More Like this

1. Tethering-facilitated DNA ‘opening’ and complementary roles of β-hairpin motifs in the Rad4/XPC DNA damage sensor protein

   https://doi.org/10.1093/nar/gkaa909  

   Paul, Debamita ; Mu, Hong ; Tavakoli, Amirrasoul ; Dai, Qing ; Chen, Xuejing ; Chakraborty, Sagnik ; He, Chuan ; Ansari, Anjum ; Broyde, Suse ; Min, Jung-Hyun ( October 2020 , Nucleic Acids Research)

   null (Ed.)

   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 cells. 

   more » « less
2. Quantifying site-specific chromatin mechanics and DNA damage response

   https://doi.org/10.1038/s41598-018-36343-x  

   Whitefield, Daniel B. ; Spagnol, Stephen T. ; Armiger, Travis J. ; Lan, Li ; Dahl, Kris Noel ( December 2018 , Scientific Reports)

   DNA double-strand breaks pose a direct threat to genomic stability. Studies of DNA damage and chromatin dynamics have yielded opposing results that support either increased or decreased chromatin motion after damage. In this study, we independently measure the dynamics of transcriptionally active or repressed chromatin regions using particle tracking microrheology. We find that the baseline motion of transcriptionally repressed regions of chromatin are significantly less mobile than transcriptionally active chromatin, which is statistically similar to the bulk motion of chromatin within the nucleus. Site specific DNA damage using KillerRed tags induced in loci within repressed chromatin causes an increased motion, while loci within transcriptionally active regions remains unchanged at similar time scales. We also observe a time-dependent response associated with a further increase in chromatin decondensation. Global induction of damage with bleocin displays similar trends of chromatin decondensation and increased mobility only at 53BP1-labeled damage sites but not at non-damaged sites, indicating that chromatin dynamics are tightly regulated locally after damage. These results shed light on the evolution of the local and global DNA damage response associated with chromatin remodeling and dynamics, with direct implications for their role in repair.

    

   more » « less
3. Cryo-EM structure of TFIIH/Rad4–Rad23–Rad33 in damaged DNA opening in nucleotide excision repair

   https://doi.org/10.1038/s41467-021-23684-x  

   van Eeuwen, Trevor ; Shim, Yoonjung ; Kim, Hee Jong ; Zhao, Tingting ; Basu, Shrabani ; Garcia, Benjamin A. ; Kaplan, Craig D. ; Min, Jung-Hyun ; Murakami, Kenji ( June 2021 , Nature Communications)

   The versatile nucleotide excision repair (NER) pathway initiates as the XPC–RAD23B–CETN2 complex first recognizes DNA lesions from the genomic DNA and recruits the general transcription factor complex, TFIIH, for subsequent lesion verification. Here, we present a cryo-EM structure of an NER initiation complex containing Rad4–Rad23-Rad33 (yeast homologue of XPC–RAD23B–CETN2) and 7-subunit coreTFIIH assembled on a carcinogen-DNA adduct lesion at 3.9–9.2 Å resolution. A ~30-bp DNA duplex could be mapped as it straddles between Rad4 and the Ssl2 (XPB) subunit of TFIIH on the 3' and 5' side of the lesion, respectively. The simultaneous binding with Rad4 and TFIIH was permitted by an unwinding of DNA at the lesion. Translocation coupled with torque generation by Ssl2 and Rad4 would extend the DNA unwinding at the lesion and deliver the damaged strand to Rad3 (XPD) in an open form suitable for subsequent lesion scanning and verification.

    

   more » « less
4. The Human DNA Mismatch Repair Protein MSH3 Contains Nuclear Localization and Export Signals That Enable Nuclear-Cytosolic Shuttling in Response to Inflammation

   https://doi.org/10.1128/MCB.00029-20  

   Tseng-Rogenski, Stephanie S. ; Munakata, Koji ; Choi, Daniel Y. ; Martin, Paul K. ; Mehta, Supal ; Koi, Minoru ; Zheng, Wei ; Zhang, Yang ; Carethers, John M. ( June 2020 , Molecular and Cellular Biology)

   null (Ed.)

   ABSTRACT Inactivation of DNA mismatch repair propels colorectal cancer (CRC) tumorigenesis. CRCs exhibiting e levated m icrosatellite a lterations at s elected t etranucleotide repeats (EMAST) show reduced nuclear MutS homolog 3 (MSH3) expression with surrounding inflammation and portend poor patient outcomes. MSH3 reversibly exits from the nucleus to the cytosol in response to the proinflammatory cytokine interleukin-6 (IL-6), suggesting that MSH3 may be a shuttling protein. In this study, we manipulated three putative nuclear localization (NLS1 to -3) and two potential nuclear export signals (NES1 and -2) within MSH3. We found that both NLS1 and NLS2 possess nuclear import function, with NLS1 responsible for nuclear localization within full-length MSH3. We also found that NES1 and NES2 work synergistically to maximize nuclear export, with both being required for IL-6-induced MSH3 export. We examined a 27-bp deletion (Δ27bp) within the polymorphic exon 1 that occurs frequently in human CRC cells and neighbors NLS1. With oxidative stress, MSH3 with this deletion (Δ27bp MSH3) localizes to the cytoplasm, suggesting that NLS1 function in Δ27bp MSH3 is compromised. Overall, MSH3’s shuttling in response to inflammation enables accumulation in the cytoplasm; reduced nuclear MSH3 increases EMAST and DNA damage. We suggest that polymorphic sequences adjacent to NLS1 may enhance cytosolic retention, which has clinical implications for inflammation-associated neoplastic processes. 

   more » « less
5. Light-induced modulation of DNA recognition by the Rad4/XPC damage sensor protein

   https://doi.org/10.1039/d0cb00192a  

   Tavakoli, Amirrasoul ; Paul, Debamita ; Mu, Hong ; Kuchlyan, Jagannath ; Baral, Saroj ; Ansari, Anjum ; Broyde, Suse ; Min, Jung-Hyun ( April 2021 , RSC Chemical Biology)

   null (Ed.)

   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. 

   more » « less