Quantification of dye-mediated photodamage during single-molecule DNA imaging

Tycon, Michael A.; Dial, Catherine F.; Faison, Keia; Melvin, W. Lee; Fecko, Christopher J.

doi:10.1016/j.ab.2012.03.021

Cited by 34 publications

(35 citation statements)

References 24 publications

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“…To that end, we systematically tested three strategies for conjugating QDs to UV-DDB and proceeded with a His-Ab conjugation strategy that enabled us to conjugate UV-DDB to SA-QDs using a biotinylated pentaHis antibody, while retaining DNA damage binding activity (SI Materials and Methods, section 1.2.3) (29). We incubated QD UV-DDB with undamaged λ-DNA tightropes or λ-DNA containing on average one lesion per 2,200 bp (SI Materials and Methods, section 2) and observed these interactions in a time window of 900 s. Imaging was performed in the absence of YOYO-1 dye to minimize potential double-strand breaks in the DNA tightropes (SI Materials and Methods, section 1.2.4) (38). We observed the binding of UV-DDB to DNA ( Fig.…”

Section: Resultsmentioning

confidence: 99%

Single-molecule analysis reveals human UV-damaged DNA-binding protein (UV-DDB) dimerizes on DNA via multiple kinetic intermediates

Ghodke

Wang

Hsieh

et al. 2014

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

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How human DNA repair proteins survey the genome for UVinduced photoproducts remains a poorly understood aspect of the initial damage recognition step in nucleotide excision repair (NER). To understand this process, we performed single-molecule experiments, which revealed that the human UV-damaged DNA-binding protein (UV-DDB) performs a 3D search mechanism and displays a remarkable heterogeneity in the kinetics of damage recognition. Our results indicate that UV-DDB examines sites on DNA in discrete steps before forming long-lived, nonmotile UV-DDB dimers (DDB1-DDB2) 2 at sites of damage. Analysis of the rates of dissociation for the transient binding molecules on both undamaged and damaged DNA show multiple dwell times over three orders of magnitude: 0.3-0.8, 8.1, and 113-126 s. These intermediate states are believed to represent discrete UV-DDB conformers on the trajectory to stable damage detection. DNA damage promoted the formation of highly stable dimers lasting for at least 15 min. The xeroderma pigmentosum group E (XP-E) causing K244E mutant of DDB2 found in patient XP82TO, supported UV-DDB dimerization but was found to slide on DNA and failed to stably engage lesions. These findings provide molecular insight into the loss of damage discrimination observed in this XP-E patient. This study proposes that UV-DDB recognizes lesions via multiple kinetic intermediates, through a conformational proofreading mechanism.DNA damage recognition | single-molecule tracking | DNA tightrope | human nucleotide excision repair U nrepaired photoproducts in the genome arising from exposure to UV irradiation can be highly mutagenic and three pathways have evolved in mammalian cells to process these lesions, which include (i) global genomic repair, (ii) transcription-coupled repair, and (iii) translesion synthesis (1-5). During global genomic repair, cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts [(6-4) photoproducts] are repaired by the nucleotide excision repair (NER) pathway that recognizes and excises bulky helix distorting lesions in the genome (6, 7). The recognition of CPD lesions in UV-damaged chromatin is mediated by UV-damaged DNAbinding protein (UV-DDB), composed of the tightly associated heterodimer of damage-specific DNA binding protein (DDB) 1 (p127) and DDB2 (p48) (5,8). Following surveillance and CPD identification by UV-DDB, NER proceeds via lesion handover to XPC-hHR23B-centrin2 (XPC) followed by damage verification, helix opening and stabilizing of the repair intermediates, dual incision of the DNA in the context of the lesion, repair synthesis, and DNA ligation (7). In contrast to global genomic repair, transcription-coupled repair is initiated when CPD lesions in transcribed chromatin cause stalling of RNA polymerases (3). In mammalian NER, these two pathways converge after damage detection and are orchestrated by over 30 different gene products (9). Deficiencies in the molecular functions in seven of these NER proteins lead to various forms of the autosomal recessive disor...

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Section: Resultsmentioning

confidence: 99%

Single-molecule analysis reveals human UV-damaged DNA-binding protein (UV-DDB) dimerizes on DNA via multiple kinetic intermediates

Ghodke

Wang

Hsieh

et al. 2014

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

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“…DNA photodamage depends linearly on the power used to excite fluorescent intercalators in its DNA-bound state (Tycon et al 2012) and consequently affects the total time a single DNA molecule can be imaged without degradation. We characterized the DNA photodamage dependence on laser power at fixed SYTOX green and YOYO-1 concentrations (40 nM) by measuring the cumulative probability of DNA breakage and the number of DNA molecules broken (1) to experimental data.…”

Section: Light-induced Dna Cleavage By Sytox Greenmentioning

confidence: 99%

The fluorescence properties and binding mechanism of SYTOX green, a bright, low photo-damage DNA intercalating agent

2015

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DNA intercalators are widely used in cancer therapeutics, to probe protein-DNA interactions and to investigate the statistical-mechanical properties of DNA. Here, we employ single-molecule fluorescence microscopy, magnetic tweezers, and ensemble-binding assays to investigate the fluorescence properties and binding mechanism of SYTOX green, a DNA labeling dye previously used for staining dead cells and becoming of common use for single-molecule methodologies. Specifically, we show that SYTOX green presents several advantages with respect to other dyes: (1) binds DNA rapidly and with high affinity; (2) has a good signal-to-noise ratio even at low concentrations; (3) exhibits a low photobleaching rate; and (4) induces lower light-induced DNA degradation. Finally, we show that SYTOX green is a DNA intercalator that binds DNA cooperatively with a binding site of 3.5 bp, increasing the DNA length upon binding by 43%, while not affecting its mechanical properties.

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“…In particular, excitation of intercalating stain has been shown to induce DNA cleavage through freeradical formation (27). However, at the low intensity of 532-nm illumination required for excitation of the intercalating stain and photo-activation of Cy5 (0.49 W$cm À2 at 532 nm), no DNA degradation was detected for tens of minutes.…”

Section: Discussionmentioning

confidence: 99%

Single-Molecule Imaging at High Fluorophore Concentrations by Local Activation of Dye

Geertsema

Schulte

Spenkelink

et al. 2015

Biophysical Journal

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Single-molecule fluorescence microscopy is a powerful tool for observing biomolecular interactions with high spatial and temporal resolution. Detecting fluorescent signals from individual labeled proteins above high levels of background fluorescence remains challenging, however. For this reason, the concentrations of labeled proteins in in vitro assays are often kept low compared to their in vivo concentrations. Here, we present a new fluorescence imaging technique by which single fluorescent molecules can be observed in real time at high, physiologically relevant concentrations. The technique requires a protein and its macromolecular substrate to be labeled each with a different fluorophore. Making use of short-distance energy-transfer mechanisms, only the fluorescence from those proteins that bind to their substrate is activated. This approach is demonstrated by labeling a DNA substrate with an intercalating stain, exciting the stain, and using energy transfer from the stain to activate the fluorescence of only those labeled DNA-binding proteins bound to the DNA. Such an experimental design allowed us to observe the sequence-independent interaction of Cy5-labeled interferon-inducible protein 16 with DNA and the sliding via one-dimensional diffusion of Cy5-labeled adenovirus protease on DNA in the presence of a background of hundreds of nanomolar Cy5 fluorophore.

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Quantification of dye-mediated photodamage during single-molecule DNA imaging

Cited by 34 publications

References 24 publications

Single-molecule analysis reveals human UV-damaged DNA-binding protein (UV-DDB) dimerizes on DNA via multiple kinetic intermediates

Single-molecule analysis reveals human UV-damaged DNA-binding protein (UV-DDB) dimerizes on DNA via multiple kinetic intermediates

The fluorescence properties and binding mechanism of SYTOX green, a bright, low photo-damage DNA intercalating agent

Single-Molecule Imaging at High Fluorophore Concentrations by Local Activation of Dye

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