Biophysical characterization of DNA repair complex TFIIH

National Institute Of Diabetes And Digestive And Kidney Diseases

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Abstract

The protein factors involved in NER have been known for thirty years. Beyond the core factors XPA through XPG, which encompass fourteen active subunits, the three-subunit single-strand DNA binding protein RPA is also essential. These seventeen subunits can identify and verify DNA lesions — not mismatched base pairs nor oxidized or deaminated bases — and excise a 25-30 nt DNA segment containing the lesion. However, decades of intensive research have primarily characterized only individual components. We began working on NER in 2009, starting by over-expressing and purifying the TFIIH complex (composed of ten subunits) as well as its seven-subunit core (Core7). We characterized the ATPase and helicase activities (XPB and XPD) and examined lesion recognition activity of Core7 and TFIIH. We worked hard to obtain the purest and most active TFIIH, and our findings were not published in Molecular Cell until 2015. With the generational change of postdoctoral fellows and cryoEM revolution, we expanded our studies to include XPA, RPA, XPC, XPF and XPG. One of the most significant advances we achieved was the identification and construction of lesion-containing oligonucleotide DNA substrates. These substrates offer a more efficient alternative to the previously used laboriously constructed internally 32P-labeled lesion-containing plasmids. Additionally, we successfully reconstituted an efficient lesion-excision reaction using our purified NER proteins. Previously, NER analysis had been heavily dependent on cell nuclear extracts, supplemented with purified NER components and plasmid DNA, with only a small percentage of substrate being converted to product. Once we obtained the highly efficient NER system, composed of 17 purified protein subunits, we were able to capture a series of NER structures at resolution of 3.2 - 3.8 Å. These structures represent sequential steps of the NER process: (a) initial lesion recognition by XPC and TFIIH, (b) recruitment of XPA and realignment of the DNA lesion relative to the XPB and XPD helicases, (c) recruitment of XPF and RPA in the presence of ATP, leading to the formation of a 16-nt bubble, (d) arrival of XPG and displacement of lesion-bound XPC, and (e) full displacement of XPC and engagement of XPA, XPB, XPD, XPF, XPG, RPA and five supporting subunits of TFIIH for the DNA bubble enlargement. Our detailed structural and biochemical data explain the function of each component and their inter-dependence in the NER process and also reveal many protein interfaces for intervention to enhance the efficacy of chemotherapy. The dramatic conformational changes in these NER steps (the last three currently have been written up for publication) are truly breathtaking. The latest NER findings are written in a manuscript. The functional interpretations of our structures are supported by existing mutations found in Xeroderma Pigmentosum (XP) patients or cell-based mutagenic and functional analyses. The manuscript is under review currently.