DNA–Protein Interactions Studied Directly Using Single Molecule Fluorescence Imaging of Quantum Dot Tagged Proteins Moving on DNA Tightropes

Springall, Luke; Inchingolo, Alessio V.; Kad, Neil M.

doi:10.1007/978-1-4939-3631-1_11

Cited by 11 publications

(17 citation statements)

References 6 publications

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“…To investigate this, we used a single molecule DNA tightrope assay (23) (Figure 2). Conjugation of a fluorescent quantum dot (QDot) to the poly-histidine purification tag on the p44/p62 complex (24) was achieved using an anti-His IgG antibody. Substantial binding of p44/p62 to dsDNA was observed, and of these approximately 80% could diffuse (n = 599 total) providing the first direct evidence that these factors are able to bind DNA independently of XPD.…”

Section: The P44/p62 Complex Directly Binds Dnamentioning

confidence: 99%

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The TFIIH subunits p44/p62 act as a damage sensor during nucleotide excision repair

Barnett

Kuper

Koelmel

et al. 2019

Preprint

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11Nucleotide excision repair (NER) protects the genome following exposure to 12 diverse types of DNA damage, including UV light and chemotherapeutics. 13 Mutations in human NER genes lead to diseases such as xeroderma 14 pigmentosum and Cockayne syndrome 1 . In eukaryotes, the major transcription 15 factor TFIIH is the central hub of NER. The core components of TFIIH include 16 the helicases XPB, XPD, and the five core 'structural' subunits 2-6 . Two of these 17 core-TFIIH proteins, p44 and p62 remain relatively unstudied; although p44 is 18 known to regulate the helicase activity of XPD during NER 7-9 . p62's role is 19 thought to be structural 10 ; however, a recent cryo-EM structure 11 shows p44, 20 p62, and XPD making contacts with each other, implying a more extensive role 21 in DNA repair beyond the structural integrity of TFIIH. Here, we show that p44 22 stimulates XPD's ATPase, but upon encountering DNA damage further 23 stimulation is only observed when p62 is in the ternary complex. More 24 significantly, we show that the p44/p62 complex binds DNA independently of 25 XPD and diffuses along its backbone, indicating a novel DNA-binding entity in 26 TFIIH. These data support a role for p44/p62 in TFIIH's mechanism of damage 27 detection. This revises our understanding of TFIIH and prompts more extensive 28 investigation of all of the core subunits, for an active role during both DNA 29 repair and transcription. 30 2 Results and Discussion 31 32 XPD's ATPase is stimulated by p44/p62 33 p44 contacts both p62 and XPD in TFIIH 3,11,12 , and mutations in XPD that 34 disrupt p44 or p62 binding cause defects in NER and result in disease 3,7,8,12 . To 35 investigate if p44/p62 was able to stimulate the ATPase of XPD, the turnover of 36 ATP in the presence of different DNA substrates was measured using an 37 NADH-coupled assay. 38 In the absence of p44 and p62, XPD's ATPase activity is slow even in the 39 presence of single-stranded DNA (0.043 s -1 ). However, with a p44 fragment 40 (residues 1-285 (N-p44)) containing the von Willebrand domain, XPD's 41 ATPase was significantly stimulated in the presence of both double-and single-42 stranded DNA 8 (~0.03 s -1 to 0.136 s -1 and 0.504 s -1 respectively p < 0.05 43 (Figure 1)). No further acceleration of the ATPase was observed with full 44 length p44 co-expressed in complex with p62 (p44/p62). However, remarkably, 45 when damage (a fluorescein moiety shown to proxy for damage 13 ) was 46 introduced into a dsDNA substrate, p44/p62 accelerated XPD's ATPase two-47 101 p44/p62 complex conjugated to a QDot, the diffusion constant appears limited 102 by rotation-coupled diffusion around the backbone of the DNA helix 20 . This is 103 consistent with the inability for complexes to pass one another on the DNA.104 105In summary, we present the first mechanistic characterisation of the non-106 helicase TFIIH subunits p44/p62. Complexes formed by these two proteins 107 were observed to bind and slide on dsDNA. Our bulk phase ATPase and 108 helicase data indicate that p44/p62 ...

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Section: The P44/p62 Complex Directly Binds Dnamentioning

confidence: 99%

“…For a detailed protocol see (24). p44/p62 interactions with DNA were studied in imaging buffer (20 mM Tris pH 8.0, 10 mM KCl (100 mM for high salt), 5 mM MgCl2, 1 mM TCEP).…”

Section: Single Molecule Dna Tightrope Assaymentioning

confidence: 99%

The TFIIH subunits p44/p62 act as a damage sensor during nucleotide excision repair

Barnett

Kuper

Koelmel

et al. 2019

Preprint

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“…This provides confirmatory evidence that cFos can bind DNA independently of AP-1 partners, indicating the existence of a new and potentially important member of the AP-1 transcription factor family. In addition, our approaches provide quantitative characterisation of the target search mechanisms employed by each protein combination (25,26). These physical properties affect their search capabilities, and we also suggest a mechanism for the differential affinities for target and nontarget sequences.…”

Section: Introductionmentioning

confidence: 90%

“…Flow cells and DNA tightropes were constructed as described previously (26). All experiments were performed in a buffer composed of 150 mM KCl, 50 mM Tris and 10 mM MgCl2 (pH 7.5), termed HSABC.…”

Section: Single Molecule Dna Tightrope Assaymentioning

confidence: 99%

Single molecule imaging reveals the collective and independent search mechanisms of cFos and cJun on DNA

Leech

Don

Mason

et al. 2020

Preprint

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AP-1 proteins are members of the basic leucine zipper (bZIP) protein family of dimeric transcription factors, responsible for controlling many integral cellular processes. These proteins form dimers with each other, and their aberrant expression can lead to a number of cancer types. The oncogenic transcription factor AP-1 binds its target TRE site (5’TCA[G/C]TGA), however the physical mechanism of how this is achieved is not understood. Such an understanding is essential to know how these proteins function, and could offer the potential to uncover new drug targets. The archetypal AP-1 complex is formed by cFos and cJun, which heterodimerise via their bZIP domains. Here, we set out to investigate how these proteins interact with DNA using a real-time single molecule fluorescence imaging approach. Using DNA tightropes as a substrate, we determine that the AP-1 bZIP dimers cJun:cFos and cJun:cJun rapidly scan DNA using a 1D diffusional search with an average diffusion constant of 0.14 µm2s-1 and 0.26 µm2s-1 respectively. Remarkably, we also found that cFos was able to bind to and diffuse on DNA (0.29 µm2s-1) both as a monomer and homodimer. Periods of diffusion were punctuated by pauses, suggesting a mechanism for how AP-1 may rapidly find its target sites on DNA. Taken together the results we have obtained indicate a considerably more complex and graded interaction between cFos, cJun and DNA than has been reported previously.

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“…RTFs were suspended between 5 µm silica beads (MicroSil™ microspheres, Bang Laboratories) coated in poly-L-lysine adhered to coverslip of a microfluidic flow cell (Figure 1a), as detailed in (19). These RTF tightropes were illuminated using a continuous wave variable power 20 mW 488 nm DPSS laser (JDSU), focused off-centre at the back-focal plane of a 100x objective (1.45 NA) to generate an obliquely angled field (20)(21)(22), custom-built into an Olympus IX50 microscope.…”

Section: Creating and Imaging Thin Filament Tightropesmentioning

confidence: 99%

Single molecule imaging reveals the concerted release of myosin from regulated thin filaments

Inchingolo¹,

Mihailescu²,

Dai³

et al. 2018

Preprint

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Regulated thin filaments (RTFs) tightly control striated muscle contraction through calcium binding to troponin, which in turn shifts the position of tropomyosin on actin to expose myosin binding sites. The binding of the first myosin holds tropomyosin in a position such that more myosin binding sites on actin are available, resulting in cooperative activation. Troponin and tropomyosin also act to turn off the thin filament; however, this is antagonized by the high local concentration of myosin, questioning how the thin filament relaxes. To provide molecular details of deactivation we use the RTF tightrope assay, in which single RTFs are suspended between pedestals above a microscope coverslip surface. Single molecule imaging of GFP tagged myosin-S1 (S1-GFP) is used to follow the activation of RTF tightropes. In sub-maximal activation conditions, S1-GFP molecules bind forming metastable clusters, from which release and rebinding of S1-GFP leads to prolonged activation in these regions. Because the RTFs are not fully active we are able to directly observe deactivation in real time. Using a Reversible Jump Markov Chain Monte Carlo model we are able to dynamically assess the fate of active regions. This analysis reveals that myosin binding occurs in a stochastic stepwise fashion; however, an unexpectedly large probability of multiple simultaneous detachments is observed. This suggests that deactivation of the thin filament is a coordinated, active process.

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DNA–Protein Interactions Studied Directly Using Single Molecule Fluorescence Imaging of Quantum Dot Tagged Proteins Moving on DNA Tightropes

Cited by 11 publications

References 6 publications

The TFIIH subunits p44/p62 act as a damage sensor during nucleotide excision repair

The TFIIH subunits p44/p62 act as a damage sensor during nucleotide excision repair

Single molecule imaging reveals the collective and independent search mechanisms of cFos and cJun on DNA

Single molecule imaging reveals the concerted release of myosin from regulated thin filaments

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