skip to main content
Explore Scholarly Publications and Datasets in the NSF-PAR

  • NSF Public Access
  • Search Results
  • Accepted Manuscript: Tethering-facilitated DNA ‘opening’ and complementary roles of β-hairpin motifs in the Rad4/XPC DNA damage sensor protein
Title: Tethering-facilitated DNA ‘opening’ and complementary roles of β-hairpin motifs in the Rad4/XPC DNA damage sensor protein
Abstract XPC/Rad4 initiates eukaryotic nucleotide excision repair on structurally diverse helix-destabilizing/distorting DNA lesions by selectively ‘opening’ these sites while rapidly diffusing along undamaged DNA. Previous structural studies showed that Rad4, when tethered to DNA, could also open undamaged DNA, suggesting a ‘kinetic gating’ mechanism whereby lesion discrimination relied on efficient opening versus diffusion. However, solution studies in support of such a mechanism were lacking and how ‘opening’ is brought about remained unclear. Here, we present crystal structures and fluorescence-based conformational analyses on tethered complexes, showing that Rad4 can indeed ‘open’ undamaged DNA in solution and that such ‘opening’ can largely occur without one or the other of the β-hairpin motifs in the BHD2 or BHD3 domains. Notably, the Rad4-bound ‘open’ DNA adopts multiple conformations in solution notwithstanding the DNA’s original structure or the β-hairpins. Molecular dynamics simulations reveal compensatory roles of the β-hairpins, which may render robustness in dealing with and opening diverse lesions. Our study showcases how fluorescence-based studies can be used to obtain information complementary to ensemble structural studies. The tethering-facilitated DNA ‘opening’ of undamaged sites and the dynamic nature of ‘open’ DNA may shed light on how the protein functions within and beyond nucleotide excision repair in more » cells. « less
Authors:
; ; ; ; ; ; ; ; ;
Award ID(s):
1715649
Publication Date:
NSF-PAR ID:
10294029
Journal Name:
Nucleic Acids Research
Volume:
48
Issue:
21
Page Range or eLocation-ID:
12348 to 12364
ISSN:
0305-1048
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; ; ; ; ; ( , RSC Chemical Biology)
    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 themore »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.« less
  2. ; ( , Journal of Biological Chemistry)
    The adenine, cytosine, and guanine bases of DNA are susceptible to alkylation by the aldehyde products of lipid peroxidation and by the metabolic byproducts of vinyl chloride pollutants. The resulting adducts spontaneously cyclize to form harmful etheno lesions. Cells employ a variety of DNA repair pathways to protect themselves from these pro-mutagenic modifications. Human alkyladenine DNA glycosylase (AAG) is thought to initiate base excision repair of both 1, N 6 -ethenoadenine (ϵA) and 1, N 2 -ethenoguanine (ϵG). However, it is not clear how AAG might accommodate ϵG in an active site that is complementary to ϵA. This prompted a thorough investigation of AAG-catalyzed excision of ϵG from several relevant contexts. Using single-turnover and multiple-turnover kinetic analyses, we found that ϵG in its natural ϵG·C context is very poorly recognized relative to ϵA·T. Bulged and mispaired ϵG contexts, which can form during DNA replication, were similarly poor substrates for AAG. Furthermore, AAG could not recognize an ϵG site in competition with excess undamaged DNA sites. Guided by previous structural studies, we hypothesized that Asn-169, a conserved residue in the AAG active-site pocket, contributes to discrimination against ϵG. Consistent with this model, the N169S variant of AAG was 7-fold more activemore »for excision of ϵG compared with the wildtype (WT) enzyme. Taken together, these findings suggest that ϵG is not a primary substrate of AAG, and that current models for etheno lesion repair in humans should be revised. We propose that other repair and tolerance mechanisms operate in the case of ϵG lesions.« less
  3. ; ; ; ; ( , Frontiers in Molecular Biosciences)
    DNA polymerase β (pol β ) is a member of the X- family of DNA polymerases that catalyze the distributive addition of nucleoside triphosphates during base excision DNA repair. Previous studies showed that the enzyme was phosphorylated in vitro with PKC at two serines (44 and 55), causing loss of DNA polymerase activity but not DNA binding. In this work, we have investigated the phosphorylation-induced conformational changes in DNA polymerase β in the presence of Mg ions. We report a comprehensive atomic resolution study of wild type and phosphorylated DNA polymerase using molecular dynamics (MD) simulations. The results are examined via novel methods of internal dynamics and energetics analysis to reveal the underlying mechanism of conformational transitions observed in DNA pol β . The results show drastic conformational changes in the structure of DNA polymerase β due to S44 phosphorylation. Phosphorylation-induced conformational changes transform the enzyme from a closed to an open structure. The dynamic cross-correlation shows that phosphorylation enhances the correlated motions between the different domains. Centrality network analysis reveals that the S44 phosphorylation causes structural rearrangements and modulates the information pathway between the Lyase domain and base pair binding domain. Further analysis of our simulations reveals that amore »critical hydrogen bond (between S44 and E335) disruption and the formation of three additional salt bridges are potential drivers of these conformational changes. In addition, we found that two of these additional salt bridges form in the presence of Mg ions on the active sites of the enzyme. These results agree with our previous study of DNA pol β S44 phosphorylation without Mg ions which predicted the deactivation of DNA pol β . However, the phase space of structural transitions induced by S44 phosphorylation is much richer in the presence of Mg ions.« less
  4. ; ( , Frontiers in Molecular Biosciences)
    High fidelity (HiFi) DNA polymerases (Pols) perform the bulk of DNA synthesis required to duplicate genomes in all forms of life. Their structural features, enzymatic mechanisms, and inherent properties are well-described over several decades of research. HiFi Pols are so accurate that they become stalled at sites of DNA damage or lesions that are not one of the four canonical DNA bases. Once stalled, the replisome becomes compromised and vulnerable to further DNA damage. One mechanism to relieve stalling is to recruit a translesion synthesis (TLS) Pol to rapidly synthesize over and past the damage. These TLS Pols have good specificities for the lesion but are less accurate when synthesizing opposite undamaged DNA, and so, mechanisms are needed to limit TLS Pol synthesis and recruit back a HiFi Pol to reestablish the replisome. The overall TLS process can be complicated with several cellular Pols, multifaceted protein contacts, and variable nucleotide incorporation kinetics all contributing to several discrete substitution (or template hand-off) steps. In this review, we highlight the mechanistic differences between distributive equilibrium exchange events and concerted contact-dependent switching by DNA Pols for insertion, extension, and resumption of high-fidelity synthesis beyond the lesion.
  5. ; ; ( , Plants)
    Substitution rates in plant mitochondrial genes are extremely low, indicating strong selective pressure as well as efficient repair. Plant mitochondria possess base excision repair pathways; however, many repair pathways such as nucleotide excision repair and mismatch repair appear to be absent. In the absence of these pathways, many DNA lesions must be repaired by a different mechanism. To test the hypothesis that double-strand break repair (DSBR) is that mechanism, we maintained independent self-crossing lineages of plants deficient in uracil-N-glycosylase (UNG) for 11 generations to determine the repair outcomes when that pathway is missing. Surprisingly, no single nucleotide polymorphisms (SNPs) were fixed in any line in generation 11. The pattern of heteroplasmic SNPs was also unaltered through 11 generations. When the rate of cytosine deamination was increased by mitochondrial expression of the cytosine deaminase APOBEC3G, there was an increase in heteroplasmic SNPs but only in mature leaves. Clearly, DNA maintenance in reproductive meristem mitochondria is very effective in the absence of UNG while mitochondrial genomes in differentiated tissue are maintained through a different mechanism or not at all. Several genes involved in DSBR are upregulated in the absence of UNG, indicating that double-strand break repair is a general system of repairmore »in plant mitochondria. It is important to note that the developmental stage of tissues is critically important for these types of experiments.« less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Save / Share this Record
  • Send to Email