Chemo-mechanical pushing of proteins along single-stranded DNA

Sokoloski, Joshua E.; Козлов, А. Г.; Galletto, Roberto; Lohman, Timothy M.

doi:10.1073/pnas.1602878113

Cited by 50 publications

(73 citation statements)

References 69 publications

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“…Recent studies have shown that S. cerevisiae Pif1, and the E. coli UvrD and Rep helicases can push isolated E. coli SSB tetramers along ssDNA (Sokoloski et al, 2016). However, it remained unclear how helicases might behave when the ssDNA is fully coated by single strand binding proteins.…”

Section: Discussionmentioning

confidence: 99%

Yeast Srs2 Helicase Promotes Redistribution of Single-Stranded DNA-Bound RPA and Rad52 in Homologous Recombination Regulation

et al. 2017

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Summary Srs2 is a Super-Family 1 helicase that promotes genome stability by dismantling toxic DNA recombination intermediates. However, the mechanisms by which Srs2 remodels or resolves recombination intermediates remain poorly understood. Here, single molecule imaging is used to visualize Srs2 in real time as it acts on single-stranded DNA (ssDNA) bound by protein factors that function in recombination. We demonstrate that Srs2 is highly processive and translocates rapidly (~170 nucleotides per second) in the 3′→5′ direction along ssDNA saturated with replication protein A (RPA). We show that RPA is evicted from DNA during the passage of Srs2. Remarkably, Srs2 also readily removes the recombination mediator Rad52 from RPA-ssDNA, and in doing so promotes rapid redistribution of both Rad52 and RPA. These findings have important mechanistic implications for understanding how Srs2 and related nucleic acid motor proteins resolve potentially pathogenic nucleoprotein intermediates.

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Section: Discussionmentioning

confidence: 99%

Yeast Srs2 Helicase Promotes Redistribution of Single-Stranded DNA-Bound RPA and Rad52 in Homologous Recombination Regulation

et al. 2017

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“…Its ATPdependent activities include translocation along single-stranded (ss) DNA (12)(13)(14)(15)(16), duplex DNA unwinding (17)(18)(19)(20)(21)(22), displacement of RecA filaments from ssDNA (10,11), and pushing of proteins along ssDNA (23).…”

mentioning

confidence: 99%

Large Domain Movements Upon UvrD Dimerization and Helicase Activation

Nguyen

Ordabayev

Sokoloski

et al. 2018

Biophysical Journal

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Escherichia coli UvrD DNA helicase functions in several DNA repair processes. As a monomer, UvrD can translocate rapidly and processively along ssDNA; however, the monomer is a poor helicase. To unwind duplex DNA in vitro, UvrD needs to be activated either by self-assembly to form a dimer or by interaction with an accessory protein. However, the mechanism of activation is not understood. UvrD can exist in multiple conformations associated with the rotational conformational state of its 2B subdomain, and its helicase activity has been correlated with a closed 2B conformation. Using single-molecule total internal reflection fluorescence microscopy, we examined the rotational conformational states of the 2B subdomain of fluorescently labeled UvrD and their rates of interconversion. We find that the 2B subdomain of the UvrD monomer can rotate between an open and closed conformation as well as two highly populated intermediate states. The binding of a DNA substrate shifts the 2B conformation of a labeled UvrD monomer to a more open state that shows no helicase activity. The binding of a second unlabeled UvrD shifts the 2B conformation of the labeled UvrD to a more closed state resulting in activation of helicase activity. Binding of a monomer of the structurally similar Escherichia coli Rep helicase does not elicit this effect. This indicates that the helicase activity of a UvrD dimer is promoted via direct interactions between UvrD subunits that affect the rotational conformational state of its 2B subdomain.single molecule | conformational heterogeneity | DNA motors | DNA repair U vrD from Escherichia coli is a nonhexameric SF1A DNA helicase/translocase, structurally similar to E. coli Rep and Bacillus stearothermophilus PcrA, which is involved in many aspects of genome maintenance (1, 2), including DNA repair (3, 4), replication (5-8), and recombination (7, 9-11). Its ATPdependent activities include translocation along single-stranded (ss) DNA (12-16), duplex DNA unwinding (17-22), displacement of RecA filaments from ssDNA (10, 11), and pushing of proteins along ssDNA (23).The activities of UvrD can be modulated in vitro by its assembly state and/or by binding partners (1). UvrD monomers can translocate directionally (3′ to 5′) along ssDNA [∼190 nucleotides (nts) s −1 ] (12, 13, 15, 16), in a reaction that is tightly coupled to ATP hydrolysis (14). However, UvrD monomers show little helicase activity (12,16,21,24,25). In the absence of accessory proteins, helicase activity in vitro requires formation of a UvrD dimer (16,21,(25)(26)(27). The dimer unwinds dsDNA with rates of ∼70 base pair (bp) s −1 (20,21,24,27). Rep and PcrA monomers also show no helicase activity and require oligomerization or an accessory protein for helicase activity in vitro (28-31).UvrD, Rep, and PcrA all contain four subdomains (1A, 2A, 1B, and 2B) (2,(32)(33)(34)(35), and the 2B subdomain can undergo a substantial rotation about a hinge region connecting it to the 2A subdomain (2,22,(32)(33)(34)(35)(36)(37). A structure of an apo UvrD monome...

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“…For instance, smFRET studies have shown that SSB can diffuse along ssDNA, and this process may be involved in the displacement of SSB from ssDNA during the assembly of the RecA presynaptic complex 80 . SSB can even be pushed along ssDNA through the action of the E. coli helicases Rep and UvrD, which suggests a role for these helicases in assisting the reorganization or removal of SSB from DNA repair intermediates 81 . A TIRFM study looking at time-resolved widefield images of SSB bound to ssDNA revealed that SSB can form reversible long-range contacts that can lead to extensive condensation of SSB-bound ssDNA intermediates 82 .…”

Section: Sm-based Insights Into Recombinationmentioning

confidence: 99%

A change of view: homologous recombination at single-molecule resolution

2017

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Genetic recombination occurs in all organisms and is vital for genome stability. Indeed, in humans, aberrant recombination can lead to diseases such as cancer. Our understanding of homologous recombination is built upon more than a century of scientific inquiry, but achieving a more complete picture using ensemble biochemical and genetic approaches is hampered by population heterogeneity and transient recombination intermediates. Recent advances in single-molecule and super-resolution microscopy methods help to overcome these limitations and have led to new and refined insights into recombination mechanisms, including a detailed understanding of DNA helicase function and synaptonemal complex structure. The ability to view cellular processes at single-molecule resolution promises to transform our understanding of recombination and related processes.

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Chemo-mechanical pushing of proteins along single-stranded DNA

Cited by 50 publications

References 69 publications

Yeast Srs2 Helicase Promotes Redistribution of Single-Stranded DNA-Bound RPA and Rad52 in Homologous Recombination Regulation

Yeast Srs2 Helicase Promotes Redistribution of Single-Stranded DNA-Bound RPA and Rad52 in Homologous Recombination Regulation

Large Domain Movements Upon UvrD Dimerization and Helicase Activation

A change of view: homologous recombination at single-molecule resolution

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