High‐throughput single‐molecule studies of protein–DNA interactions

Robison, Aaron D.; Finkelstein, Ilya J.

doi:10.1016/j.febslet.2014.05.021

Cited by 17 publications

(19 citation statements)

References 98 publications

(122 reference statements)

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“…The flowcell was coated with a surfacepassivating fluid lipid bilayer, and the DNA substrate was affixed to the bilayer via a biotin-streptavidin linkage. Nanofabricated chrome barriers were used to organize thousands of DNA molecules for high-throughput data collection and analysis (28)(29)(30). As hExo1 has been reported to preferentially load on 3′-ssDNA ends (18,(31)(32)(33), we prepared a λ-phage DNA that contained a 72-nt 3′-polyT overhang.…”

Section: Resultsmentioning

confidence: 99%

Single-molecule imaging reveals the mechanism of Exo1 regulation by single-stranded DNA binding proteins

Myler

Gallardo

Zhou

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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104

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Exonuclease 1 (Exo1) is a 5′→3′ exonuclease and 5′-flap endonuclease that plays a critical role in multiple eukaryotic DNA repair pathways. Exo1 processing at DNA nicks and double-strand breaks creates long stretches of single-stranded DNA, which are rapidly bound by replication protein A (RPA) and other single-stranded DNA binding proteins (SSBs). Here, we use single-molecule fluorescence imaging and quantitative cell biology approaches to reveal the interplay between Exo1 and SSBs. Both human and yeast Exo1 are processive nucleases on their own. RPA rapidly strips Exo1 from DNA, and this activity is dependent on at least three RPA-encoded single-stranded DNA binding domains. Furthermore, we show that ablation of RPA in human cells increases Exo1 recruitment to damage sites. In contrast, the sensor of single-stranded DNA complex 1-a recently identified human SSB that promotes DNA resection during homologous recombination-supports processive resection by Exo1. Although RPA rapidly turns over Exo1, multiple cycles of nuclease rebinding at the same DNA site can still support limited DNA processing. These results reveal the role of single-stranded DNA binding proteins in controlling Exo1-catalyzed resection with implications for how Exo1 is regulated during DNA repair in eukaryotic cells.A ll DNA maintenance processes require nucleases, which enzymatically cleave the phosphodiester bonds in nucleic acids. Exo1, a member of the Rad2 family of nucleases, participates in DNA mismatch repair (MMR), double-strand break (DSB) repair, nucleotide excision repair (NER), and telomere maintenance (1-3). Exo1 is the only nuclease implicated in MMR, where its 5ʹ to 3ʹ exonuclease activity is used to remove long tracts of mismatch-containing single-stranded DNA (ssDNA) (2, 4-7). In addition, functionally deficient Exo1 variants have been identified in familial colorectal cancers, and Exo1-null mice exhibit a significant increase in tumor development, decreased lifespan, and sterility (8, 9). Exo1 also promotes DSB repair via homologous recombination (HR) by processing the free DNA ends to generate kilobase-length ssDNA resection products (1, 10-12). The resulting ssDNA is paired with a homologous DNA sequence located on a sister chromatid, and the missing genetic information is then restored via DNA synthesis. The central role of Exo1 in DNA repair is highlighted by the large set of genetic interactions between Exo1 and nearly all other DNA maintenance and metabolism pathways (13).Exo1 generates long tracts of ssDNA in both MMR and DSB repair (3). This ssDNA is rapidly bound by replication protein A (RPA), a ubiquitous heterotrimeric protein that participates in all DNA transactions that generate ssDNA intermediates (14). RPA protects the ssDNA from degradation, participates in DNA damage response signaling, and acts as a loading platform for downstream DSB repair proteins (15-17). RPA also coordinates DNA resection by removing secondary ssDNA structures and by modulating the Bloom syndrome, RecQ helicase-like (BLM)/ DNA2-and Ex...

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Section: Resultsmentioning

confidence: 99%

Single-molecule imaging reveals the mechanism of Exo1 regulation by single-stranded DNA binding proteins

Myler

Gallardo

Zhou

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

104

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“…This method was recently used to demonstrate that break-induced replication, a double strand break-repair pathway used to repair one-ended breaks, proceeds via a conservative mode of inheritance unlike the canonical semi-conservative DNA synthesis [9]. Other single-molecule techniques include the use of “DNA curtains” wherein individual DNA molecules are tethered at one end to help understand how proteins traverse the length of DNA; tethered particle motion to study the change in DNA length upon interaction with enzymes; and the use of molecular tweezers to study the mechanical properties of DNA-protein and protein-protein interactions [reviewed in [10]].…”

Section: Dna Repair - the Current Eramentioning

confidence: 99%

The Journey of DNA Repair

Saini

2015

Trends in Cancer

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“…Experimental detection on protein searching motions or 1-D diffusion along DNA have provided evidence on the facilitated diffusion (7)(8)(9)(10)(11)(12). Nevertheless, as protein movements for base pair (bp) distances on DNA can take place as fast as microseconds, tracking the 1-D protein diffusion at such a high temporal and spatial resolution remains technically challenging (13)(14)(15)(16).…”

Section: Introductionmentioning

confidence: 99%

Revealing Atomic-scale Molecular Diffusion of a Plant Transcription Factor WRKY domain protein along DNA

Dai

et al. 2020

Preprint

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designed the study. Liqiang Dai conducted the MD simulations and coarse-grained simulations and analyzed the related data. Yongping Xu solved the crystal structure of WRKY-DNA complex and conducted ITC experiment. Zhenwei Du conducted the single-molecule fluorescence experiments of WRKY diffusing on DNA. AbstractTranscription factor (TF) target search on genome is highly essential for gene expression and regulation. High-resolution determination of TF diffusion along DNA remains technically challenging. Here we constructed a TF model system of the plant WRKY domain protein in complex with DNA from crystallography and demonstrated microsecond diffusion dynamics of WRKY on the DNA employing all-atom molecular dynamics (MD) simulations. Notably, we found that WRKY preferentially binds to the Crick strand of DNA with significantly stronger energetic association than to the Watson strand. The preferential binding becomes highly prominent from non-specific to specific DNA binding, but less distinct from static binding to diffusive movements of WRKY on the DNA. Remarkably, without employing acceleration forces or bias, we captured a complete one-base pair (bp) stepping cycle of WRKY tracking along major groove of DNA with homogenous (AT)n sequence, as individual protein-DNA contacts break and reform at the binding interface. Continuous tracking of WRKY forward or backward, with occasional sliding as well as strand crossing to the minor groove of DNA, have also been captured in the simulation. The processive diffusion of WRKY had been confirmed by accompanied single-molecule fluorescence assays and coarsegrained (CG) structural simulations. The study thus provides unprecedented structural dynamics details on the TF diffusion, suggests how TF possibly approaches to gene target, and supports further high-precision experimental follow-up. The stochastic movements revealed in the TF diffusion also provide general clues on how other nucleic acid walkers step and slide along DNA. Significance StatementHow transcription factors search for target genes impact on how quickly and accurately the genes are transcribed and expressed. To locate target sufficiently fast, 1D diffusion of the protein along DNA appears essential. Experimentally, it remains challenging to determine diffusional steps of protein on DNA. Here, we report all-atom equilibrium simulations of a WRKY protein binding and diffusing on DNA, revealing structural dynamics details which have not been identified previously. We unprecedently demonstrate a complete stepping cycle of the protein for one base pair on DNA within microseconds, along with stochastic stepping or sliding, directional switching, and strand crossing. Additionally, we have found preferential DNA strand association of WRKY. These suggest how protein factors approach toward target DNA sequences.

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High‐throughput single‐molecule studies of protein–DNA interactions

Cited by 17 publications

References 98 publications

Single-molecule imaging reveals the mechanism of Exo1 regulation by single-stranded DNA binding proteins

Single-molecule imaging reveals the mechanism of Exo1 regulation by single-stranded DNA binding proteins

The Journey of DNA Repair

Revealing Atomic-scale Molecular Diffusion of a Plant Transcription Factor WRKY domain protein along DNA

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