UC Riverside

The superfamily II (SF2) DNA helicase XPB is an essential protein involved in twobiologically important processes: transcription and DNA repair. In eukaryotes, XPB is the largest subunit of the TFIIH complex and is essentially required in both transcription initiation and the nucleotide excision repair (NER) pathway. Previous structural and biochemical data of TFIIH all have showed that eukaryotic XPB binds and translocates on the double-stranded (ds) DNA for DNA unwinding, strikingly different from other conventional DNA helicases that requires a single-stranded (ss) DNA extension for strand separation. XPB was proposed to work as a “molecular wrench” or dsDNA translocase and use its ATPase activity for achieving its DNA opening function in transcription. However, it still remains elusive how this unconventional enzyme achieves DNA opening in the context of NER. XPB also plays a key role in coordinating the DNA incision by nucleases like XPF or XPG, and the underlying mechanisms are unclear. In archaea, there is no TFIIH-like complex. XPB is in complex with a nuclease called Bax1 for DNAunwinding and incision, but how this helicase-nuclease complex binds, opens and cleaves DNA is unknown. The objective of my research is to obtain structures and biochemical data of XPB with nuclease/DNA substrates for better understanding the DNA opening mechanisms of XPB and the crosstalk between XPB and nuclease for damage incision. A combination of biochemical, structural, and molecular biology techniques has been employed throughout to achieve these goals. Mutational and biochemical analyses based on crystal structures of archaeal XPB-Bax1 complexes from Sulfurisphaera tokodaii (St) and Archaeogloubus fulgidus (Af) demonstrates that this helicase-nuclease complex is a dynamic machinery and the activities of XPB are regulated by protein-protein interactions. Crystal structures of the StXPB-Bax1ΔC with and without DNA substrate reveal how the XPB-Bax1 complex interacts with the repair bubble DNA, and adopts different conformations in DNA-free and DNA-bound states. Biochemical and mutational data confirmed that the conserved RED and ThM motifs of XPB play key roles in DNA binding and XPB activities. My investigation uncovered the unconventional helicase activity of archaeal XPB using its ThM motif to unzip DNA while translocating in 3'-5' direction on the duplex. Biochemical data together with structure comparison of the StXPB-Bax1ΔC-DNA ternary complex with the cryo-EM structure of human TFIIH-XPA suggest human XPB cooperates with XPA for the initial DNA opening around the lesion. In sum, my findings provide new insights into the molecular mechanisms of XPB-mediated DNA binding and opening during the repair bubble formation, and how XPB and nucleases may coordinate DNA incision in archaeal and eukaryotic NER.