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Structural and Functional Characterization of the Archaeal DNA Repair Helicase XPB and its Interactions With the Endonuclease Bax1


The XPB helicase is a critical factor for transcription and nucleotide excision repair (NER). As the largest subunit of the TFIIH general transcription factor complex, it establishes multiple interactions with other proteins to maintain TFIIH assembly and promotes pre-incision complex (PIC) formation during NER. XPB utilizes ATP hydrolyzing and unidirectional 3’-5’ helicase activities in concert with the 5’-3’ helicase XPD subunit of TFIIH during NER to open the DNA helix around bulky lesions for subsequent removal by downstream NER factors. Structure solutions of XPB have suggested a unique “rotation and push” mechanism adopted by this unconventional Superfamily 2 (SF2) helicase to mediate DNA unwinding by ATP-driven conformational changes in its domain orientations from an inactive open to the active closed form. The objective of this dissertation is to obtain “molecular snapshots” of XPB domain rotation induced by ATP binding and hydrolysis through a multi-disciplinary approach including X-ray crystallography and small angle X-ray scattering (SAXS). Our SAXS data obtained for a homologous archaeal XPB protein from Archaeoglobus fulgidus (AfXPB) in the absence and presence of an ATP non-hydrolyzable analog confirmed the conversion from the open conformation observed previously in crystals to the closed conformation previously proposed for ATP-bound XPB helicase. Furthermore, the crystallographic results obtained for a homologous archaeal XPB protein from Sulfolobus tokodaii (StXPB) reveal intermediate stages of the XPB domain rotation from the open to closed form. In addition, the structural and functional investigation of the interactions of XPB with the archaeal Bax1 endonuclease revealed that the XPB C-terminal helicase domain 2 (HD2) and Thumb (ThM) domains exclusively interact with Bax1, potentially explaining the observed stimulatory effects on XPB DNA-dependent ATPase activity. Our investigation suggests the XPB N-terminal HD1 and damage recognition domain (DRD) disrupt Bax1 dimerization, likely acting as a control mechanism to switch Bax1 from a proposed homodimeric Holliday junction resolvase to heterodimeric NER endonuclease. Homology modeling with DNA suggests how the XPB:Bax1 heterodimer recognizes, unwinds, and cleaves kinked DNA in a concerted mechanism. The archaeal complex will provide a structural framework for investigating eukaryotic XPB’s interactions with various repair factors during NER.

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