Lawrence Berkeley National Laboratory

Nucleotide excision repair (NER) is vital for genome integrity. Yet, our understanding of the complex NER protein machinery remains incomplete. Combining cryo-EM and XL-MS data with AlphaFold2 predictions, we build an integrative model of the NER pre-incision complex(PInC). Here TFIIH serves as a molecular ruler, defining the DNA bubble size and precisely positioning the XPG and XPF nucleases for incision. Using simulations and graph theoretical analyses, we unveil PInCs assembly, global motions, and partitioning into dynamic communities. Remarkably, XPG caps XPDs DNA-binding groove and bridges both junctions of the DNA bubble, suggesting a novel coordination mechanism of PInCs dual incision. XPA rigging interlaces XPF/ERCC1 with RPA, XPD, XPB, and 5 ssDNA, exposing XPAs crucial role in licensing the XPF/ERCC1 incision. Mapping disease mutations onto our models reveals clustering into distinct mechanistic classes, elucidating xeroderma pigmentosum and Cockayne syndrome disease etiology.