Fork sensing and strand switching control antagonistic activities of RecQ helicases
TL;DR: 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.
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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.
Abstract: 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.
113 citations
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.
Abstract: 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.
80 citations
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.
Abstract: 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.
64 citations
Cites background from "Fork sensing and strand switching c..."
...In agreement with this no direction reversals during unwinding (i.e. rezipping) that could originate from strand–switches were observed (Dessinges et al., 2004; Klaue et al., 2013)....
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...rezipping) that could originate from strand–switches were observed (Dessinges et al., 2004; Klaue et al., 2013)....
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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.
Abstract: 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.
61 citations
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.
56 citations
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TL;DR: Biochemical analyses indicate that Topoisomerase 3α functions together with a RecQ DNA helicase and a third partner, RMI1/BLAP75, in the resolution step of homologous recombination in a process called Holliday Junction dissolution in eukaryotes, and suggests the dissolution of double Holliday Junctions via a hemicatenane intermediate is indeed an indispensable step of meiotic recombination.
Abstract: Topoisomerases are enzymes with crucial functions in DNA metabolism. They are ubiquitously present in prokaryotes and eukaryotes and modify the steady-state level of DNA supercoiling. Biochemical analyses indicate that Topoisomerase 3a (TOP3a) functions together with a RecQ DNA helicase and a third partner, RMI1/BLAP75, in the resolution step of homologous recombination in a process called Holliday Junction dissolution in eukaryotes. Apart from that, little is known about the role of TOP3a in higher eukaryotes, as knockout mutants show early lethality or strong developmental defects. Using a hypomorphic insertion mutant of Arabidopsis thaliana (top3a-2), which is viable but completely sterile, we were able to define three different functions of the protein in mitosis and meiosis. The top3a-2 line exhibits fragmented chromosomes during mitosis and sensitivity to camptothecin, suggesting an important role in chromosome segregation partly overlapping with that of type IB topoisomerases. Furthermore, AtTOP3a, together with AtRECQ4A and AtRMI1, is involved in the suppression of crossover recombination in somatic cells as well as DNA repair in both mammals and A. thaliana. Surprisingly, AtTOP3a is also essential for meiosis. The phenotype of chromosome fragmentation, bridges, and telophase I arrest can be suppressed by AtSPO11 and AtRAD51 mutations, indicating that the protein is required for the resolution of recombination intermediates. As Atrmi1 mutants have a similar meiotic phenotype to Attop3a mutants, both proteins seem to be involved in a mechanism safeguarding the entangling of homologous chromosomes during meiosis. The requirement of AtTOP3a and AtRMI1 in a late step of meiotic recombination strongly hints at the possibility that the dissolution of double Holliday Junctions via a hemicatenane intermediate is indeed an indispensable step of meiotic recombination.
92 citations
TL;DR: The results show that neither illumination nor software algorithms limit the speed of real-time particle tracking with CMOS technology.
Abstract: We introduce optical fiber illumination for real-time tracking of optically trapped micrometer-sized particles with microsecond time resolution. Our light source is a high-radiance mercury arc lamp and a 600 μm optical fiber for short-distance illumination of the sample cell. Particle tracking is carried out with a software implemented cross-correlation algorithm following image acquisition from a CMOS camera. Our image data reveals that fiber illumination results in a signal-to-noise ratio usually one order of magnitude higher compared to standard Kohler illumination. We demonstrate position determination of a single optically trapped colloid with up to 10,000 frames per second over hours. We calibrate our optical tweezers and compare the results with quadrant photo diode measurements. Finally, we determine the positional accuracy of our setup to 2 nm by calculating the Allan variance. Our results show that neither illumination nor software algorithms limit the speed of real-time particle tracking with CMOS technology.
80 citations
TL;DR: Single‐molecule fluorescence resonance energy transfer microscopy is utilized to examine the behaviour of Bloom syndrome gene encodes a member of the RecQ family of 3′–5′ DNA helicases and shows that a monomeric BLM can ‘measure’ how many base pairs it has unwound, and once it has unraveled a critical length, it reverses the unwinding reaction through strand switching and translocating on the opposing strand.
Abstract: Bloom syndrome (BS) is a rare genetic disorder characterized by genomic instability and a high predisposition to cancer. The gene defective in BS, BLM, encodes a member of the RecQ family of 3'-5' DNA helicases, and is proposed to function in recombinational repair during DNA replication. Here, we have utilized single-molecule fluorescence resonance energy transfer microscopy to examine the behaviour of BLM on forked DNA substrates. Strikingly, BLM unwound individual DNA molecules in a repetitive manner, unwinding a short length of duplex DNA followed by rapid reannealing and reinitiation of unwinding in several successions. Our results show that a monomeric BLM can 'measure' how many base pairs it has unwound, and once it has unwound a critical length, it reverses the unwinding reaction through strand switching and translocating on the opposing strand. Repetitive unwinding persisted even in the presence of hRPA, and interaction between wild-type BLM and hRPA was necessary for unwinding reinitiation on hRPA-coated DNA. The reported activities may facilitate BLM processing of stalled replication forks and illegitimately formed recombination intermediates.
76 citations
TL;DR: In this paper, the authors examined four partners of the two isoforms of Brca2 identified in Arabidopsis (AtRad51, AtDmc1, and two AtDss1 isoforms).
Abstract: The Arabidopsis (Arabidopsis thaliana) orthologs of Brca2, a protein whose mutations are involved in breast cancer in humans, were previously shown to be essential at meiosis. In an attempt to better understand the Brca2-interacting properties, we examined four partners of the two isoforms of Brca2 identified in Arabidopsis (AtRad51, AtDmc1, and two AtDss1 isoforms). The two Brca2 and the two Dss1 isoforms are named AtBrca2(IV), AtBrca2(V), AtDss1(I), and AtDss1(V) after their chromosomal localization. We first show that both AtBrca2 proteins can interact with either AtRad51 or AtDmc1 in vitro, and that the N-terminal region of AtBrca2 is responsible for these interactions. More specifically, the BRC motifs (so called because iterated in the Brca2 protein) in Brca2 are involved in these interactions: BRC motif number 2 (BRC2) alone can interact with AtDmc1, whereas BRC motif number 4 (BRC4) recognizes AtRad51. The human Rad51 and Dmc1 proteins themselves can interact with either the complete (HsRad51) or a shorter version of AtBrca2 (HsRad51 or HsDmc1) that comprises all four BRC motifs. We also identified two Arabidopsis isoforms of Dss1, another known partner of Brca2 in other organisms. Although all four Brca2 and Dss1 proteins are much conserved, AtBrca2(IV) interacts with only one of these AtDss1 proteins, whereas AtBrca2(V) interacts with both of them. Finally, we show for the first time that an AtBrca2 protein could bind two different partners at the same time: AtRad51 and AtDss1(I), or AtDmc1 and AtDss1(I).
76 citations
TL;DR: In this paper, the UvsW DNA helicase in cooperation with the T4 holoenzyme can overcome leading-strand lesion damage by a pseudostochastic process, periodically forming and migrating a four-way Holliday junction.
Abstract: The restart of a stalled replication fork is a major challenge for DNA replication. Depending on the nature of the damage, different repair processes might be triggered; one is template switching, which is a bypass of a leading-strand lesion via fork regression. Using magnetic tweezers to study the T4 bacteriophage enzymes, we have reproduced in vitro the complete process of template switching. We show that the UvsW DNA helicase in cooperation with the T4 holoenzyme can overcome leading-strand lesion damage by a pseudostochastic process, periodically forming and migrating a four-way Holliday junction. The initiation of the repair process requires partial replisome disassembly via the departure of the replicative helicase. The results support the role of fork regression pathways in DNA repair.
76 citations