2020
DOI: 10.1038/s41467-020-15168-1
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Single molecule analysis reveals monomeric XPA bends DNA and undergoes episodic linear diffusion during damage search

Abstract: 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 non… Show more

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Cited by 18 publications
(20 citation statements)
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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%
See 1 more Smart Citation
“…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%
“…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%
“…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%