Particle tracking with ultra-high resolution in optical and magnetic tweezers has so far relied on laser detection through photodiodes. Here, Huhle et al. demonstrate three-dimensional particle tracking with Angstrom accuracy and real-time GPU-accelerated data processing at kHz rates using camera-based imaging.

/pdf/camera-based-three-dimensional-real-time-particle-tracking-3zih50b99d.pdf

Camera-based three-dimensional real-time particle tracking at kHz rates and Ångström accuracy

Bloom syndrome is an autosomal recessive disorder caused by mutations in the RecQ family helicase BLM that is associated with growth retardation and predisposition to cancer. BLM helicase has a high specificity for non-canonical G-quadruplex (G4) DNA structures, which are formed by G-rich DNA strands and play an important role in the maintenance of genomic integrity. Here we used single-molecule FRET to define the mechanism of interaction of BLM helicase with intra-stranded G4 structures. We show that the activity of BLM is substrate dependent, and highly regulated by a short-strand DNA (ssDNA) segment that separates the G4 motif from double-stranded DNA. We demonstrate cooperativity between the RQC and HRDC domains of BLM during binding and unfolding of the G4 structure, where the RQC domain interaction with G4 is stabilized by HRDC binding to ssDNA. We present a model that proposes a unique role for G4 structures in modulating the activity of DNA processing enzymes.

/pdf/mechanistic-insight-into-the-interaction-of-blm-helicase-5frxtbl6kr.pdf

Mechanistic insight into the interaction of BLM helicase with intra-strand G-quadruplex structures.

Human DNA2 (hDNA2) contains both a helicase and a nuclease domain within the same polypeptide. The nuclease of hDNA2 is involved in a variety of DNA metabolic processes. Little is known about the role of the hDNA2 helicase. Using bulk and single-molecule approaches, we show that hDNA2 is a processive helicase capable of unwinding kilobases of dsDNA in length. The nuclease activity prevents the engagement of the helicase by competing for the same substrate, hence prominent DNA unwinding by hDNA2 alone can only be observed using the nuclease-deficient variant. We show that the helicase of hDNA2 functionally integrates with BLM or WRN helicases to promote dsDNA degradation by forming a heterodimeric molecular machine. This collectively suggests that the hDNA2 motor promotes the enzyme's capacity to degrade dsDNA in conjunction with BLM or WRN and thus promote the repair of broken DNA.

/pdf/human-dna2-possesses-a-cryptic-dna-unwinding-activity-that-i4n17szlan.pdf

Human DNA2 possesses a cryptic DNA unwinding activity that functionally integrates with BLM or WRN helicases

A prerequisite for DNA replication is the unwinding of duplex DNA catalyzed by a replicative hexameric helicase. Despite a growing body of research, key elements of helicase mechanism remain under substantial debate. In particular, the number of DNA strands encircled by the helicase ring during unwinding and the ring orientation at the replication fork completely contrast in contemporary mechanistic models. Here we use single-molecule and ensemble assays to address these questions for the papillomavirus E1 helicase. We find that E1 unwinds DNA with a strand-exclusion mechanism, with the N-terminal side of the helicase ring facing the replication fork. We show that E1 generates strikingly heterogeneous unwinding patterns stemming from varying degrees of repetitive movements, which is modulated by the DNA-binding domain. Together, our studies reveal previously unrecognized dynamic facets of replicative helicase unwinding mechanisms.

https://www.pnas.org/content/pnas/111/9/E827.full.pdf

Dynamic look at DNA unwinding by a replicative helicase

https://www.cell.com/biophysj/pdf/S0006-3495(15)00389-6.pdf

Extending the Range for Force Calibration in Magnetic Tweezers

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.

/pdf/fork-sensing-and-strand-switching-control-antagonistic-2d4ysby15q.pdf

Alicja Kozikowska

Papers

Fork sensing and strand switching control antagonistic activities of RecQ helicases