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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. 

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2. Evidence for intrinsic DNA dynamics and deformability in damage sensing by the Rad4/XPC nucleotide excision repair complex

   Baral, S. ; Chakraborty, S. ; Steinbach, P. J. ; Paul, D. ; Min, J.-H. ; Ansari, A. ( April 2024 , bioRxiv)

   Altered DNA dynamics at lesion sites are implicated in how DNA repair proteins pause and identify damage within genomic DNA. We examined DNA dynamics in the context of damage recognition by Rad4 (yeast ortholog of XPC), which recognizes diverse lesions from environmental mutagens and initiates nucleotide excision repair. Previous studies with a cytosine-analog FRET pair placed on either side of 3 base-pair (bp) mismatched sites – recognized specifically by Rad4 in vitro – unveiled severely deformed DNA even without Rad4 (Chakraborty et al. (2018) Nucleic Acid Res. 46: 1240-1255). Here, using laser T-jump, we revealed the timescales of these spontaneous deformations. 3-bp AT-rich nonspecific sites, whether matched or mismatched, exhibited conformational dynamics primarily within the T-jump observation window (~20 µs – <100 ms), albeit with some amplitude in unresolved (<20 µs) kinetics. The amplitudes of the “missing” fast kinetics increased dramatically for mismatched specific sites, which were further distinguished by additional “missing” amplitude in slow (>100 ms) kinetics at elevated temperatures. We posit that the rapid (µs-ms) fluctuations help stall a diffusing protein at AT-rich/damaged sites and that the >100-ms kinetics reflect a propensity for specific DNA to adopt unwound/bent conformations that may resemble Rad4-bound structures. These studies provide compelling evidence for unusual DNA dynamics and deformability that likely govern how Rad4 senses DNA damage. 

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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.

    

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4. A scanning-to-incision switch in TFIIH-XPG induced by DNA damage licenses nucleotide excision repair

   https://doi.org/10.1093/nar/gkac1095  

   Bralić, Amer ; Tehseen, Muhammad ; Sobhy, Mohamed A ; Tsai, Chi-Lin ; Alhudhali, Lubna ; Yi, Gang ; Yu, Jina ; Yan, Chunli ; Ivanov, Ivaylo ; Tsutakawa, Susan E ; et al ( December 2022 , Nucleic Acids Research)

   Abstract Nucleotide excision repair (NER) is critical for removing bulky DNA base lesions and avoiding diseases. NER couples lesion recognition by XPC to strand separation by XPB and XPD ATPases, followed by lesion excision by XPF and XPG nucleases. Here, we describe key regulatory mechanisms and roles of XPG for and beyond its cleavage activity. Strikingly, by combing single-molecule imaging and bulk cleavage assays, we found that XPG binding to the 7-subunit TFIIH core (coreTFIIH) stimulates coreTFIIH-dependent double-strand (ds)DNA unwinding 10-fold, and XPG-dependent DNA cleavage by up to 700-fold. Simultaneous monitoring of rates for coreTFIIH single-stranded (ss)DNA translocation and dsDNA unwinding showed XPG acts by switching ssDNA translocation to dsDNA unwinding as a likely committed step. Pertinent to the NER pathway regulation, XPG incision activity is suppressed during coreTFIIH translocation on DNA but is licensed when coreTFIIH stalls at the lesion or when ATP hydrolysis is blocked. Moreover, ≥15 nucleotides of 5′-ssDNA is a prerequisite for efficient translocation and incision. Our results unveil a paired coordination mechanism in which key lesion scanning and DNA incision steps are sequentially coordinated, and damaged patch removal is only licensed after generation of ≥15 nucleotides of 5′-ssDNA, ensuring the correct ssDNA bubble size before cleavage. 

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5. Mechanism of Rad26-assisted rescue of stalled RNA polymerase II in transcription-coupled repair

   https://doi.org/10.1038/s41467-021-27295-4  

   Yan, Chunli ; Dodd, Thomas ; Yu, Jina ; Leung, Bernice ; Xu, Jun ; Oh, Juntaek ; Wang, Dong ; Ivanov, Ivaylo ( December 2021 , Nature Communications)

   Transcription-coupled repair is essential for the removal of DNA lesions from the transcribed genome. The pathway is initiated by CSB protein binding to stalled RNA polymerase II. Mutations impairing CSB function cause severe genetic disease. Yet, the ATP-dependent mechanism by which CSB powers RNA polymerase to bypass certain lesions while triggering excision of others is incompletely understood. Here we build structural models of RNA polymerase II bound to the yeast CSB ortholog Rad26 in nucleotide-free and bound states. This enables simulations and graph-theoretical analyses to define partitioning of this complex into dynamic communities and delineate how its structural elements function together to remodel DNA. We identify an allosteric pathway coupling motions of the Rad26 ATPase modules to changes in RNA polymerase and DNA to unveil a structural mechanism for CSB-assisted progression past less bulky lesions. Our models allow functional interpretation of the effects of Cockayne syndrome disease mutations.

    

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