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Open Access Publications from the University of California

Scholarly Works (6 results)

  • Thesis
  • Peer Reviewed
UC Riverside Electronic Theses and Dissertations (2016)

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.

    Cover page: Structural and Functional Characterization of the Archaeal DNA Repair Helicase XPB and its Interactions With the Endonuclease Bax1
    • Article
    • Peer Reviewed
    UC Riverside Previously Published Works (2020)
    Nucleotide excision repair (NER) is a major DNA repair pathway for a variety of DNA lesions. XPB plays a key role in DNA opening at damage sites and coordinating damage incision by nucleases. XPB is conserved from archaea to human. In archaea, XPB is associated with a nuclease Bax1. Here we report crystal structures of XPB in complex with Bax1 from Archaeoglobus fulgidus (Af) and Sulfolobus tokodaii (St). These structures reveal for the first time four domains in Bax1, which interacts with XPB mainly through its N-terminal domain. A Cas2-like domain likely helps to position Bax1 at the forked DNA allowing the nuclease domain to incise one arm of the fork. Bax1 exists in monomer or homodimer but forms a heterodimer exclusively with XPB. StBax1 keeps StXPB in a closed conformation and stimulates ATP hydrolysis by XPB while AfBax1 maintains AfXPB in the open conformation and reduces its ATPase activity. Bax1 contains two distinguished nuclease active sites to presumably incise DNA damage. Our results demonstrate that protein-protein interactions regulate the activities of XPB ATPase and Bax1 nuclease. These structures provide a platform to understand the XPB-nuclease interactions important for the coordination of DNA unwinding and damage incision in eukaryotic NER.
      Cover page: Structural basis of the XPB–Bax1 complex as a dynamic helicase–nuclease machinery for DNA repair
      • Article
      • Peer Reviewed
      UC Riverside Previously Published Works (2020)
      Nucleotide excision repair (NER) removes various DNA lesions caused by UV light and chemical carcinogens. The DNA helicase XPB plays a key role in DNA opening and coordinating damage incision by nucleases during NER, but the underlying mechanisms remain unclear. Here, we report crystal structures of XPB from Sulfurisphaera tokodaii (St) bound to the nuclease Bax1 and their complex with a bubble DNA having one arm unwound in the crystal. StXPB and Bax1 together spirally encircle 10 base pairs of duplex DNA at the double-/single-stranded (ds-ss) junction. Furthermore, StXPB has its ThM motif intruding between the two DNA strands and gripping the 3'-overhang while Bax1 interacts with the 5'-overhang. This ternary complex likely reflects the state of repair bubble extension by the XPB and nuclease machine. ATP binding and hydrolysis by StXPB could lead to a spiral translocation along dsDNA and DNA strand separation by the ThM motif, revealing an unconventional DNA unwinding mechanism. Interestingly, the DNA is kept away from the nuclease domain of Bax1, potentially preventing DNA incision by Bax1 during repair bubble extension.
        Cover page: Structural basis of the XPB helicase–Bax1 nuclease complex interacting with the repair bubble DNA
        • Article
        • Peer Reviewed
        UC Riverside Previously Published Works (2018)
        Much remains to be understood about the kinetics and thermodynamics of DNA helicase binding and activity. Here, we utilize probe-modified DNA monolayers on multiplexed gold electrodes as a sensitive recognition element and morphologically responsive transducer of helicase-DNA interactions. The electrochemical signals from these devices are highly sensitive to structural distortion of the DNA produced by the helicases. We used this DNA electrochemistry to distinguish the details of the DNA interactions of three distinct XPB helicases, which belong to the superfamily-2 of helicases. Clear changes in DNA melting temperature and duplex stability were observed upon helicase binding, shifts that could not be observed with conventional UV-visible absorption measurements. Binding dissociation constants were estimated in the range from 10 to 50 nM and correlated with observations of activity. ATP-stimulated DNA unwinding activity was also followed, revealing exponential time scales and distinct time constants associated with conventional and molecular wrench modes of operation further confirmed by crystal structures. These devices thus provide a sensitive measure of the structural thermodynamics and kinetics of helicase-DNA interactions.
          Cover page: Application of Electrochemical Devices to Characterize the Dynamic Actions of Helicases on DNA
          • Article
          • Peer Reviewed
          UC Riverside Previously Published Works (2018)
          PINA is a novel ATPase and DNA helicase highly conserved in Archaea, the third domain of life. The PINA from Sulfolobus islandicus (SisPINA) forms a hexameric ring in crystal and solution. The protein is able to promote Holliday junction (HJ) migration and physically and functionally interacts with Hjc, the HJ specific endonuclease. Here, we show that SisPINA has direct physical interaction with Hjm (Hel308a), a helicase presumably targeting replication forks. In vitro biochemical analysis revealed that Hjm, Hjc, and SisPINA are able to coordinate HJ migration and cleavage in a concerted way. Deletion of the carboxyl 13 amino acid residues impaired the interaction between SisPINA and Hjm. Crystal structure analysis showed that the carboxyl 70 amino acid residues fold into a type II KH domain which, in other proteins, functions in binding RNA or ssDNA. The KH domain not only mediates the interactions of PINA with Hjm and Hjc but also regulates the hexameric assembly of PINA. Our results collectively suggest that SisPINA, Hjm and Hjc work together to function in replication fork regression, HJ formation and HJ cleavage.
            Cover page: The archaeal ATPase PINA interacts with the helicase Hjm via its carboxyl terminal KH domain remodeling and processing replication fork and Holliday junction
            • Article
            • Peer Reviewed
            UC Riverside Previously Published Works (2017)
            Holliday junction (HJ) is a hallmark intermediate in DNA recombination and must be processed by dissolution (for double HJ) or resolution to ensure genome stability. Although HJ resolvases have been identified in all domains of life, there is a long-standing effort to search in prokaryotes and eukarya for proteins promoting HJ migration. Here, we report the structural and functional characterization of a novel ATPase, Sulfolobus islandicusPilT N-terminal-domain-containing ATPase (SisPINA), encoded by the gene adjacent to the resolvase Hjc coding gene. PINA is conserved in archaea and vital for S. islandicus viability. Purified SisPINA forms hexameric rings in the crystalline state and in solution, similar to the HJ migration helicase RuvB in Gram-negative bacteria. Structural analysis suggests that ATP binding and hydrolysis cause conformational changes in SisPINA to drive branch migration. Further studies reveal that SisPINA interacts with SisHjc and coordinates HJ migration and cleavage.
              Cover page: Structure and Function of a Novel ATPase that Interacts with Holliday Junction Resolvase Hjc and Promotes Branch Migration
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