Single-Molecule Analysis Reveals Differential Effect of ssDNA-Binding Proteins on DNA Translocation by XPD Helicase

Honda, Masayoshi; Park, Jeehae; Pugh, Robert A.; Ha, Taekjip; Spies, Maria

doi:10.1016/j.molcel.2009.07.003

Cited by 77 publications

(131 citation statements)

References 47 publications

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“…More recently, wild-type recombinant FacXPD was shown to translocate on RPA-coated ssDNA. 35 From our studies of the FANCJ-A349P mutant, we determined that this particular mutation interferes with a critical step downstream of DNA translocation that is required for disruption of standard Watson-Crick hydrogen bonds between base pairs of B-form DNA as well as alternate Hoogsteen hydrogen bonds between guanine residues of the G-tetrad stack. From steady-state measurements, we determined that the mutant protein can hydrolyze ATP and translocate along the ssDNA lattice but was impaired in its ability to destabilize protein-DNA interactions.…”

Section: Discussionmentioning

confidence: 99%

Fanconi anemia group J mutation abolishes its DNA repair function by uncoupling DNA translocation from helicase activity or disruption of protein-DNA complexes

Sommers

Suhasini

et al. 2010

Blood

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Section: Discussionmentioning

confidence: 99%

Fanconi anemia group J mutation abolishes its DNA repair function by uncoupling DNA translocation from helicase activity or disruption of protein-DNA complexes

Sommers

Suhasini

et al. 2010

Blood

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“…All experiments were conducted in reaction buffer containing 50 mM Tris-Cl (pH 7.5), 150 mM sodium chloride, 0.1 mg/ml bovine serum albumin, 0.8% glucose, and 1 mM dithiothreitol. The oxygen scavenging system used was described previously (40,41). In addition, 12 mM Trolox (6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid; Sigma) was prepared in the presence of 12 mM sodium hydroxide and incubated at room temperature under fluorescent light with continuous rotation for 3-4 days.…”

mentioning

confidence: 99%

The Proliferating Cell Nuclear Antigen (PCNA)-interacting Protein (PIP) Motif of DNA Polymerase η Mediates Its Interaction with the C-terminal Domain of Rev1

Boehm

Powers

Kondratick

et al. 2016

Journal of Biological Chemistry

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Y-family DNA polymerases, such as polymerase , polymerase , and polymerase , catalyze the bypass of DNA damage during translesion synthesis. These enzymes are recruited to sites of DNA damage by interacting with the essential replication accessory protein proliferating cell nuclear antigen (PCNA) and the scaffold protein Rev1. In most Y-family polymerases, these interactions are mediated by one or more conserved PCNA-interacting protein (PIP) motifs that bind in a hydrophobic pocket on the front side of PCNA as well as by conserved Rev1-interacting region (RIR) motifs that bind in a hydrophobic pocket on the C-terminal domain of Rev1. Yeast polymerase , a prototypical translesion synthesis polymerase, binds both PCNA and Rev1. It possesses a single PIP motif but not an RIR motif. Here we show that the PIP motif of yeast polymerase mediates its interactions both with PCNA and with Rev1. Moreover, the PIP motif of polymerase binds in the hydrophobic pocket on the Rev1 C-terminal domain. We also show that the RIR motif of human polymerase and the PIP motif of yeast Msh6 bind both PCNA and Rev1. Overall, these findings demonstrate that PIP motifs and RIR motifs have overlapping specificities and can interact with both PCNA and Rev1 in structurally similar ways. These findings also suggest that PIP motifs are a more versatile protein interaction motif than previously believed. Proliferating cell nuclear antigen (PCNA)2 is an essential protein that functions in DNA replication, repair, recombination, damage tolerance, and cell cycle control. It is a homotrimer that forms a ring-shaped clamp, which slides along DNA (1-3). Many proteins involved in DNA metabolism, the maintenance of genome stability, and cell cycle control possess PCNA-interacting protein (PIP) motifs that mediate their binding to PCNA (1, 4 -6). These PIP motifs contain two adjacent aromatic residues, which bind in a hydrophobic pocket on the front face of the PCNA ring (2, 4), and the binding of these PIP motifs to PCNA represents a major point of regulation for these proteins.Among the proteins that are regulated by PCNA are the Y-family DNA polymerases such as DNA polymerase (pol ), DNA polymerase (pol ), DNA polymerase (pol ), and Rev1 (7-12). These polymerases bypass DNA damage during translesion DNA synthesis in order to allow for continuous progression of the replication fork (13-16). Pol , for example, catalyzes the bypass of UV-induced thymine-thymine dimers and 8-oxoguanine lesions (17, 18). Defects in pol cause the variant form of xeroderma pigmentosum, a genetic disorder characterized by sensitivity to sunlight and a high incidence of skin cancers (19,20). Rev1, by contrast, bypasses damaged guanines and abasic sites (21-24).In addition to its catalytic function, Rev1 plays an important non-catalytic role in translesion synthesis by binding to other polymerases, like pol , pol , and pol , and acting as a scaffold to recruit them to PCNA (25-29). Many Y-family polymerases, such as mammalian pol , pol , and pol , possess short, conserved Rev1...

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“…FeS cluster dependent quenching of Cy3 and Cy5 can also be exploited at the singlemolecule level using total internal reflection microscopy (TIRM) [3]. The most informative single-molecule TIRM experiments follow FRET between donor and acceptor fluorophores (such as Cy3 and Cy5) site-specifically incorporated into proteins and/or nucleic acids [36,37].…”

Section: Helicase Characterization Assaysmentioning

confidence: 99%

“…The presence of iron-sulfur (FeS) clusters in the Rad3 family helicases [1] led to development of a number of fluorescence based assays to characterize the enzymes' ability to bind to and recognize DNA substrates [2], and observe translocation on a DNA lattice [3]. FeS clusters in these enzymes serve as endogenous quenchers of a wide spectral range of fluorophores which can be used as reporters for monitoring helicase-DNA interactions.…”

Section: Introductionmentioning

confidence: 99%

“…FeS clusters in these enzymes serve as endogenous quenchers of a wide spectral range of fluorophores which can be used as reporters for monitoring helicase-DNA interactions. This effect has been used to develop new assays for ensemble and single-molecule characterization of Rad3 helicases [2][3][4]. Moreover, combining FeS cluster-mediated fluorescence quenching with Föster resonance energy transfer (FRET) [5,6] between a pair of fluorophores allows for expansion of these analyses to pseudo three color measurements to monitor complex nucleoprotein interactions.…”

Section: Introductionmentioning

confidence: 99%

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Ensemble and single-molecule fluorescence-based assays to monitor DNA binding, translocation, and unwinding by iron–sulfur cluster containing helicases

Pugh

Honda

Spies

2010

Methods

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Many quantitative approaches for analysis of helicase-nucleic acid interactions require a robust and specific signal, which reports on the presence of the helicase and its position on a nucleic acid lattice. Since 2006, iron-sulfur (FeS) clusters have been found in a number of helicases. They serve as endogenous quenchers of Cy3 and Cy5 fluorescence which can be exploited to characterize FeS containing helicases both in ensemble-based assays and at the single-molecule level. Synthetic oligonucleotides site-specifically labeled with either Cy3 or Cy5 can be used to create a variety of DNA substrates that can be used to characterized DNA binding, as well as helicase translocation and unwinding. Equilibrium binding affinities for ssDNA, duplex and branched DNA substrates can be determined using bulk assays. Identification of preferred cognate substrates, and the orientation and position of the helicase when bound to DNA can also be determined by taking advantage of the intrinsic quencher in the helicase. At the single-molecule level, real-time observation of the helicase translocating along DNA either towards the dye or away from the dye can be used to determine the rate of translocation by the helicase on ssDNA and its orientation when bound to DNA. The use of duplex substrates can reveal the rate of unwinding and processivity of the helicase. Finally, the FeS cluster can be used to visualize protein-protein interactions, and to examine the interplay between helicases and other DNA binding proteins on the same DNA substrate.

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Single-Molecule Analysis Reveals Differential Effect of ssDNA-Binding Proteins on DNA Translocation by XPD Helicase

Cited by 77 publications

References 47 publications

Fanconi anemia group J mutation abolishes its DNA repair function by uncoupling DNA translocation from helicase activity or disruption of protein-DNA complexes

Fanconi anemia group J mutation abolishes its DNA repair function by uncoupling DNA translocation from helicase activity or disruption of protein-DNA complexes

The Proliferating Cell Nuclear Antigen (PCNA)-interacting Protein (PIP) Motif of DNA Polymerase η Mediates Its Interaction with the C-terminal Domain of Rev1

Ensemble and single-molecule fluorescence-based assays to monitor DNA binding, translocation, and unwinding by iron–sulfur cluster containing helicases

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