Kinetic and structural mechanism for DNA unwinding by a non-hexameric helicase

Carney, Sean P.; Wen, Ming‐Shien; Whitley, Kevin D.; Jia, Haifeng; Lohman, Timothy M.; Luthey‐Schulten, Zaida; Chemla, Yann R.

doi:10.1038/s41467-021-27304-6

Cited by 15 publications

(10 citation statements)

References 67 publications

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“…The trajectories still show discrete fluorescence drops within one frame, suggesting a dissociative process that is completed within 30 ms. UvrD loading on the free 3′-DNA end and unwinding DNA towards the surface within 30 ms would correspond to a rate of 80 000 nt·s −1 . Since this high rate would be in stark contradiction to the literature ( 15 , 37 , 39 , 40 ), we conclude that UvrD is loading in close proximity to the surface and exhibits an enzymatic activity different to DNA unwinding.…”

Section: Resultscontrasting

confidence: 99%

Rapid single-molecule characterisation of enzymes involved in nucleic-acid metabolism

Mueller

Fitschen

Shirbini

et al. 2022

Nucleic Acids Research

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The activity of enzymes is traditionally characterised through bulk-phase biochemical methods that only report on population averages. Single-molecule methods are advantageous in elucidating kinetic and population heterogeneity but are often complicated, time consuming, and lack statistical power. We present a highly-generalisable and high-throughput single-molecule assay to rapidly characterise proteins involved in DNA metabolism. The assay exclusively relies on changes in total fluorescence intensity of surface-immobilised DNA templates as a result of DNA synthesis, unwinding or digestion. Combined with an automated data-analysis pipeline, our method provides enzymatic activity data of thousands of molecules in less than an hour. We demonstrate our method by characterising three fundamentally different enzyme activities: digestion by the phage λ exonuclease, synthesis by the phage Phi29 polymerase, and unwinding by the E. coli UvrD helicase. We observe the previously unknown activity of the UvrD helicase to remove neutravidin bound to 5′-, but not 3′-ends of biotinylated DNA.

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Section: Resultscontrasting

confidence: 99%

Rapid single-molecule characterisation of enzymes involved in nucleic-acid metabolism

Mueller

Fitschen

Shirbini

et al. 2022

Nucleic Acids Research

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show abstract

“…We The trajectories still show discrete fluorescence drops within one frame, suggesting a dissociative process that is completed within 30 ms. UvrD loading on the free 3’-DNA end and unwinding DNA towards the surface within 30 ms would correspond to a rate of 80,000 nt·s -1 . Since this high rate would be in stark contradiction to the literature [14], [30], [32], [33], we conclude that UvrD is loading in close proximity to the surface and exhibits an enzymatic activity different to DNA unwinding.…”

Section: Resultscontrasting

confidence: 98%

Rapid single-molecule characterisation of nucleic-acid enzymes

Mueller

Fitschen

Shirbini

et al. 2022

Preprint

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The activity of enzymes is traditionally characterised through bulk-phase biochemical methods that only report on population averages. Single-molecule methods are advantageous in elucidating kinetic and population heterogeneity but are often complicated, time consuming, and lacking statistical power. We present a highly generalisable and high-throughput single-molecule assay to rapidly characterise proteins involved in DNA metabolism. The assay exclusively relies on changes in total fluorescence intensity of surface-immobilised DNA templates as a result of DNA synthesis, unwinding or digestion. Combined with an automated data-analysis pipeline, our method provides enzymatic activity data of thousands of molecules in less than an hour. We demonstrate our method by characterising three fundamentally different nucleic-acid enzyme activities: digestion by the phage λ exonuclease, synthesis by the phage Phi29 polymerase, and unwinding by the E. coli UvrD helicase. We observe a previously unknown activity of the UvrD helicase to remove proteins tightly bound to the ends of DNA.

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“…Owing to contributions from ATP binding, hydrolysis, and release, which may even be rate-limiting for the overall cycle, the translocation speed of Prp43 is likely lower than the maximum value of ~600 bp/s estimated from our simulations. Notably, assuming some reduction of the translocation speed from ATP binding, hydrolysis and release, our value is in reasonable agreement with translocation speeds of 100 to 300 bp/s observed for other helicases 51 – 53 .…”

Section: Resultssupporting

confidence: 90%

Continuous millisecond conformational cycle of a DEAH box helicase reveals control of domain motions by atomic-scale transitions

Becker

Hub

2023

Commun Biol

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Helicases are motor enzymes found in every living organism and viruses, where they maintain the stability of the genome and control against false recombination. The DEAH-box helicase Prp43 plays a crucial role in pre-mRNA splicing in unicellular organisms by translocating single-stranded RNA. The molecular mechanisms and conformational transitions of helicases are not understood at the atomic level. We present a complete conformational cycle of RNA translocation by Prp43 in atomic detail based on molecular dynamics simulations. To enable the sampling of such complex transition on the millisecond timescale, we combined two enhanced sampling techniques, namely simulated tempering and adaptive sampling guided by crystallographic data. During RNA translocation, the center-of-mass motions of the RecA-like domains followed the established inchworm model, whereas the domains crawled along the RNA in a caterpillar-like movement, suggesting an inchworm/caterpillar model. However, this crawling required a complex sequence of atomic-scale transitions involving the release of an arginine finger from the ATP pocket, stepping of the hook-loop and hook-turn motifs along the RNA backbone, and several others. These findings highlight that large-scale domain dynamics may be controlled by complex sequences of atomic-scale transitions.

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Kinetic and structural mechanism for DNA unwinding by a non-hexameric helicase

Cited by 15 publications

References 67 publications

Rapid single-molecule characterisation of enzymes involved in nucleic-acid metabolism

Rapid single-molecule characterisation of enzymes involved in nucleic-acid metabolism

Rapid single-molecule characterisation of nucleic-acid enzymes

Continuous millisecond conformational cycle of a DEAH box helicase reveals control of domain motions by atomic-scale transitions

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