Studying protein-DNA interactions using atomic force microscopy

Beckwitt, Emily C.; Kong, Muwen; Houten, Bennett Van

doi:10.1016/j.semcdb.2017.06.028

Cited by 54 publications

(33 citation statements)

References 146 publications

(154 reference statements)

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“…Furthermore, at higher XPA concentrations, a second band of higher molecular weight appeared, indicating binding of an additional XPA protein. It is important to note that any affinity of XPA for DNA ends could obscure EMSA results in terms of (a) specificity, as end-binding (a common feature of DNA-binding proteins 38,39 ) would increase overall binding on both substrates, thereby lowering the apparent specificity for the lesion, and (b) stoichiometry, as separate XPA proteins bound to the lesion and the end of the DNA would migrate the same as a true dimer in the gel. If XPA has fairly high non-specific binding affinity, then embedding a lesion in a long stretch of non-damaged DNA might pose difficulties in damage recognition.…”

Section: Resultsmentioning

confidence: 99%

Single molecule analysis reveals monomeric XPA bends DNA and undergoes episodic linear diffusion during damage search

et al. 2020

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Nucleotide excision repair (NER) removes a wide range of DNA lesions, including UVinduced photoproducts and bulky base adducts. XPA is an essential protein in eukaryotic NER, although reports about its stoichiometry and role in damage recognition are controversial. Here, by PeakForce Tapping atomic force microscopy, we show that human XPA binds and bends DNA by ∼60°as a monomer. Furthermore, we observe XPA specificity for the helix-distorting base adduct N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene over nondamaged dsDNA. Moreover, single molecule fluorescence microscopy reveals that DNAbound XPA exhibits multiple modes of linear diffusion between paused phases. The presence of DNA damage increases the frequency of pausing. Truncated XPA, lacking the intrinsically disordered N-and C-termini, loses specificity for DNA lesions and shows less pausing on damaged DNA. Our data are consistent with a working model in which monomeric XPA bends DNA, displays episodic phases of linear diffusion along DNA, and pauses in response to DNA damage.

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Section: Resultsmentioning

confidence: 99%

Single molecule analysis reveals monomeric XPA bends DNA and undergoes episodic linear diffusion during damage search

et al. 2020

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“…It allows for visualization of these molecules and its complexes deposited over a surface with nanometer resolution, preserving their native structure [44]. AFM has been utilized to characterize protein-protein and protein-DNA interactions and conformations; it also allows determining the specificity of proteins binding to specific sites on DNA [45,46]. Due to these features, AFM was used in this work to explore the changes occurring on the surface of the quartz crystal during DNA immobilization and hybridization processes.…”

Section: Real-time Quantitative Polymerase Chain Reactionmentioning

confidence: 99%

NLRP3, IL-1β, and Caspase-1 Gene Transcript Identification and Expression by QCM-D in Obese Children

et al. 2019

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Background. Obesity in children is highly prevalent in Mexican population. Adipose tissue has been related to specific pro- and anti-inflammatory cytokine and inflammasome gene and protein expression patterns. Actually, there is no existing biosensor for detecting gene expression patterns in children with obesity. The quartz crystal microbalance with dissipation monitoring (QCM-D) has been used as a transducer for DNA biosensor design. Results. In this study, the gene expression pattern of IL-1β, NLRP3, and CASPASE-1 in children with obesity was successfully determined by means of QCM-D. Gene expression patterns were validated with those obtained by quantitative polymerase chain reaction (qPCR), a validated molecular biology technique for gene expression quantification. QCM-D analysis of the detected mass corresponding results for each of the genes showed a major detected mass for IL-1β, followed by similar NLRP3 and constitutive gene 18S deposited mass and a smaller deposited mass for CASPASE-1. Surprisingly, when comparing mRNA gene expression results for NLRP3, IL-1β, and CASPASE-1 obtained with qPCR and QCM-D, similar patterns were found, revealing greatest expression of IL-1β, followed by NLRP3, with CASPASE-1 being the molecule of least expression in the group of children with obesity. AFM images illustrate the step-by-step changes that took place on the quartz surface. Conclusions. QCM-D proved successfully for determining the gene transcripts and expression of NLRP3, IL-1β, and CASPASE-1 in children with obesity, with similar results validated by qPCR. “QCM-D decreases detection costs compared with a validated molecular biology technique.” The QCM-D biosensor developed by our group was successful for gene expression determination; in the future, it can be used for molecular diagnosis.

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“…91,96 AFM studies with specific IHF-DNA complexes revealed single broad distributions peaked at bending angles of ~120-130°, with a range that covered bending angles from ~80° to ~160°. 27,33 These AFM studies were done with other IHF-DNA complexes; we are unaware of similar studies with IHF-H'. Notably, the range of bent conformations observed in AFM covers what we expect for the low-FRET state (with one site kinked) and for the high-FRET state (with both sites kinked).…”

Section: Discussionmentioning

confidence: 99%

“…and an LAS Award for Faculty in the Sciences from the University of Illinois at Chicago (to A.A.) This work was also supported by the Intramural Research Program of the National Institutes of Health (NIH, CIT). 33…”

Section: Acknowledgmentsmentioning

confidence: 99%

“…The lifetime approach has a few advantages over single-molecule assays of FRET distributions. 31 Measurements are done under solution conditions and thus avoid many of the issues that may come with immobilization of protein-DNA complexes as needed for AFM studies [32][33] or single-molecule FRET (smFRET) studies, [34][35][36][37] although smFRET on freely diffusing molecules do alleviate immobilization issues, as showcased in a series of elegant papers by Eaton and co-workers [38][39][40][41] on the dynamics and distributions during protein folding. More important, fluorescence decay curves measured in bulk solution provide significantly better resolution of the shape of the FRET efficiency distributions than can be obtained from the few-to-tens thousands molecules that are sampled in single-molecule studies.…”

Section: Introductionmentioning

confidence: 99%

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Static Kinks or Flexible Hinges: Conformational Distributions of Bent DNA Bound to Integration Host Factor Mapped by Fluorescence Lifetime Measurements

Connolly

Arra

Zvoda

et al. 2018

Preprint

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Gene regulation depends on proteins that bind to specific DNA sites. Such specific recognition often involves severe DNA deformations including sharp kinks. It has been unclear how rigid or flexible these protein-induced kinks are. Here, we investigated the dynamic nature of DNA in complex with integration host factor (IHF), a nucleoid-associated architectural protein known to bend one of its cognate sites (35 base pair H') into a U-turn by kinking DNA at two sites. We utilized fluorescence lifetime based FRET spectroscopy to map the distribution of bent conformations in various IHF-DNA complexes. Our results reveal a surprisingly dynamic specific complex: while 80% of the IHF-H' population exhibited FRET efficiency consistent with the crystal structure, 20% exhibited FRET efficiency indicative of unbent or partially bent DNA. This conformational flexibility is modulated by sequence variations in the cognate site. In another site (H1) that lacks an A-tract of H' on one side of the binding site, the population in the fully U-bent conformation decreased to 36%, as did the extent of bending. A similar decrease in the U-bent population was observed with a single base mutation in H' in a consensus region on the other side. Taken together, these results provide important insights into the finely tuned interactions between IHF and its cognate sites that keep the DNA bent (or not), and yield quantitative data on the dynamic equilibrium between different DNA conformations (kinked or not kinked) that depend sensitively on DNA sequence and deformability. Notably, the difference in dynamics between IHF-H' and IHF-H1 reflects the different roles of these complexes in their natural context, in the phage lambda "intasome" (the complex that integrates phage lambda into the E. coli chromosome).

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Studying protein-DNA interactions using atomic force microscopy

Cited by 54 publications

References 146 publications

Single molecule analysis reveals monomeric XPA bends DNA and undergoes episodic linear diffusion during damage search

Single molecule analysis reveals monomeric XPA bends DNA and undergoes episodic linear diffusion during damage search

NLRP3, IL-1β, and Caspase-1 Gene Transcript Identification and Expression by QCM-D in Obese Children

Static Kinks or Flexible Hinges: Conformational Distributions of Bent DNA Bound to Integration Host Factor Mapped by Fluorescence Lifetime Measurements

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