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Light-induced modulation of DNA recognition by the Rad4/XPC damage sensor protein.

ABSTRACT: Biomolecular structural changes upon binding/unbinding are key to their functions. However, characterization of such dynamical processes is difficult as it requires ways to rapidly and specifically trigger the assembly/disassembly as well as ways to monitor the resulting changes over time. Recently, various chemical strategies have been developed to use light to trigger changes in oligonucleotide structures, and thereby their activities. Here we report that photocleavable DNA can be used to modulate the DNA binding of the Rad4/XPC DNA repair complex using light. Rad4/XPC specifically recognizes diverse helix-destabilizing/distorting lesions including bulky organic adduct lesions and functions as a key initiator for the eukaryotic nucleotide excision repair (NER) pathway. We show that the 6-nitropiperonyloxymethyl (NPOM)-modified DNA is recognized by the Rad4 protein as a specific substrate and that the specific binding can be abolished by light-induced cleavage of the NPOM group from DNA in a dose-dependent manner. Fluorescence lifetime-based analyses of the DNA conformations suggest that free NPOM-DNA retains B-DNA-like conformations despite its bulky NPOM adduct, but Rad4-binding causes it to be heterogeneously distorted. Subsequent extensive conformational searches and molecular dynamics simulations demonstrate that NPOM in DNA can be housed in the major groove of the DNA, with stacking interactions among the nucleotide pairs remaining largely unperturbed and thus retaining overall B-DNA conformation. Our work suggests that photoactivable DNA may be used as a DNA lesion surrogate to study DNA repair mechanisms such as nucleotide excision repair.

SUBMITTER: Tavakoli A

PROVIDER: S-EPMC8142930 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Kinetic gating mechanism of DNA damage recognition by Rad4/XPC.

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Sequence specificity, energetics and mechanism of mismatch recognition by DNA damage sensing protein Rad4/XPC.

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Modulation of DNA damage/DNA repair capacity by XPC polymorphisms.

Project description:XPC, a key protein in the nucleotide excision repair (NER) pathway, recognizes damaged DNA and initiates NER. Genetic variations in the XPC gene might be associated with altered DNA repair capacities (DRC). In this study, we genotyped three XPC polymorphisms, Ala499Val (C-->T), PAT (-/+) and Lys939Gln (A-->C), and measured the DNA damage/DRC by alkaline comet assay challenged by BPDE and gamma-radiation in 476 healthy subjects. We also evaluated the associations between DNA damage/DRC and genotypes of XPC polymorphisms. Compared with the XPC Lys939Gln homozygous wild type (AA) subjects, subjects with the variant alleles (AC and CC) had significantly higher DNA damages induced by BPDE (Median and 95% confidence interval [CI]: 3.16 (3.01-3.44) vs. 2.88 (2.51-3.05), P=0.01), and gamma-radiation (4.18 (3.94-4.44) vs. 3.71 (3.49-4.04), P=0.01). However, subjects with the variant alleles (CT and TT) of Ala499Val exhibited a 8.6% and 13.1% decrease in DNA damages induced by BPDE (P=0.05) and gamma-radiation (P=0.001), respectively. Significant correlations were found between genotypes and induced DNA damages in XPC Lys939Gln (For BPDE: R=0.12, P=0.01; for gamma-radiation: R=0.094, P=0.046) and Ala499Val (For BPDE: R=-0.11, P=0.03; for gamma-radiation: R=-0.16, P=0.0009). The haplotypes "T-A" (in the order of Ala499Val-PAT-Lys939Gln) was associated with the lowest DNA damages. Our results suggested that the DRC of host cells might be modulated by specific XPC polymorphisms.

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Recognition of DNA damage by XPC coincides with disruption of the XPC-RAD23 complex.

Project description:The recognition of helix-distorting deoxyribonucleic acid (DNA) lesions by the global genome nucleotide excision repair subpathway is performed by the XPC-RAD23-CEN2 complex. Although it has been established that Rad23 homologs are essential to protect XPC from proteasomal degradation, it is unclear whether RAD23 proteins have a direct role in the recognition of DNA damage. In this paper, we show that the association of XPC with ultraviolet-induced lesions was impaired in the absence of RAD23 proteins. Furthermore, we show that RAD23 proteins rapidly dissociated from XPC upon binding to damaged DNA. Our data suggest that RAD23 proteins facilitate lesion recognition by XPC but do not participate in the downstream DNA repair process.

| S-EPMC3308700 | biostudies-literature

Single-Molecule Imaging Reveals that Rad4 Employs a Dynamic DNA Damage Recognition Process.

Project description:Nucleotide excision repair (NER) is an evolutionarily conserved mechanism that processes helix-destabilizing and/or -distorting DNA lesions, such as UV-induced photoproducts. Here, we investigate the dynamic protein-DNA interactions during the damage recognition step using single-molecule fluorescence microscopy. Quantum dot-labeled Rad4-Rad23 (yeast XPC-RAD23B ortholog) forms non-motile complexes or conducts a one-dimensional search via either random diffusion or constrained motion. Atomic force microcopy analysis of Rad4 with the β-hairpin domain 3 (BHD3) deleted reveals that this motif is non-essential for damage-specific binding and DNA bending. Furthermore, we find that deletion of seven residues in the tip of β-hairpin in BHD3 increases Rad4-Rad23 constrained motion at the expense of stable binding at sites of DNA lesions, without diminishing cellular UV resistance or photoproduct repair in vivo. These results suggest a distinct intermediate in the damage recognition process during NER, allowing dynamic DNA damage detection at a distance.

| S-EPMC5123691 | biostudies-literature

Impact of DNA sequences on DNA 'opening' by the Rad4/XPC nucleotide excision repair complex.

Project description:Rad4/XPC recognizes diverse DNA lesions to initiate nucleotide excision repair (NER). However, NER propensities among lesions vary widely and repair-resistant lesions are persistent and thus highly mutagenic. Rad4 recognizes repair-proficient lesions by unwinding ('opening') the damaged DNA site. Such 'opening' is also observed on a normal DNA sequence containing consecutive C/G's (CCC/GGG) when tethered to Rad4 to prevent protein diffusion. However, it was unknown if such tethering-facilitated DNA 'opening' could occur on any DNA or if certain structures/sequences would resist being 'opened'. Here, we report that DNA containing alternating C/G's (CGC/GCG) failed to be opened even when tethered; instead, Rad4 bound in a 180°-reversed manner, capping the DNA end. Fluorescence lifetime studies of DNA conformations in solution showed that CCC/GGG exhibits local pre-melting that is absent in CGC/GCG. In MD simulations, CGC/GCG failed to engage Rad4 to promote 'opening' contrary to CCC/GGG. Altogether, our study illustrates how local sequences can impact DNA recognition by Rad4/XPC and how certain DNA sites resist being 'opened' even with Rad4 held at that site indefinitely. The contrast between CCC/GGG and CGC/GCG sequences in Rad4-DNA recognition may help decipher a lesion's mutagenicity in various genomic sequence contexts to explain lesion-determined mutational hot and cold spots.

| S-EPMC8934541 | biostudies-literature

Structure and mechanism of pyrimidine-pyrimidone (6-4) photoproduct recognition by the Rad4/XPC nucleotide excision repair complex.

Project description:Failure in repairing ultraviolet radiation-induced DNA damage can lead to mutations and cancer. Among UV-lesions, the pyrimidine-pyrimidone (6-4) photoproduct (6-4PP) is removed from the genome much faster than the cyclobutane pyrimidine dimer (CPD), owing to the more efficient recognition of 6-4PP by XPC-RAD23B, a key initiator of global-genome nucleotide excision repair (NER). Here, we report a crystal structure of a Rad4-Rad23 (yeast XPC-Rad23B ortholog) bound to 6-4PP-containing DNA and 4-μs molecular dynamics (MD) simulations examining the initial binding of Rad4 to 6-4PP or CPD. This first structure of Rad4/XPC bound to a physiological substrate with matched DNA sequence shows that Rad4 flips out both 6-4PP-containing nucleotide pairs, forming an 'open' conformation. The MD trajectories detail how Rad4/XPC initiates 'opening' 6-4PP: Rad4 initially engages BHD2 to bend/untwist DNA from the minor groove, leading to unstacking and extrusion of the 6-4PP:AA nucleotide pairs towards the major groove. The 5' partner adenine first flips out and is captured by a BHD2/3 groove, while the 3' adenine extrudes episodically, facilitating ensuing insertion of the BHD3 β-hairpin to open DNA as in the crystal structure. However, CPD resists such Rad4-induced structural distortions. Untwisting/bending from the minor groove may be a common way to interrogate DNA in NER.

| S-EPMC6614856 | biostudies-literature

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