capacity to degrade ds DNA in conjunction with BLMor WRN and thus promote the repair of broken DNA.
Journal ArticleDOI
Dynamic look at DNA unwinding by a replicative helicase
Seung Jae Lee,Salman Syed,Eric J. Enemark,Eric J. Enemark,Stephen Schuck,Arne Stenlund,Taekjip Ha,Leemor Joshua-Tor,Leemor Joshua-Tor,Leemor Joshua-Tor +9 more
TL;DR: The findings reveal that E1 employs a strand exclusion mechanism to unwind DNA with the N-terminal side leading at the replication fork, and DNA unwinding by E1 is modulated by the origin-recognition domain, suggesting a previously unsuspected role for this domain in regulating helicase activity.
Journal ArticleDOI
Extending the Range for Force Calibration in Magnetic Tweezers
Peter Daldrop,Hergen Brutzer,Alexander Huhle,Dominik J. Kauert,Ralf Seidel,Ralf Seidel,Ralf Seidel +6 more
TL;DR: The force calibration based on the long pendulum geometry will facilitate high-resolution magnetic-tweezers experiments that rely on short molecules and large forces, as well as highly parallelized measurements that use low frame rates.
References
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Journal ArticleDOI
Biochemical Characterization of AtRECQ3 Reveals Significant Differences Relative to Other RecQ Helicases
TL;DR: In this article, the authors performed biochemical analysis of AtRECQ3, expanded upon a previous analysis of atRECQ2, and compared their properties, showing that they are 3′ to 5′ helicases with similar activities on partial duplex DNA.
Journal ArticleDOI
Erratum: SSB protein diffusion on single-stranded DNA stimulates RecA filament formation (Nature (2009) 461 (1092-1097))
Book ChapterDOI
Purification and Characterization of RecQ Helicases of Plants
TL;DR: In this chapter the initial steps that will provide the basis for a proper biochemical characterization are given and its purification with the help of two affinity tags and the preparation of a model DNA substrate for the strand displacement assay are described.