skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Structure of the C-Terminal Half of UvrC Reveals an RNase H Endonuclease Domain with an Argonaute-like Catalytic Triad

Abstract

Removal and repair of DNA damage by the nucleotide excision repair pathway requires two sequential incision reactions, which are achieved by the endonuclease UvrC in eubacteria. Here, we describe the crystal structure of the C-terminal half of UvrC, which contains the catalytic domain responsible for 5' incision and a helix-hairpin-helix-domain that is implicated in DNA binding. Surprisingly, the 5' catalytic domain shares structural homology with RNase H despite the lack of sequence homology and contains an uncommon DDH triad. The structure also reveals two highly conserved patches on the surface of the protein, which are not related to the active site. Mutations of residues in one of these patches led to the inability of the enzyme to bind DNA and severely compromised both incision reactions. Based on our results, we suggest a model of how UvrC forms a productive protein-DNA complex to excise the damage from DNA.

Authors:
; ; ; ; ; ;
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL) National Synchrotron Light Source
Sponsoring Org.:
Doe - Office Of Science
OSTI Identifier:
930301
Report Number(s):
BNL-81006-2008-JA
Journal ID: ISSN 0261-4189; EMJODG; TRN: US200822%%1457
DOE Contract Number:
DE-AC02-98CH10886
Resource Type:
Journal Article
Resource Relation:
Journal Name: EMBO Journal; Journal Volume: 26
Country of Publication:
United States
Language:
English
Subject:
36 MATERIALS SCIENCE; CRYSTAL STRUCTURE; DAMAGE; DNA; DNA DAMAGES; ENDONUCLEASES; ENZYMES; EXCISION REPAIR; MUTATIONS; NUCLEOTIDES; REMOVAL; REPAIR; RESIDUES; SURFACES; national synchrotron light source

Citation Formats

Karakas,E., Truglio, J., Croteau, D., Rhau, B., Wang, L., Van Houten, B., and Kisker, C. Structure of the C-Terminal Half of UvrC Reveals an RNase H Endonuclease Domain with an Argonaute-like Catalytic Triad. United States: N. p., 2007. Web. doi:10.1038/sj.emboj.7601497.
Karakas,E., Truglio, J., Croteau, D., Rhau, B., Wang, L., Van Houten, B., & Kisker, C. Structure of the C-Terminal Half of UvrC Reveals an RNase H Endonuclease Domain with an Argonaute-like Catalytic Triad. United States. doi:10.1038/sj.emboj.7601497.
Karakas,E., Truglio, J., Croteau, D., Rhau, B., Wang, L., Van Houten, B., and Kisker, C. Mon . "Structure of the C-Terminal Half of UvrC Reveals an RNase H Endonuclease Domain with an Argonaute-like Catalytic Triad". United States. doi:10.1038/sj.emboj.7601497.
@article{osti_930301,
title = {Structure of the C-Terminal Half of UvrC Reveals an RNase H Endonuclease Domain with an Argonaute-like Catalytic Triad},
author = {Karakas,E. and Truglio, J. and Croteau, D. and Rhau, B. and Wang, L. and Van Houten, B. and Kisker, C.},
abstractNote = {Removal and repair of DNA damage by the nucleotide excision repair pathway requires two sequential incision reactions, which are achieved by the endonuclease UvrC in eubacteria. Here, we describe the crystal structure of the C-terminal half of UvrC, which contains the catalytic domain responsible for 5' incision and a helix-hairpin-helix-domain that is implicated in DNA binding. Surprisingly, the 5' catalytic domain shares structural homology with RNase H despite the lack of sequence homology and contains an uncommon DDH triad. The structure also reveals two highly conserved patches on the surface of the protein, which are not related to the active site. Mutations of residues in one of these patches led to the inability of the enzyme to bind DNA and severely compromised both incision reactions. Based on our results, we suggest a model of how UvrC forms a productive protein-DNA complex to excise the damage from DNA.},
doi = {10.1038/sj.emboj.7601497},
journal = {EMBO Journal},
number = ,
volume = 26,
place = {United States},
year = {Mon Jan 01 00:00:00 EST 2007},
month = {Mon Jan 01 00:00:00 EST 2007}
}
  • Highlights: • Crystal structure of the C-terminal (CT) domain of Swt1 was determined at 2.3 Å. • Structure of the CT domain was identified as HEPN domain superfamily member. • Low-resolution envelope of Swt1 full-length in solution was analyzed by SAXS. • The middle and CT domains gave good fit to SAXS structural model. - Abstract: Swt1 is an RNA endonuclease that plays an important role in quality control of nuclear messenger ribonucleoprotein particles (mRNPs) in eukaryotes; however, its structural details remain to be elucidated. Here, we report the crystal structure of the C-terminal (CT) domain of Swt1 from Saccharomycesmore » cerevisiae, which shares common characteristics of higher eukaryotes and prokaryotes nucleotide binding (HEPN) domain superfamily. To study in detail the full-length protein structure, we analyzed the low-resolution architecture of Swt1 in solution using small angle X-ray scattering (SAXS) method. Both the CT domain and middle domain exhibited a good fit upon superimposing onto the molecular envelope of Swt1. Our study provides the necessary structural information for detailed analysis of the functional role of Swt1, and its importance in the process of nuclear mRNP surveillance.« less
  • The UvrC protein is one of three subunits of the Escherichia coli repair enzyme (A)BC excinuclease. This subunit is thought to have at least one of the active sites for nucleophilic attack on the phosphodiester bonds of damaged DNA. To localize the active site, mutant UvrC proteins were constructed by linker-scanning and deletion mutagenesis. In vivo studies revealed that the C-terminal 314 amino acids of the 610-amino acid UvrC protein were sufficient to confer UV resistance to cells lacking the uvrC gene. The portion of the uvrC gene encoding the C-terminal half of the protein was fused to the 3{prime}more » end of the E. coli malE gene (which encodes maltose binding protein), and the fusion protein MBP-C314C was purified and characterized. The fusion protein, in combination with UvrA and UvrB subunits, reconstituted the excinuclease activity that incised the eighth phosphodiester bond 5{prime} and the fourth phosphodiester bond 3{prime} to a psoralen-thymine adduct. These results suggest that the C-terminal 314 amino acids of UvrC constitute a functional domain capable of interacting with the UvrB-damaged DNA complex and of inducing the two phosphodiester bond incisions characteristic of (A)BC excinuclease.« less
  • Phospholipase C-β (PLCβ) is directly activated by Gα q, but the molecular basis for how its distal C-terminal domain (CTD) contributes to maximal activity is poorly understood. Herein we present both the crystal structure and cryo-EM three-dimensional reconstructions of human full-length PLCβ3 in complex with mouse Gα q. The distal CTD forms an extended monomeric helical bundle consisting of three antiparallel segments with structural similarity to membrane-binding bin-amphiphysin-Rvs (BAR) domains. Sequence conservation of the distal CTD suggests putative membrane and protein interaction sites, the latter of which bind the N-terminal helix of Gα q in both the crystal structure andmore » cryo-EM reconstructions. Functional analysis suggests that the distal CTD has roles in membrane targeting and in optimizing the orientation of the catalytic core at the membrane for maximal rates of lipid hydrolysis.« less
  • No abstract prepared.