Single-Molecule Imaging Reveals that Rad4 Employs a Dynamic DNA Damage Recognition Process

Kong, Muwen; Liu, Huan; Chen, Xuejing; Driscoll, Katherine; Mao, Peng; Böhm, Stefanie; Kad, Neil M.; Watkins, Simon C.; Bernstein, Kara A.; Wyrick, John J.; Min, Jo Young; Houten, Bennett Van

doi:10.1016/j.molcel.2016.09.005

Cited by 82 publications

(110 citation statements)

References 53 publications

(93 reference statements)

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“…1 Single molecule studies have been employed to study the dynamics of various NER factors including XPC, XPD, and RPA. 26−29 …”

Section: Introductionmentioning

confidence: 99%

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Nucleotide Excision Repair Lesion-Recognition Protein Rad4 Captures a Pre-Flipped Partner Base in a Benzo[a]pyrene-Derived DNA Lesion: How Structure Impacts the Binding Pathway

Mu¹,

Geacintov²,

Min

et al. 2017

Chem. Res. Toxicol.

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The xeroderma pigmentosum C protein complex (XPC) recognizes a variety of environmentally induced DNA lesions and is the key in initiating their repair by the nucleotide excision repair (NER) pathway. When bound to a lesion, XPC flips two nucleotide pairs that include the lesion out of the DNA duplex, yielding a productively bound complex that can lead to successful lesion excision. Interestingly, the efficiencies of NER vary greatly among different lesions, influencing their toxicity and mutagenicity in cells. Though differences in XPC binding may influence NER efficiency, it is not understood whether XPC utilizes different mechanisms to achieve productive binding with different lesions. Here, we investigated the well-repaired 10R-(+)-cis-anti-benzo[a]pyrene-N2-dG (cis-B[a]P-dG) DNA adduct in a duplex containing normal partner C opposite the lesion. This adduct is derived from the environmental pro-carcinogen benzo[a]pyrene and is likely to be encountered by NER in the cell. We have extensively investigated its binding to the yeast XPC orthologue, Rad4, using umbrella sampling with restrained molecular dynamics simulations and free energy calculations. The NMR solution structure of this lesion in duplex DNA has shown that the dC complementary to the adducted dG is flipped out of the DNA duplex in the absence of XPC. However, it is not known whether the “pre-flipped” base would play a role in its recognition by XPC. Our results show that Rad4 first captures the displaced dC, which is followed by a tightly coupled lesion-extruding pathway for productive binding. This binding path differs significantly from the one deduced for the small cis-syn cyclobutane pyrimidine dimer lesion opposite mismatched thymines [MuH.MuH.26270861Biochemistry20155452637]. The possibility of multiple paths that lead to productive binding to XPC is consistent with the versatile lesion recognition by XPC that is required for successful NER.

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“…1 Single molecule studies have been employed to study the dynamics of various NER factors including XPC, XPD, and RPA. 26−29 …”

Section: Introductionmentioning

confidence: 99%

“…40 Single-molecule fluorescence microscopy studies have provided further support for the “twist-open” and the “kinetic gating” lesion recognition mechanisms. 29 …”

Section: Introductionmentioning

confidence: 99%

Nucleotide Excision Repair Lesion-Recognition Protein Rad4 Captures a Pre-Flipped Partner Base in a Benzo[a]pyrene-Derived DNA Lesion: How Structure Impacts the Binding Pathway

Mu¹,

Geacintov²,

Min

et al. 2017

Chem. Res. Toxicol.

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“…In a recently published study, we characterized the diffusive behavior of quantum dot-labeled Rad4-Rad23 (the yeast homolog of human XPC-HR23B, Figure 2A), the damage sensor in yeast nucleotide excision repair (NER), using the single-molecule DNA tightrope assay (Kong et al, 2016). It was shown that Rad4-Rad23 utilizes the facilitated diffusion mechanism (See Section 4 for in-depth discussion on the topic) to search for lesions in DNA.…”

Section: Diffusion Of a Dna Repair Proteinmentioning

confidence: 99%

“…His-tagged Rad4-Rad23 (yellow, pink, cyan, and blue) is labeled with streptavidin (red)-coated quantum dot (green) through a His antibody (His-Ab)-biotin conjugate (gray). Adapted from Kong et al , (2016) Mol Cell , 64 , 376 – 387 with permission.…”

Section: Figurementioning

confidence: 99%

Rad4 recognition-at-a-distance: Physical basis of conformation-specific anomalous diffusion of DNA repair proteins

Kong

Houten

2017

Progress in Biophysics and Molecular Biology

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Since Robert Brown’s first observations of random walks by pollen particles suspended in solution, the concept of diffusion has been subject to countless theoretical and experimental studies in diverse fields from finance and social sciences, to physics and biology. Diffusive transport of macromolecules in cells is intimately linked to essential cellular functions including nutrient uptake, signal transduction, gene expression, as well as DNA replication and repair. Advancement in experimental techniques has allowed precise measurements of these diffusion processes. Mathematical and physical descriptions and computer simulations have been applied to model complicated biological systems in which anomalous diffusion, in addition to simple Brownian motion, was observed. The purpose of this review is to provide an overview of the major physical models of anomalous diffusion and corresponding experimental evidence on the target search problem faced by DNA-binding proteins, with an emphasis on DNA repair proteins and the role of anomalous diffusion in DNA target recognition.

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“…A recent report 9 revealed that the yeast protein Rad4, which is involved in nucleotide-excision repair, does not directly bind damaged sites, but instead diffuses for up to 1 kilobase around the damage, providing a dynamic platform for the recruitment of other repair proteins. Furthermore, the protein Mfd, which dislodges RNA polymerase enzymes that become stalled while transcribing DNA, then scouts ahead for DNA damage on the transcribed strand 10 .…”

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confidence: 99%

Clamping down on copy errors

Kad

Houten

2016

Nature

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Single-Molecule Imaging Reveals that Rad4 Employs a Dynamic DNA Damage Recognition Process

Cited by 82 publications

References 53 publications

Nucleotide Excision Repair Lesion-Recognition Protein Rad4 Captures a Pre-Flipped Partner Base in a Benzo[a]pyrene-Derived DNA Lesion: How Structure Impacts the Binding Pathway

Nucleotide Excision Repair Lesion-Recognition Protein Rad4 Captures a Pre-Flipped Partner Base in a Benzo[a]pyrene-Derived DNA Lesion: How Structure Impacts the Binding Pathway

Rad4 recognition-at-a-distance: Physical basis of conformation-specific anomalous diffusion of DNA repair proteins

Clamping down on copy errors

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