The UvrD303 Hyper-helicase Exhibits Increased Processivity

Meiners, Matthew J.; Tahmaseb, Kambiz; Matson, Steven W.

doi:10.1074/jbc.m114.565309

Cited by 5 publications

(9 citation statements)

References 43 publications

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“…The properties obtained from the transition pathway are consistent with the single-molecule data ( Comstock et al, 2015 ) as well as mutagenesis studies ( Meiners et al, 2014 ). Firstly, we carried out equilibrium simulations of UvrD site-specifically labeled with FRET dye pair AlexaF555/AlexaF647 for both the closed state and the tilted state.…”

Section: Introductionsupporting

confidence: 81%

Free-energy simulations reveal molecular mechanism for functional switch of a DNA helicase

Whitley

Chemla

et al. 2018

eLife

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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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Section: Introductionsupporting

confidence: 81%

Free-energy simulations reveal molecular mechanism for functional switch of a DNA helicase

Whitley

Chemla

et al. 2018

eLife

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“…It has been proposed that the active form of UvrD is a dimer that is pre-assembled in solution in absence of DNA ( 17 ), however, all crystal structures published to date are monomeric. Domain 2B has been proposed to have a regulatory role, modulating helicase activity ( 14 , 27 ).…”

Section: Introductionmentioning

confidence: 99%

Taking a molecular motor for a spin: helicase mechanism studied by spin labeling and PELDOR

Constantinescu-Aruxandei

Petrovic-Stojanovska

Schiemann

et al. 2015

Nucleic Acids Res

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The complex molecular motions central to the functions of helicases have long attracted attention. Protein crystallography has provided transformative insights into these dynamic conformational changes, however important questions about the true nature of helicase configurations during the catalytic cycle remain. Using pulsed EPR (PELDOR or DEER) to measure interdomain distances in solution, we have examined two representative helicases: PcrA from superfamily 1 and XPD from superfamily 2. The data show that PcrA is a dynamic structure with domain movements that correlate with particular functional states, confirming and extending the information gleaned from crystal structures and other techniques. XPD in contrast is shown to be a rigid protein with almost no conformational changes resulting from nucleotide or DNA binding, which is well described by static crystal structures. Our results highlight the complimentary nature of PELDOR to crystallography and the power of its precision in understanding the conformational changes relevant to helicase function.

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“…In uvrd303 , a hyperactive helicase variant, two aspartate residues (D403, D404) are mutated to alanine ( Fig 5A )[ 61 – 63 ]. They reside in the 2B domain and are thought to participate in controlling the transition between the ‘closed’ and ‘open’ conformations of this domain ( Fig 5B )[ 61 ]. The two aspartates are conserved in UvrD, Rep and PcrA, but only the first aspartate residue is conserved in Srs2 (D437).…”

Section: Resultsmentioning

confidence: 99%

“…In the presence of DNA, the 2B domain adopts the closed conformation ( Fig 5B )[ 67 – 69 ]. Deletion of the 2B domain stimulates the DNA unwinding activity of both UvrD and Rep suggesting that the 2B domain is inhibitory to unwinding activity[ 58 , 60 , 61 ]. uvrD303 is a hyperactive DNA unwinding variant of UvrD carrying two mutations in the 2B domain.…”

Section: Discussionmentioning

confidence: 99%

“…uvrD303 is a hyperactive DNA unwinding variant of UvrD carrying two mutations in the 2B domain. D403 and D404 are mutated to alanine residues in this UvrD variant[ 61 ] and these residues are highly conserved in the Srs2 homologs ( Fig 5A ) . Since only the first aspartate residue is conserved in Srs2, we generated the Srs2 DK-AA variant to investigate the role of the 2B domain.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Rad51 Nucleoprotein Filament Disassembly Captured Using Fluorescent Plasmodium falciparum SSB as a Reporter for Single-Stranded DNA

et al. 2016

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Single-stranded DNA binding (SSB) proteins coordinate DNA replication, repair, and recombination and are critical for maintaining genomic integrity. SSB binds to single-stranded DNA (ssDNA) rapidly and with very high affinity making it a useful molecular tool to detect free ssDNA in solution. We have labeled SSB from Plasmodium falciparum (Pf-SSB) with the MDCC (7-diethylamino-3-((((2-maleimidyl)ethyl)amino)-carbonyl)coumarin) fluorophore which yields a four-fold increase in fluorescence upon binding to ssDNA. Pf-SSBMDCC binding to DNA is unaffected by NaCl or Mg2+ concentration and does not display salt-dependent changes in DNA binding modes or cooperative binding on long DNA substrates. These features are unique to Pf-SSB, making it an ideal tool to probe the presence of free ssDNA in any biochemical reaction. Using this Pf-SSBMDCC probe as a sensor for free ssDNA, we have investigated the clearing of preformed yeast Rad51 nucleoprotein filaments by the Srs2 helicase during HR. Our studies provide a rate for the disassembly of the Rad51 filament by full length Srs2 on long ssDNA substrates. Mutations in the conserved 2B domain in the homologous bacterial UvrD, Rep and PcrA helicases show an enhancement of DNA unwinding activity, but similar mutations in Srs2 do not affect its DNA unwinding or Rad51 clearing properties. These studies showcase the utility of the Pf-SSB probe in mechanistic investigation of enzymes that function in DNA metabolism.

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The UvrD303 Hyper-helicase Exhibits Increased Processivity

Cited by 5 publications

References 43 publications

Free-energy simulations reveal molecular mechanism for functional switch of a DNA helicase

Free-energy simulations reveal molecular mechanism for functional switch of a DNA helicase

Taking a molecular motor for a spin: helicase mechanism studied by spin labeling and PELDOR

Rad51 Nucleoprotein Filament Disassembly Captured Using Fluorescent Plasmodium falciparum SSB as a Reporter for Single-Stranded DNA

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