Kata Sarlós

Processive translocation mechanism of the human Bloom’s syndrome helicase along single-stranded DNA

BLM, one of the human RecQ helicases, plays a fundamental role in homologous recombination-based error-free DNA repair pathways, which require its translocation and DNA unwinding activities. Although translocation is essential in vivo during DNA repair processes and it provides a framework for more complex activities of helicases, including strand separation and nucleoprotein displacement, its mechanism has not been resolved for any human DNA helicase. Here, we present a quantitative model for the translocation of a monomeric form of BLM along ssDNA. We show that BLM performs translocation at a low adenosine triphosphate (ATP) coupling ratio (1 ATP consumed/1 nucleotide traveled) and moderate processivity (with a mean number of 50 nucleotides traveled in a single run). We also show that the rate-limiting step of the translocation cycle is a transition between two ADP-bound enzyme states. Via opening of the helicase core, this structural change may drive the stepping of BLM along the DNA track by a directed inchworm mechanism. The data also support the conclusion that BLM performs double-stranded DNA unwinding by fully active duplex destabilization.

Complex activities of the human Bloom's syndrome helicase are encoded in a core region comprising the RecA and Zn-binding domains

Blooms syndrome DNA helicase (BLM), a member of the RecQ family, is a key player in homologous recombination (HR)-based error-free DNA repair processes. During HR, BLM exerts various biochemical activities including single-stranded (ss) DNA translocation, separation and annealing of complementary DNA strands, disruption of complex DNA structures (e.g. displacement loops) and contributes to quality control of HR via clearance of Rad51 nucleoprotein filaments. We performed a quantitative mechanistic analysis of truncated BLM constructs that are shorter than the previously identified minimal functional module. Surprisingly, we found that a BLM construct comprising only the two conserved RecA domains and the Zn2+-binding domain (residues 642–1077) can efficiently perform all mentioned HR-related activities. The results demonstrate that the Zn2+-binding domain is necessary for functional interaction with DNA. We show that the extensions of this core, including the winged-helix domain and the strand separation hairpin identified therein in other RecQ-family helicases, are not required for mechanochemical activity per se and may instead play modulatory roles and mediate protein–protein interactions.

RecQ helicase translocates along single-stranded DNA with a moderate processivity and tight mechanochemical coupling

Maintenance of genome integrity is the major biological role of RecQ-family helicases via their participation in homologous recombination (HR)-mediated DNA repair processes. RecQ helicases exert their functions by using the free energy of ATP hydrolysis for mechanical movement along DNA tracks (translocation). In addition to the importance of translocation per se in recombination processes, knowledge of its mechanism is necessary for the understanding of more complex translocation-based activities, including nucleoprotein displacement, strand separation (unwinding), and branch migration. Here, we report the key properties of the ssDNA translocation mechanism of Escherichia coli RecQ helicase, the prototype of the RecQ family. We monitored the pre–steady-state kinetics of ATP hydrolysis by RecQ and the dissociation of the enzyme from ssDNA during single-round translocation. We also gained information on the translocation mechanism from the ssDNA length dependence of the steady-state ssDNA-activated ATPase activity. We show that RecQ occludes 18 ± 2 nt on ssDNA during translocation. The hydrolysis of ATP is noncooperative in the presence of ssDNA, indicating that RecQ active sites work independently during translocation. In the applied conditions, the enzyme hydrolyzes 35 ± 4 ATP molecules per second during ssDNA translocation. RecQ translocates at a moderate processivity, with a mean run length of 100–320 nt on ssDNA. The determined tight mechanochemical coupling of 1.1 ± 0.2 ATP consumed per nucleotide traveled indicates an inchworm-type mechanism.

A Nucleotide-dependent and HRDC Domain-dependent Structural Transition in DNA-bound RecQ Helicase

Background: The mechanistic role of DNA-induced structural changes in RecQ helicases is largely unexplored. Results: DNA interaction of RecQ helicase depends on the nucleotide state of the enzyme and the presence of an intact HRDC domain. Conclusion: We identified a structural transition of the RecQ-DNA complex that is linked to the mechanoenzymatic cycle. Significance: This transition contributes to translocation along DNA and genome-maintaining activities. The allosteric communication between the ATP- and DNA-binding sites of RecQ helicases enables efficient coupling of ATP hydrolysis to translocation along single-stranded DNA (ssDNA) and, in turn, the restructuring of multistranded DNA substrates during genome maintenance processes. In this study, we used the tryptophan fluorescence signal of Escherichia coli RecQ helicase to decipher the kinetic mechanism of the interaction of the enzyme with ssDNA. Rapid kinetic experiments revealed that ssDNA binding occurs in a two-step mechanism in which the initial binding step is followed by a structural transition of the DNA-bound helicase. We found that the nucleotide state of RecQ greatly influences the kinetics of the detected structural transition, which leads to a high affinity DNA-clamped state in the presence of the nucleotide analog ADP-AlF4. The DNA binding mechanism is largely independent of ssDNA length, indicating the independent binding of RecQ molecules to ssDNA and the lack of significant DNA end effects. The structural transition of DNA-bound RecQ was not detected when the ssDNA binding capability of the helicase-RNase D C-terminal domain was abolished or the domain was deleted. The results shed light on the nature of conformational changes leading to processive ssDNA translocation and multistranded DNA processing by RecQ helicases.

Shuttling along DNA and directed processing of D-loops by RecQ helicase support quality control of homologous recombination

Significance RecQ helicase and its eukaryotic homologs are thought to play crucial roles in the quality control of homologous recombination (HR)-based DNA repair. These enzymes have multiple functions in processes that can either promote or suppress HR. A major role suggested for RecQ is the selective inhibition of illegitimate recombination events that could lead to loss of genome integrity. How can RecQ enzymes perform an exceptionally wide range of activities and selectively inhibit potentially harmful recombination events? Here, we propose a model in which the conserved domain architecture of RecQ senses and responds to the geometry of DNA substrates to achieve HR quality control. Cells must continuously repair inevitable DNA damage while avoiding the deleterious consequences of imprecise repair. Distinction between legitimate and illegitimate repair processes is thought to be achieved in part through differential recognition and processing of specific noncanonical DNA structures, although the mechanistic basis of discrimination remains poorly defined. Here, we show that Escherichia coli RecQ, a central DNA recombination and repair enzyme, exhibits differential processing of DNA substrates based on their geometry and structure. Through single-molecule and ensemble biophysical experiments, we elucidate how the conserved domain architecture of RecQ supports geometry-dependent shuttling and directed processing of recombination-intermediate [displacement loop (D-loop)] substrates. Our study shows that these activities together suppress illegitimate recombination in vivo, whereas unregulated duplex unwinding is detrimental for recombination precision. Based on these results, we propose a mechanism through which RecQ helicases achieve recombination precision and efficiency.

Streamlined determination of processive run length and mechanochemical coupling of nucleic acid motor activities

Quantitative determination of enzymatic rates, processivity and mechanochemical coupling is a key aspect in characterizing nucleotide triphosphate (NTP)-driven nucleic acid motor enzymes, for both basic research and technological applications. Here, we present a streamlined analytical method suitable for the determination of all key functional parameters based on measurement of NTP hydrolysis during interaction of motor enzymes with the nucleic acid track. The proposed method utilizes features of kinetic time courses of NTP hydrolysis that have not been addressed in previous analyses, and also accounts for the effect of protein traps used in kinetic experiments on processivity. This analysis is suitable for rapid and precise assessment of the effects of mutations, physical conditions, binding partners and other effectors on the functioning of translocases, helicases, polymerases and other NTP-consuming processive nucleic acid motors.

Mechanism of RecQ helicase mechanoenzymatic coupling reveals that the DNA interactions of the ADP-bound enzyme control translocation run terminations.

The processing of various DNA structures by RecQ helicases is crucial for genome maintenance in both bacteria and eukaryotes. RecQ helicases perform active destabilization of DNA duplexes, based on tight coupling of their ATPase activity to moderately processive translocation along DNA strands. Here, we determined the ATPase kinetic mechanism of E. coli RecQ helicase to reveal how mechanoenzymatic coupling is achieved. We found that the interaction of RecQ with DNA results in a drastic acceleration of the rate-limiting ATP cleavage step, which occurs productively due to subsequent rapid phosphate release. ADP release is not rate-limiting and ADP-bound RecQ molecules make up a small fraction during single-stranded DNA translocation. However, the relatively rapid release of the ADP-bound enzyme from DNA causes the majority of translocation run terminations (i.e. detachment from the DNA track). Thus, the DNA interactions of ADP-bound RecQ helicase, probably dependent on DNA structure, will mainly determine translocation processivity and may control the outcome of DNA processing. Comparison with human Blooms syndrome (BLM) helicase reveals that similar macroscopic parameters are achieved by markedly different underlying mechanisms of RecQ homologs, suggesting diversity in enzymatic tuning.