Xeroderma pigmentosum (XP) group B (XPB) protein is a DNA helicase required for both transcription and nucleotide excision repair (NER), a major DNA repair pathway that removes a variety of DNA lesions. Mutations in the XPB gene are associated with skin cancer predisposition, premature aging, and neurodegeneration. In order to solve two long-standing problems concerning NER: how XPB initiates dsDNA unwinding at the damage site and how the opening of damaged DNA is coordinated with the dual incision, we plan to achieve the following three specific aims:
Aim1 : To dissect the mechanism of DNA unwinding by XPB during NER. We will determine the crystal structures of human XPB in complex with dsDNA and DNA with a bubble containing a lesion such as (6-4) pyrimidine-pyrimidone dimer. The dynamic XPB-DNA interactions and functional conformations of XPB will be further analyzed by small angle X-ray scattering (SAXS) experiments. These structural models will be validated by selective mutagenesis for in vitro biochemical assays and cell-based imaging experiments to test the role of XPB during NER in living cells.
Aim2 : To illustrate the XPB-XPF interaction that regulates the 5'-incison by endonuclease ERCC1-XPF. XPB plays a role in regulating the dual incision since phosphorylation at residue Ser751 of XPB inhibits the 5'- incison of the damaged DNA by ERCC1-XPF. We have purified a complex of human XPB C-terminal half with the N-terminal half of XPF for structural and functional analyses. A novel hypothesis how phosphorylation at Ser751 of XPB inhibits 5'-incision by XPF will be tested by selective mutagenesis for in vitro biochemical assays and cell-based imaging experiments for damage incision in the living cells.
Aim3 : To illustrate the structural and functional changes induced by disease-causing xpb mutations. There are only three amino acid substitution mutations identified so far in the literatures: F99S mutation causes XP, T119P mutation causes Trichothiodystrophy (TTD), and XP11BE mutation causes XP/Cockayne Syndrome (CS) complex. XP11BE is a frame-shift mutation that changes the last 42 residues at the C-terminus of XPB. It has been known that XP11BE mutation abolishes the interaction motif of XPB with XPF leading to XP phenotype. Our preliminary results indicate that change of the last 42 amino acid residues reduces the solubility of XPB mutant compared to the wild type XPB, therefore leading to limited cellular level of TFIIH which in turn causes reduced recovery RNA synthesis, the hall mark of CS phenotype. There is little information about why the adjacent mutations F99S and T119P cause different phenotypes. In order to examine these two mutations at chemical and atomic level, XPB recombinant proteins bearing F99S and T119P mutations will be prepared for structural and functional analysis. These results will be very important for understanding the pathogenic mechanism of these mutations and will have significant medical implications.
This project will examine the structure-function relationship of XPB, an important helicase required for both transcription and DNA repair, two fundamental biological processes important to human healthcare. Mutations in XPB cause three inherited diseases with manifestations of high risks of skin cancer, neurodegeneration, and premature aging, etc. The goal of this research is to understand the molecular basis for the normal XPB helicase activities and for their breakdown in human disease phenotypes associated with defects in XPB helicase. These studies are necessary for understanding how cells repair DNA damage linked to genomic instabilities and may lead to novel methods to treat human diseases.
| Zhai, Binyuan; DuPrez, Kevin; Han, Xiaoyun et al. (2018) The archaeal ATPase PINA interacts with the helicase Hjm via its carboxyl terminal KH domain remodeling and processing replication fork and Holliday junction. Nucleic Acids Res 46:6627-6641 |
| Kahanda, Dimithree; DuPrez, Kevin T; Hilario, Eduardo et al. (2018) Application of Electrochemical Devices to Characterize the Dynamic Actions of Helicases on DNA. Anal Chem 90:2178-2185 |
| Zhai, Binyuan; DuPrez, Kevin; Doukov, Tzanko I et al. (2017) Structure and Function of a Novel ATPase that Interacts with Holliday Junction Resolvase Hjc and Promotes Branch Migration. J Mol Biol 429:1009-1029 |
