PcrA Helicase Dismantles RecA Filaments by Reeling in DNA in Uniform Steps

doi:10.1016/J.CELL.2010.07.016

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PcrA Helicase Dismantles RecA Filaments by Reeling in DNA in Uniform Steps

Journal Article•DOI•

PcrA Helicase Dismantles RecA Filaments by Reeling in DNA in Uniform Steps

Jeehae Park¹, Sua Myong¹, Anita Niedziela-Majka², Kyung S. Lee¹, Jin Yu³, Timothy M. Lohman², Taekjip Ha - Show less +3 more•Institutions (3)

University of Illinois at Urbana–Champaign¹, Washington University in St. Louis², University of California, Berkeley³

20 Aug 2010-Cell (Cell Press)-Vol. 142, Iss: 4, pp 544-555

TL;DR: It is discovered that PcrA preferentially translocates on the DNA lagging strand instead of unwinding the template duplex, suggesting a mode of action for eliminating potentially deleterious recombination intermediates.

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About: This article is published in Cell.The article was published on 2010-08-20 and is currently open access. It has received 166 citations till now. The article focuses on the topics: PcrA.

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Journal Article•DOI•

Probing cellular protein complexes using single-molecule pull-down

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Ankur Jain¹, Ruijie Liu¹, Biswarathan Ramani¹, Edwin Arauz¹, Yuji Ishitsuka², Yuji Ishitsuka¹, Kaushik Ragunathan¹, Jeehae Park¹, Jie Chen¹, Yang Kevin Xiang¹, Taekjip Ha¹, Taekjip Ha² - Show less +8 more•Institutions (2)

University of Illinois at Urbana–Champaign¹, Howard Hughes Medical Institute²

26 May 2011-Nature

TL;DR: An assay that combines the principles of a conventional pull-down assay with single-molecule fluorescence microscopy and enables direct visualization of individual cellular protein complexes is described, which can reveal how many proteins and of which kinds are present in the in vivo complex.

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Abstract: Proteins perform most cellular functions in macromolecular complexes. The same protein often participates in different complexes to exhibit diverse functionality. Current ensemble approaches of identifying cellular protein interactions cannot reveal physiological permutations of these interactions. Here we describe a single-molecule pull-down (SiMPull) assay that combines the principles of a conventional pull-down assay with single-molecule fluorescence microscopy and enables direct visualization of individual cellular protein complexes. SiMPull can reveal how many proteins and of which kinds are present in the in vivo complex, as we show using protein kinase A. We then demonstrate a wide applicability to various signalling proteins found in the cytosol, membrane and cellular organelles, and to endogenous protein complexes from animal tissue extracts. The pulled-down proteins are functional and are used, without further processing, for single-molecule biochemical studies. SiMPull should provide a rapid, sensitive and robust platform for analysing protein assemblies in biological pathways.

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378 citations

Journal Article•DOI•

Protein induced fluorescence enhancement as a single molecule assay with short distance sensitivity

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Helen Hwang¹, Hajin Kim¹, Sua Myong•Institutions (1)

University of Illinois at Urbana–Champaign¹

03 May 2011-Proceedings of the National Academy of Sciences of the United States of America

TL;DR: The high spatio-temporal resolution data and sensitivity to short distances combined with the ability to bypass protein labeling makes this assay an effective alternative or a complement to FRET.

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Abstract: Single-molecule FRET has been widely used for monitoring protein–nucleic acids interactions. Direct visualization of the interactions, however, often requires a site-specific labeling of the protein, which can be circuitous and inefficient. In addition, FRET is insensitive to distance changes in the 0–3-nm range. Here, we report a systematic calibration of a single molecule fluorescence assay termed protein induced fluorescence enhancement. This method circumvents protein labeling and displays a marked distance dependence below the 4-nm distance range. The enhancement of fluorescence is based on the photophysical phenomenon whereby the intensity of a fluorophore increases upon proximal binding of a protein. Our data reveals that the method can resolve as small as a single base pair distance at the extreme vicinity of the fluorophore, where the enhancement is maximized. We demonstrate the general applicability and distance sensitivity using (a) a finely spaced DNA ladder carrying a restriction site for BamHI, (b) RNA translocation by DExH enzyme RIG‐I, and (c) filament dynamics of RecA on single-stranded DNA. The high spatio-temporal resolution data and sensitivity to short distances combined with the ability to bypass protein labeling makes this assay an effective alternative or a complement to FRET.

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225 citations

Cites background from "PcrA Helicase Dismantles RecA Filam..."

...This photophysical effect was originally employed in stop flow measurement (9) for monitoring directional movement of DNAmotor proteins in ensemble (10–13), for following the dynamics of DNA and RNA polymerases on DNA (14, 15), and for detecting the motion of helicases on RNA or DNA at the single-molecule level (16, 17)....
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Journal Article•DOI•

Protein induced fluorescence enhancement (PIFE) for probing protein–nucleic acid interactions

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Helen Hwang¹, Sua Myong•Institutions (1)

University of Illinois at Urbana–Champaign¹

28 Jan 2014-Chemical Society Reviews

TL;DR: This review provides an overview of a single molecule fluorescence method, termed "protein induced fluorescence enhancement" (PIFE), which employs a single dye attached to DNA or RNA to which an unlabeled protein is applied.

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Abstract: Single molecule studies of protein–nucleic acid interactions shed light on molecular mechanisms and kinetics involved in protein binding, translocation, and unwinding of DNA and RNA substrates. In this review, we provide an overview of a single molecule fluorescence method, termed “protein induced fluorescence enhancement” (PIFE). Unlike FRET where two dyes are required, PIFE employs a single dye attached to DNA or RNA to which an unlabeled protein is applied. We discuss both ensemble and single molecule studies in which PIFE was utilized.

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187 citations

Journal Article•DOI•

Replication-transcription conflicts in bacteria

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Houra Merrikh¹, Yan Zhang², Alan D. Grossman¹, Jue D. Wang²•Institutions (2)

Massachusetts Institute of Technology¹, Baylor College of Medicine²

06 Jun 2012-Nature Reviews Microbiology

TL;DR: Findings are summarized which shed light on the significance of the problem and on how bacterial cells deal with unwanted encounters between the replication and transcription machineries.

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Abstract: DNA replication and transcription use the same template and occur concurrently in bacteria. The lack of temporal and spatial separation of these two processes leads to their conflict, and failure to deal with this conflict can result in genome alterations and reduced fitness. In recent years major advances have been made in understanding how cells avoid conflicts between replication and transcription and how such conflicts are resolved when they do occur. In this Review, we summarize these findings, which shed light on the significance of the problem and on how bacterial cells deal with unwanted encounters between the replication and transcription machineries.

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185 citations

Journal Article•DOI•

Regulating the chromatin landscape: structural and mechanistic perspectives.

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Blaine Bartholomew¹•Institutions (1)

University of Texas MD Anderson Cancer Center¹

06 Jun 2014-Annual Review of Biochemistry

TL;DR: A large family of chromatin remodelers that noncovalently modify chromatin is crucial in cell development and differentiation and is being identified as targets in human diseases by NGS (next-generation sequencing).

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Abstract: A large family of chromatin remodelers that noncovalently modify chromatin is crucial in cell development and differentiation. They are often the targets of cancer, neurological disorders, and other human diseases. These complexes alter nucleosome positioning, higher-order chromatin structure, and nuclear organization. They also assemble chromatin, exchange out histone variants, and disassemble chromatin at defined locations. We review aspects of the structural organization of these complexes, the functional properties of their protein domains, and variation between complexes. We also address the mechanistic details of these complexes in mobilizing nucleosomes and altering chromatin structure. A better understanding of these issues will be vital for further analyses of subunits of these chromatin remodelers, which are being identified as targets in human diseases by NGS (next-generation sequencing).

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185 citations

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Journal Article•DOI•

Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor

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Taekjip Ha¹, Th. Enderle¹, D. F. Ogletree¹, Daniel S. Chemla¹, Daniel S. Chemla², Paul R. Selvin¹, Paul R. Selvin², Shimon Weiss¹ - Show less +4 more•Institutions (2)

Lawrence Berkeley National Laboratory¹, University of California, Berkeley²

25 Jun 1996-Proceedings of the National Academy of Sciences of the United States of America

TL;DR: In this article, the sensitivity of fluorescence resonance energy transfer (FRET) was extended to the single molecule level by measuring energy transfer between a single donor and a single acceptor fluorophore.

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Abstract: We extend the sensitivity of fluorescence resonance energy transfer (FRET) to the single molecule level by measuring energy transfer between a single donor fluorophore and a single acceptor fluorophore. Near-field scanning optical microscopy (NSOM) is used to obtain simultaneous dual color images and emission spectra from donor and acceptor fluorophores linked by a short DNA molecule. Photodestruction dynamics of the donor or acceptor are used to determine the presence and efficiency of energy transfer. The classical equations used to measure energy transfer on ensembles of fluorophores are modified for single-molecule measurements. In contrast to ensemble measurements, dynamic events on a molecular scale are observable in single pair FRET measurements because they are not canceled out by random averaging. Monitoring conformational changes, such as rotations and distance changes on a nanometer scale, within single biological macromolecules, may be possible with single pair FRET.

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1,172 citations

Journal Article•DOI•

Structure and mechanism of helicases and nucleic acid translocases.

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Martin R. Singleton¹, Mark S. Dillingham, Dale B. Wigley•Institutions (1)

London Research Institute¹

19 Jun 2007-Annual Review of Biochemistry

TL;DR: This review sets out to define a nomenclature for helicase and translocase enzymes based on current knowledge of sequence, structure, and mechanism, and delineate six superfamilies of enzymes, with examples of crystal structures where available.

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Abstract: Helicases and translocases are a ubiquitous, highly diverse group of proteins that perform an extraordinary variety of functions in cells. Consequently, this review sets out to define a nomenclature for these enzymes based on current knowledge of sequence, structure, and mechanism. Using previous definitions of helicase families as a basis, we delineate six superfamilies of enzymes, with examples of crystal structures where available, and discuss these structures in the context of biochemical data to outline our present understanding of helicase and translocase activity. As a result, each superfamily is subdivided, where appropriate, on the basis of mechanistic understanding, which we hope will provide a framework for classification of new superfamily members as they are discovered and characterized.

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1,145 citations

"PcrA Helicase Dismantles RecA Filam..." refers background in this paper

...Helicases are ubiquitous enzymes that are involved in nearly all aspects of nucleic acid metabolism in living organisms (Lohman and Bjornson, 1996; Pyle, 2008; Singleton et al., 2007)....
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...Most proteins that belong to this class are also ‘‘translocases’’ that move directionally along a DNA or RNA track, although translocation activity is necessary but not sufficient for duplex DNA unwinding (Lohman et al., 2008; Singleton et al., 2007)....
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...Helicases/translocases belonging to superfamily 1 (SF1) are among the most extensively characterized of these enzymes (Singleton et al., 2007)....
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...Rep, PcrA, and UvrD are structurally similar SF1A helicases (Korolev et al., 1997; Lee and Yang, 2006; Singleton et al., 2007; Velankar et al., 1999)....
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Journal Article•DOI•

Crystal structures of complexes of PcrA DNA helicase with a DNA substrate indicate an inchworm mechanism.

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Sameer Velankar¹, Panos Soultanas¹, Mark S. Dillingham¹, H.S. Subramanya¹, Dale B. Wigley¹ - Show less +1 more•Institutions (1)

University of Oxford¹

02 Apr 1999-Cell

TL;DR: Two different structures of PcrA DNA helicase complexed with the same single strand tailed DNA duplex are determined, providing snapshots of different steps on the catalytic pathway, providing evidence against an "active rolling" model for helicase action but are instead consistent with an "inchworm" mechanism.

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759 citations

"PcrA Helicase Dismantles RecA Filam..." refers background in this paper

...Role of 2B Subdomain and Its Conformation PcrA and UvrD monomers crystallized with a 30-tailed partial duplex DNA showed that the 2B subdomain is in contact with the duplex region (Lee and Yang, 2006; Velankar et al., 1999)....
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...In the closed conformation of PcrA (Figure 7A) (Velankar et al., 1999), the 50 ssDNA tail would need to traverse approximately 34 Å to enter the 1A domain with the correct orientation (Figure 7C), whereas our tail length-dependent data suggest that translocation begins very close to the duplex region....
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...In the closed conformation of PcrA (Figure 7A) (Velankar et al., 1999), the 50 ssDNA tail would need to traverse approximately 34 Å to enter the 1A domain with the correct orientation (Figure 7C), whereas our tail length-dependent data suggest that translocation begins very close to the duplex…...
[...]
...PcrA and UvrD monomers crystallized with a 30-tailed partial duplex DNA showed that the 2B subdomain is in contact with the duplex region (Lee and Yang, 2006; Velankar et al., 1999)....
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...Rep, PcrA, and UvrD are structurally similar SF1A helicases (Korolev et al., 1997; Lee and Yang, 2006; Singleton et al., 2007; Velankar et al., 1999)....
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Journal Article•DOI•

Mechanisms of helicase-catalyzed DNA unwinding.

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Timothy M. Lohman¹, Keith P. Bjornson¹•Institutions (1)

Washington University in St. Louis¹

01 Jan 1996-Annual Review of Biochemistry

TL;DR: This review discusses mechanistic aspects of helicase-catalyzed DNA unwinding and translocation with a focus on energetic (thermodynamic), kinetic, and structural studies of the few DNA helicases for which such information is available.

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Abstract: DNA helicases are essential motor proteins that function to unwind duplex DNA to yield the transient single-stranded DNA intermediates required for replication, recombination, and repair. These enzymes unwind duplex DNA and translocate along DNA in reactions that are coupled to the binding and hydrolysis of 5'-nucleoside triphosphates (NTP). Although these enzymes are essential for DNA metabolism, the molecular details of their mechanisms are only beginning to emerge. This review discusses mechanistic aspects of helicase-catalyzed DNA unwinding and translocation with a focus on energetic (thermodynamic), kinetic, and structural studies of the few DNA helicases for which such information is available. Recent studies of DNA and NTP binding and DNA unwinding by the Escherichia coli (E. coli) Rep helicase suggest that the Rep helicase dimer unwinds DNA by an active, rolling mechanism. In fact, DNA helicases appear to be generally oligomeric (usually dimers or hexamers), which provides the helicase with multiple DNA binding sites. The apparent mechanistic similarities and differences among these DNA helicases are discussed.

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741 citations

"PcrA Helicase Dismantles RecA Filam..." refers background in this paper

...Helicases are ubiquitous enzymes that are involved in nearly all aspects of nucleic acid metabolism in living organisms (Lohman and Bjornson, 1996; Pyle, 2008; Singleton et al., 2007)....
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Journal Article•DOI•

DNA helicase Srs2 disrupts the Rad51 presynaptic filament

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Lumir Krejci¹, Stephen Van Komen¹, Ying Li², Jana Villemain¹, Mothe Sreedhar Reddy¹, Hannah L. Klein³, Thomas E. Ellenberger², Patrick Sung¹ - Show less +4 more•Institutions (3)

University of Texas Health Science Center at San Antonio¹, Harvard University², New York University³

15 May 2003-Nature

TL;DR: The role of SRS2 in recombination modulation is clarified by purifying its encoded product and examining its interactions with the Rad51 recombinase, and it is shown that Srs2 acts by dislodging Rad51 from ssDNA.

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Abstract: Mutations in the Saccharomyces cerevisiae gene SRS2 result in the yeast's sensitivity to genotoxic agents, failure to recover or adapt from DNA damage checkpoint-mediated cell cycle arrest, slow growth, chromosome loss, and hyper-recombination1,2. Furthermore, double mutant strains, with mutations in DNA helicase genes SRS2 and SGS1, show low viability that can be overcome by inactivating recombination, implying that untimely recombination is the cause of growth impairment1,3,4. Here we clarify the role of SRS2 in recombination modulation by purifying its encoded product and examining its interactions with the Rad51 recombinase. Srs2 has a robust ATPase activity that is dependent on single-stranded DNA (ssDNA) and binds Rad51, but the addition of a catalytic quantity of Srs2 to Rad51-mediated recombination reactions causes severe inhibition of these reactions. We show that Srs2 acts by dislodging Rad51 from ssDNA. Thus, the attenuation of recombination efficiency by Srs2 stems primarily from its ability to dismantle the Rad51 presynaptic filament efficiently. Our findings have implications for the basis of Bloom's and Werner's syndromes, which are caused by mutations in DNA helicases and are characterized by increased frequencies of recombination and a predisposition to cancers and accelerated ageing5.

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609 citations

"PcrA Helicase Dismantles RecA Filam..." refers background in this paper

...nation proteins, respectively, from single-stranded (ss) DNA (Antony et al., 2009; Krejci et al., 2003; Veaute et al., 2003, 2005)....
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...…both display antirecombinase activity that is related to their abilities to displace RecA and RAD51 recombi- 544 Cell 142, 544–555, August 20, 2010 ª2010 Elsevier Inc. nation proteins, respectively, from single-stranded (ss) DNA (Antony et al., 2009; Krejci et al., 2003; Veaute et al., 2003, 2005)....
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