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Structure-activity relationships at a nucleobase-stacking tryptophan required for chemomechanical coupling in the DNA resecting motor-nuclease AdnAB.

ABSTRACT: Mycobacterial AdnAB is a heterodimeric helicase-nuclease that initiates homologous recombination by resecting DNA double-strand breaks. The AdnB subunit hydrolyzes ATP to drive single-nucleotide steps of 3'-to-5' translocation of AdnAB on the tracking DNA strand via a ratchet-like mechanism. Trp325 in AdnB motif III, which intercalates into the tracking strand and makes a π stack on a nucleobase 5' of a flipped-out nucleoside, is the putative ratchet pawl without which ATP hydrolysis is mechanically futile. Here, we report that AdnAB mutants wherein Trp325 was replaced with phenylalanine, tyrosine, histidine, leucine, or alanine retained activity in ssDNA-dependent ATP hydrolysis but displayed a gradient of effects on DSB resection. The resection velocities of Phe325 and Tyr325 mutants were 90% and 85% of the wild-type AdnAB velocity. His325 slowed resection rate to 3% of wild-type and Leu325 and Ala325 abolished DNA resection. A cryo-EM structure of the DNA-bound Ala325 mutant revealed that the AdnB motif III peptide was disordered and the erstwhile flipped out tracking strand nucleobase reverted to a continuous base-stacked arrangement with its neighbors. We conclude that π stacking of Trp325 on a DNA nucleobase triggers and stabilizes the flipped-out conformation of the neighboring nucleoside that underlies formation of a ratchet pawl.

SUBMITTER: Warren GM

PROVIDER: S-EPMC8789073 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Clutch mechanism of chemomechanical coupling in a DNA resecting motor nuclease.

Project description:Mycobacterial AdnAB is a heterodimeric helicase-nuclease that initiates homologous recombination by resecting DNA double-strand breaks (DSBs). The N-terminal motor domain of the AdnB subunit hydrolyzes ATP to drive rapid and processive 3' to 5' translocation of AdnAB on the tracking DNA strand. ATP hydrolysis is mechanically productive when oscillating protein domain motions synchronized with the ATPase cycle propel the DNA tracking strand forward by a single-nucleotide step, in what is thought to entail a pawl-and-ratchet-like fashion. By gauging the effects of alanine mutations of the 16 amino acids at the AdnB-DNA interface on DNA-dependent ATP hydrolysis, DNA translocation, and DSB resection in ensemble and single-molecule assays, we gained key insights into which DNA contacts couple ATP hydrolysis to motor activity. The results implicate AdnB Trp325, which intercalates into the tracking strand and stacks on a nucleobase, as the singular essential constituent of the ratchet pawl, without which ATP hydrolysis on ssDNA is mechanically futile. Loss of Thr663 and Thr118 contacts with tracking strand phosphates and of His665 with a nucleobase drastically slows the AdnAB motor during DSB resection. Our findings for AdnAB prompt us to analogize its mechanism to that of an automobile clutch.

| S-EPMC7980473 | biostudies-literature

AdnAB: a new DSB-resecting motor-nuclease from mycobacteria.

Project description:The resection of DNA double-strand breaks (DSBs) in bacteria is a motor-driven process performed by a multisubunit helicase-nuclease complex: either an Escherichia coli-type RecBCD enzyme or a Bacillus-type AddAB enzyme. Here we identify mycobacterial AdnAB as the founder of a new family of heterodimeric helicase-nucleases with distinctive properties. The AdnA and AdnB subunits are each composed of an N-terminal UvrD-like motor domain and a C-terminal nuclease module. The AdnAB ATPase is triggered by dsDNA with free ends and the energy of ATP hydrolysis is coupled to DSB end resection by the AdnAB nuclease. The mycobacterial nonhomologous end-joining (NHEJ) protein Ku protects DSBs from resection by AdnAB. We find that AdnAB incises ssDNA by measuring the distance from the free 5' end to dictate the sites of cleavage, which are predominantly 5 or 6 nucleotides (nt) from the 5' end. The "molecular ruler" of AdnAB is regulated by ATP, which elicits an increase in ssDNA cleavage rate and a distal displacement of the cleavage sites 16-17 nt from the 5' terminus. AdnAB is a dual nuclease with a clear division of labor between the subunits. Mutations in the nuclease active site of the AdnB subunit ablate the ATP-inducible cleavages; the corresponding changes in AdnA abolish ATP-independent cleavage. Complete suppression of DSB end resection requires simultaneous mutation of both subunit nucleases. The nuclease-null AdnAB is a helicase that unwinds linear plasmid DNA without degrading the displaced single strands. Mutations of the phosphohydrolase active site of the AdnB subunit ablate DNA-dependent ATPase activity, DSB end resection, and ATP-inducible ssDNA cleavage; the equivalent mutations of the AdnA subunit have comparatively little effect. AdnAB is a novel signature of the Actinomycetales taxon. Mycobacteria are exceptional in that they encode both AdnAB and RecBCD, suggesting the existence of alternative end-resecting motor-nuclease complexes.

| S-EPMC2701575 | biostudies-literature

Chemomechanical coupling and motor cycles of myosin V.

Project description:The molecular motor myosin V has been studied extensively both in bulk and single molecule experiments. Based on the chemical states of the motor, we construct a systematic network theory that includes experimental observations about the stepping behavior of myosin V. We utilize constraints arising from nonequilibrium thermodynamics to determine motor parameters and demonstrate that the motor behavior is governed by three chemomechanical motor cycles. The competition between these cycles can be understood via the influence of external load forces onto the chemical transition rates for the binding of adenosine triphosphate and adenosine diphosphate. In addition, we also investigate the functional dependence of the mechanical stepping rates on these forces. For substall forces, the dominant pathway of the motor network is profoundly different from the one for superstall forces, which leads to a stepping behavior that is in agreement with the experimental observations. Our theory provides a unified description of the experimental data as obtained for myosin V in single motor experiments.

| S-EPMC3072604 | biostudies-literature

Characterization of the mycobacterial AdnAB DNA motor provides insights into the evolution of bacterial motor-nuclease machines.

Project description:Mycobacterial AdnAB exemplifies a family of heterodimeric motor-nucleases involved in processing DNA double strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal UvrD-like motor domain and a C-terminal RecB-like nuclease module. Here we conducted a biochemical characterization of the AdnAB motor, using a nuclease-inactivated heterodimer. AdnAB is a vigorous single strand DNA (ssDNA)-dependent ATPase (k(cat) 415 s(-1)), and the affinity of the motor for the ssDNA cofactor increases 140-fold as DNA length is extended from 12 to 44 nucleotides. Using a streptavidin displacement assay, we demonstrate that AdnAB is a 3' --> 5' translocase on ssDNA. AdnAB binds stably to DSB ends. In the presence of ATP, the motor unwinds the DNA duplex without requiring an ssDNA loading strand. We integrate these findings into a model of DSB unwinding in which the "leading" AdnB and "lagging" AdnA motor domains track in tandem, 3' to 5', along the same DNA single strand. This contrasts with RecBCD, in which the RecB and RecD motors track in parallel along the two separated DNA single strands. The effects of 5' and 3' terminal obstacles on ssDNA cleavage by wild-type AdnAB suggest that the AdnA nuclease receives and processes the displaced 5' strand, while the AdnB nuclease cleaves the displaced 3' strand. We present evidence that the distinctive "molecular ruler" function of the ATP-dependent single strand DNase, whereby AdnAB measures the distance from the 5'-end to the sites of incision, reflects directional pumping of the ssDNA through the AdnAB motor into the AdnB nuclease. These and other findings suggest a scenario for the descent of the RecBCD- and AddAB-type DSB-processing machines from an ancestral AdnAB-like enzyme.

| S-EPMC2807320 | biostudies-literature

Structures and single-molecule analysis of bacterial motor nuclease AdnAB illuminate the mechanism of DNA double-strand break resection.

Project description:Mycobacterial AdnAB is a heterodimeric helicase-nuclease that initiates homologous recombination by resecting DNA double-strand breaks (DSBs). The AdnA and AdnB subunits are each composed of an N-terminal motor domain and a C-terminal nuclease domain. Here we report cryoelectron microscopy (cryo-EM) structures of AdnAB in three functional states: in the absence of DNA and in complex with forked duplex DNAs before and after cleavage of the 5' single-strand DNA (ssDNA) tail by the AdnA nuclease. The structures reveal the path of the 5' ssDNA through the AdnA nuclease domain and the mechanism of 5' strand cleavage; the path of the 3' tracking strand through the AdnB motor and the DNA contacts that couple ATP hydrolysis to mechanical work; the position of the AdnA iron-sulfur cluster subdomain at the Y junction and its likely role in maintaining the split trajectories of the unwound 5' and 3' strands. Single-molecule DNA curtain analysis of DSB resection reveals that AdnAB is highly processive but prone to spontaneous pausing at random sites on duplex DNA. A striking property of AdnAB is that the velocity of DSB resection slows after the enzyme experiences a spontaneous pause. Our results highlight shared as well as distinctive properties of AdnAB vis-à-vis the RecBCD and AddAB clades of bacterial DSB-resecting motor nucleases.

| S-EPMC6900545 | biostudies-literature

Tight Chemomechanical Coupling of the F<sub>1</sub> Motor Relies on Structural Stability.

Project description:The F1 motor is a rotating molecular motor that ensures a tight chemomechanical coupling between ATP hydrolysis/synthesis reactions and rotation steps. However, the mechanism underlying this tight coupling remains to be elucidated. In this study, we used electrorotation in single-molecule experiments using an F1βE190D mutant to demonstrate that the stall torque was significantly smaller than the wild-type F1, indicating a loose coupling of this mutant, despite showing similar stepping torque as the wild-type. Experiments on the ATPase activity after heat treatment and gel filtration of the α3β3-subcomplex revealed the unstable structure of the βE190D mutant. Our results suggest that the tight chemomechanical coupling of the F1 motor relies on the structural stability of F1. We also discuss the difference between the stepping torque and the stall torque.

| S-EPMC7335906 | biostudies-literature

The adnAB locus, encoding a putative helicase-nuclease activity, is essential in Streptomyces.

Project description:Homologous recombination is a crucial mechanism that repairs a wide range of DNA lesions, including the most deleterious ones, double-strand breaks (DSBs). This multistep process is initiated by the resection of the broken DNA ends by a multisubunit helicase-nuclease complex exemplified by Escherichia coli RecBCD, Bacillus subtilis AddAB, and newly discovered Mycobacterium tuberculosis AdnAB. Here we show that in Streptomyces, neither recBCD nor addAB homologues could be detected. The only putative helicase-nuclease-encoding genes identified were homologous to M. tuberculosis adnAB genes. These genes are conserved as a single copy in all sequenced genomes of Streptomyces. The disruption of adnAB in Streptomyces ambofaciens and Streptomyces coelicolor could not be achieved unless an ectopic copy was provided, indicating that adnAB is essential for growth. Both adnA and adnB genes were shown to be inducible in response to DNA damage (mitomycin C) and to be independently transcribed. Introduction of S. ambofaciens adnAB genes in an E. coli recB mutant restored viability and resistance to UV light, suggesting that Streptomyces AdnAB could be a functional homologue of RecBCD and be involved in DNA damage resistance.

| S-EPMC4097579 | biostudies-literature

Design principles governing chemomechanical coupling of kinesin.

Project description:A systematic chemomechanical network model for the molecular motor kinesin is presented in this report. The network model is based on the nucleotide-dependent binding affinity of the heads to an microtubule (MT) and the asymmetries and similarities between the chemical transitions caused by the intramolecular strain between the front and rear heads. The network model allows for multiple chemomechanical cycles and takes into account all possible mechanical transitions between states in which one head is strongly bound and the other head is weakly bound to an MT. The results obtained from the model show the ATP-concentration dependence of the dominant forward stepping cycle and support a gated rear head mechanism in which the forward step is controlled by ATP hydrolysis and the resulting ADP-bound state of the rear head when the ATP level is saturated. When the ATP level is saturated, the energy from ATP hydrolysis is used to concentrate the chemical transition flux to a force-generating state that can produce the power stroke. In contrast, when the ATP level is low, the hydrolysis energy is consumed to avoid states in which the leading head is weakly bound to an MT and to inhibit frequent backward steps upon loading.

| S-EPMC5430765 | biostudies-literature

Myosin V: Chemomechanical-coupling ratchet with load-induced mechanical slip.

Project description:A chemomechanical-network model for myosin V is presented on the basis of both the nucleotide-dependent binding affinity of the head to an actin filament (AF) and asymmetries and similarity relations among the chemical transitions due to an intramolecular strain of the leading and trailing heads. The model allows for branched chemomechanical cycles and takes into account not only two different force-generating mechanical transitions between states wherein the leading head is strongly bound and the trailing head is weakly bound to the AF but also load-induced mechanical-slip transitions between states in which both heads are strongly bound. The latter is supported by the fact that ATP-independent high-speed backward stepping has been observed for myosin V, although such motility has never been for kinesin. The network model appears as follows: (1) the high chemomechanical-coupling ratio between forward step and ATP hydrolysis is achieved even at low ATP concentrations by the dual mechanical transitions; (2) the forward stepping at high ATP concentrations is explained by the front head-gating mechanism wherein the power stroke is triggered by the inorganic-phosphate (Pi) release from the leading head; (3) the ATP-binding or hydrolyzed ADP.Pi-binding leading head produces a stable binding to the AF, especially against backward loading.

| S-EPMC5647391 | biostudies-literature

Aromatic stacking between nucleobase and enzyme promotes phosphate ester hydrolysis in dUTPase.

Project description:Aromatic interactions are well-known players in molecular recognition but their catalytic role in biological systems is less documented. Here, we report that a conserved aromatic stacking interaction between dUTPase and its nucleotide substrate largely contributes to the stabilization of the associative type transition state of the nucleotide hydrolysis reaction. The effect of the aromatic stacking on catalysis is peculiar in that uracil, the aromatic moiety influenced by the aromatic interaction is relatively distant from the site of hydrolysis at the alpha-phosphate group. Using crystallographic, kinetics, optical spectroscopy and thermodynamics calculation approaches we delineate a possible mechanism by which rate acceleration is achieved through the remote π-π interaction. The abundance of similarly positioned aromatic interactions in various nucleotide hydrolyzing enzymes (e.g. most families of ATPases) raises the possibility of the reported phenomenon being a general component of the enzymatic catalysis of phosphate ester hydrolysis.

| S-EPMC2978360 | biostudies-literature

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