Abstract The hybridization and dehybridization of DNA subject to tension is relevant to fundamental genetic processes and to the design of DNA-based mechanobiology assays. While strong tension accelerates DNA melting and decelerates DNA annealing, the effects of tension weaker than 5 pN are less clear. In this study, we developed a DNA bow assay, which uses the bending rigidity of double-stranded DNA (dsDNA) to exert weak tension on a single-stranded DNA (ssDNA) target in the range of 2–6 pN. Combining this assay with single-molecule FRET, we measured the hybridization and dehybridization kinetics between a 15 nt ssDNA under tension and a 8–9 nt oligonucleotide, and found that both the hybridization and dehybridization rates monotonically increase with tension for various nucleotide sequences tested. These findings suggest that the nucleated duplex in its transition state is more extended than the pure dsDNA or ssDNA counterpart. Based on coarse-grained oxDNA simulations, we propose that this increased extension of the transition state is due to steric repulsion between the unpaired ssDNA segments in close proximity to one another. Using linear force-extension relations verified by simulations of short DNA segments, we derived analytical equations for force-to-rate conversion that are in good agreement with our measurements.
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Free-energy simulations reveal molecular mechanism for functional switch of a DNA helicase
Helicases play key roles in genome maintenance, yet it remains elusive how these enzymes change conformations and how transitions between different conformational states regulate nucleic acid reshaping. Here, we developed a computational technique combining structural bioinformatics approaches and atomic-level free-energy simulations to characterize how the Escherichia coli DNA repair enzyme UvrD changes its conformation at the fork junction to switch its function from unwinding to rezipping DNA. The lowest free-energy path shows that UvrD opens the interface between two domains, allowing the bound ssDNA to escape. The simulation results predict a key metastable 'tilted' state during ssDNA strand switching. By simulating FRET distributions with fluorophores attached to UvrD, we show that the new state is supported quantitatively by single-molecule measurements. The present study deciphers key elements for the 'hyper-helicase' behavior of a mutant and provides an effective framework to characterize directly structure-function relationships in molecular machines.
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- Award ID(s):
- 1713784
- NSF-PAR ID:
- 10109300
- Date Published:
- Journal Name:
- eLife
- Volume:
- 7
- ISSN:
- 2050-084X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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Abstract The catalytic activity of human glutathione S‐transferase A1‐1 (hGSTA1‐1), a homodimeric detoxification enzyme, is dependent on the conformational dynamics of a key C‐terminal helix α9 in each monomer. However, the structural details of how the two monomers interact upon binding of substrates is not well understood and the structure of the ligand‐free state of the hGSTA1‐1 homodimer has not been resolved. Here, we used a combination of electron paramagnetic resonance (EPR) distance measurements and weighted ensemble (WE) simulations to characterize the conformational ensemble of the ligand‐free state at the atomic level. EPR measurements reveal a broad distance distribution between a pair of Cu(II) labels in the ligand‐free state that gradually shifts and narrows as a function of increasing ligand concentration. These shifts suggest changes in the relative positioning of the two α9 helices upon ligand binding. WE simulations generated unbiased pathways for the seconds‐timescale transition between alternate states of the enzyme, leading to the generation of atomically detailed structures of the ligand‐free state. Notably, the simulations provide direct observations of negative cooperativity between the monomers of hGSTA1‐1, which involve the mutually exclusive docking of α9 in each monomer as a lid over the active site. We identify key interactions between residues that lead to this negative cooperativity. Negative cooperativity may be essential for interaction of hGSTA1‐1 with a wide variety of toxic substrates and their subsequent neutralization. More broadly, this work demonstrates the power of integrating EPR distances with WE rare‐events sampling strategy to gain mechanistic information on protein function at the atomic level.
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null (Ed.)Abstract Escherichia coli SSB (EcSSB) is a model single-stranded DNA (ssDNA) binding protein critical in genome maintenance. EcSSB forms homotetramers that wrap ssDNA in multiple conformations to facilitate DNA replication and repair. Here we measure the binding and wrapping of many EcSSB proteins to a single long ssDNA substrate held at fixed tensions. We show EcSSB binds in a biphasic manner, where initial wrapping events are followed by unwrapping events as ssDNA-bound protein density passes critical saturation and high free protein concentration increases the fraction of EcSSBs in less-wrapped conformations. By destabilizing EcSSB wrapping through increased substrate tension, decreased substrate length, and protein mutation, we also directly observe an unstable bound but unwrapped state in which ∼8 nucleotides of ssDNA are bound by a single domain, which could act as a transition state through which rapid reorganization of the EcSSB–ssDNA complex occurs. When ssDNA is over-saturated, stimulated dissociation rapidly removes excess EcSSB, leaving an array of stably-wrapped complexes. These results provide a mechanism through which otherwise stably bound and wrapped EcSSB tetramers are rapidly removed from ssDNA to allow for DNA maintenance and replication functions, while still fully protecting ssDNA over a wide range of protein concentrations.more » « less
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Abstract Bacteriophage T4 gene 32 protein (gp32) is a model single-stranded DNA (ssDNA) binding protein, essential for DNA replication. gp32 forms cooperative filaments on ssDNA through interprotein interactions between its core and N-terminus. However, detailed understanding of gp32 filament structure and organization remains incomplete, particularly for longer, biologically-relevant DNA lengths. Moreover, it is unclear how these tightly-bound filaments dissociate from ssDNA during complementary strand synthesis. We use optical tweezers and atomic force microscopy to probe the structure and binding dynamics of gp32 on long (∼8 knt) ssDNA substrates. We find that cooperative binding of gp32 rigidifies ssDNA while also reducing its contour length, consistent with the ssDNA helically winding around the gp32 filament. While measured rates of gp32 binding and dissociation indicate nM binding affinity, at ∼1000-fold higher protein concentrations gp32 continues to bind into and restructure the gp32–ssDNA filament, leading to an increase in its helical pitch and elongation of the substrate. Furthermore, the oversaturated gp32–ssDNA filament becomes progressively unwound and unstable as observed by the appearance of a rapid, noncooperative protein dissociation phase not seen at lower complex saturation, suggesting a possible mechanism for prompt removal of gp32 from the overcrowded ssDNA in front of the polymerase during replication.
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A scanning-to-incision switch in TFIIH-XPG induced by DNA damage licenses nucleotide excision repairAbstract Nucleotide excision repair (NER) is critical for removing bulky DNA base lesions and avoiding diseases. NER couples lesion recognition by XPC to strand separation by XPB and XPD ATPases, followed by lesion excision by XPF and XPG nucleases. Here, we describe key regulatory mechanisms and roles of XPG for and beyond its cleavage activity. Strikingly, by combing single-molecule imaging and bulk cleavage assays, we found that XPG binding to the 7-subunit TFIIH core (coreTFIIH) stimulates coreTFIIH-dependent double-strand (ds)DNA unwinding 10-fold, and XPG-dependent DNA cleavage by up to 700-fold. Simultaneous monitoring of rates for coreTFIIH single-stranded (ss)DNA translocation and dsDNA unwinding showed XPG acts by switching ssDNA translocation to dsDNA unwinding as a likely committed step. Pertinent to the NER pathway regulation, XPG incision activity is suppressed during coreTFIIH translocation on DNA but is licensed when coreTFIIH stalls at the lesion or when ATP hydrolysis is blocked. Moreover, ≥15 nucleotides of 5′-ssDNA is a prerequisite for efficient translocation and incision. Our results unveil a paired coordination mechanism in which key lesion scanning and DNA incision steps are sequentially coordinated, and damaged patch removal is only licensed after generation of ≥15 nucleotides of 5′-ssDNA, ensuring the correct ssDNA bubble size before cleavage.more » « less
- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.7554/eLife.34186
