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

Beckwitt, Emily C.; Jang, Sun-Bok; Detweiler, Isadora Carnaval; Kuper, Jochen; Sauer, Florian; Simon, Nina; Bretzler, Johanna; Watkins, Simon C.; Carell, Thomas; Kisker, Caroline; Houten, Bennett Van

doi:10.1038/s41467-020-15168-1

Cited by 17 publications

(20 citation statements)

References 71 publications

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“…DNA bending as an energetic test for the presence of their target sites based on altered DNA flexibility is a commonly applied strategy by proteins (reviews: 42 , 43 ). It has been described in particular for DNA repair enzymes, whose target sites typically introduce distortion or destabilization of DNA ( 30 , 36 , 44 , 45 ). The importance of DNA bending in the context of recognition of CpG sites has also been demonstrated ( 46 ).…”

Section: Discussionmentioning

confidence: 99%

“…The volumes of the complexes were derived from the centers of the Gaussians. SFM volumes (V) can be translated into approximate molecular weights (MW) of protein complexes using our previously reported SFM volume calibration: MW = (V + 5.9)/1.2 ( 35 ), which can serve as a crude size estimate for DNA bound complexes as well ( 36 ). Lengths and heights of protein peaks at the 50% positions on the DNA substrate with volumes consistent with two DNMT3A/3L heterotetramers were measured manually with Image J ( https://imagej.nih.gov/ij/ ) and Image SXM software, respectively.…”

Section: Methodsmentioning

confidence: 99%

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Structural and biochemical insight into the mechanism of dual CpG site binding and methylation by the DNMT3A DNA methyltransferase

Emperle

Bangalore

Adam

et al. 2021

Nucleic Acids Research

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DNMT3A/3L heterotetramers contain two active centers binding CpG sites at 12 bp distance, however their interaction with DNA not containing this feature is unclear. Using randomized substrates, we observed preferential co-methylation of CpG sites with 6, 9 and 12 bp spacing by DNMT3A and DNMT3A/3L. Co-methylation was favored by AT bases between the 12 bp spaced CpG sites consistent with their increased bending flexibility. SFM analyses of DNMT3A/3L complexes bound to CpG sites with 12 bp spacing revealed either single heterotetramers inducing 40° DNA bending as observed in the X-ray structure, or two heterotetramers bound side-by-side to the DNA yielding 80° bending. SFM data of DNMT3A/3L bound to CpG sites spaced by 6 and 9 bp revealed binding of two heterotetramers and 100° DNA bending. Modeling showed that for 6 bp distance between CpG sites, two DNMT3A/3L heterotetramers could bind side-by-side on the DNA similarly as for 12 bp distance, but with each CpG bound by a different heterotetramer. For 9 bp spacing our model invokes a tetramer swap of the bound DNA. These additional DNA interaction modes explain how DNMT3A and DNMT3A/3L overcome their structural preference for CpG sites with 12 bp spacing during the methylation of natural DNA.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Structural and biochemical insight into the mechanism of dual CpG site binding and methylation by the DNMT3A DNA methyltransferase

Emperle

Bangalore

Adam

et al. 2021

Nucleic Acids Research

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“…Therefore, this represents a fast and easy method to examine datasets and extract sub-kymographic features. Based on the sliding window analysis, we also extracted pause lifetimes using a method previously applied to DNA repair proteins (Nelson et al , 2019; Beckwitt et al , 2020). Much like BER proteins we observed multiple components in the paused population.…”

Section: Discussionmentioning

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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“…Samples were incubated at room temperature for 25 min and then diluted by 1:25 in AFM deposition buffer of 25 mM Hepes (pH 7.5), 25 mM NaOAc and 10 mM Mg(OAc) 2 . Samples were then transferred to freshly cleaved mica, rinsed with filtered water, dried with nitrogen gas and imaged on a MultiModeV microscope (Bruker Corporation) in ScanAsyst PeakForce Tapping (Bruker) mode in air, at a scan rate of 0.977 Hz ( 38 ). Probes had a triangular tip with a nominal radius of 2 nm on a silicon nitride cantilever (SCANASYST-AIR, Bruker).…”

Section: Methodsmentioning

confidence: 99%

AP-endonuclease 1 sculpts DNA through an anchoring tyrosine residue on the DNA intercalating loop

Hoitsma

Whitaker

Beckwitt

et al. 2020

Nucleic Acids Research

Self Cite

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Base excision repair (BER) maintains genomic stability through the repair of DNA damage. Within BER, AP-endonuclease 1 (APE1) is a multifunctional enzyme that processes DNA intermediates through its backbone cleavage activity. To accomplish these repair activities, APE1 must recognize and accommodate several diverse DNA substrates. This is hypothesized to occur through a DNA sculpting mechanism where structural adjustments of the DNA substrate are imposed by the protein; however, how APE1 uniquely sculpts each substrate within a single rigid active site remains unclear. Here, we utilize structural and biochemical approaches to probe the DNA sculpting mechanism of APE1, specifically by characterizing a protein loop that intercalates the minor groove of the DNA (termed the intercalating loop). Pre-steady-state kinetics reveal a tyrosine residue within the intercalating loop (Y269) that is critical for AP-endonuclease activity. Using X-ray crystallography and molecular dynamics simulations, we determined the Y269 residue acts to anchor the intercalating loop on abasic DNA. Atomic force microscopy reveals the Y269 residue is required for proper DNA bending by APE1, providing evidence for the importance of this mechanism. We conclude that this previously unappreciated tyrosine residue is key to anchoring the intercalating loop and stabilizing the DNA in the APE1 active site.

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

Cited by 17 publications

References 71 publications

Structural and biochemical insight into the mechanism of dual CpG site binding and methylation by the DNMT3A DNA methyltransferase

Structural and biochemical insight into the mechanism of dual CpG site binding and methylation by the DNMT3A DNA methyltransferase

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

AP-endonuclease 1 sculpts DNA through an anchoring tyrosine residue on the DNA intercalating loop

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