DNA apurinic-apyrimidinic site binding and excision by endonuclease IV

Department of Molecular Biology and Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, MB4 La Jolla, California 92037, USA.
Nature Structural & Molecular Biology (Impact Factor: 13.31). 04/2008; 15(5):515-522. DOI: 10.1038/nsmb.1414


Escherichia coli endonuclease IV is an archetype for an abasic or apurinic-apyrimidinic endonuclease superfamily crucial for DNA base excision repair. Here biochemical, mutational and crystallographic characterizations reveal a three–metal ion mechanism for damage binding and incision. The 1.10-Å resolution DNA-free and the 2.45-Å resolution DNA-substrate complex structures capture substrate stabilization by Arg37 and reveal a distorted Zn3-ligand arrangement that reverts, after catalysis, to an ideal geometry suitable to hold rather than release cleaved DNA product. The 1.45-Å resolution DNA-product complex structure shows how Tyr72 caps the active site, tunes its dielectric environment and promotes catalysis by Glu261-activated hydroxide, bound to two Zn2+ ions throughout catalysis. These structural, mutagenesis and biochemical results suggest general requirements for abasic site removal in contrast to features specific to the distinct endonuclease IV - triose phosphate isomerase (TIM) barrel and APE1 four-layer - folds of the apurinic-apyrimidinic endonuclease families.

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    • "Furthermore, in Endo IV, an unrelated endonuclease which nevertheless contains an extremely similar Zn tri-metallic binding pocket [33], mutagenesis studies produced a variant with a phosphate molecule in the binding pocket, resulting in an inactive protein. Garcin et al. concluded that the phosphate coordination in the mutant was not functional, and could be one of the factors leading to total protein inactivation, mimicking the cleaved product [33]. "
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    ABSTRACT: The multi S1/P1 nuclease AtBFN2 (EC encoded by the Arabidopsis thaliana At1g68290 gene is a glycoprotein that digests RNA, ssDNA, and dsDNA. AtBFN2 depends on three zinc ions for cleaving DNA and RNA at 39-OH to yield 59-nucleotides. In addition, AtBFN29s enzymatic activity is strongly glycan dependent. Plant Zn 2+ -dependent endonucleases present a unique fold, and belong to the Phospholipase C (PLC)/P1 nuclease superfamily. In this work, we present the first complete, ligand-free, AtBFN2 crystal structure, along with sulfate, phosphate and ssDNA co-crystal structures. With these, we were able to provide better insight into the glycan structure and possible enzymatic mechanism. In comparison with other nucleases, the AtBFN2/ligand-free and AtBFN2/PO 4 models suggest a similar, previously proposed, catalytic mechanism. Our data also confirm that the phosphate and vanadate can inhibit the enzyme activity by occupying the active site. More importantly, the AtBFN2/A 5 T structure reveals a novel and conserved secondary binding site, which seems to be important for plant Zn 2+ -dependent endonucleases. Based on these findings, we propose a rational ssDNA binding model, in which the ssDNA wraps itself around the protein and the attached surface glycan, in turn, reinforces the binding complex. Citation: Yu T-F, Maestre-Reyna M, Ko C-Y, Ko T-P, Sun Y-J, et al. (2014) Structural Insights of the ssDNA Binding Site in the Multifunctional Endonuclease AtBFN2 from Arabidopsis thaliana. PLoS ONE 9(8): e105821. doi:10.1371/journal.pone.0105821to J.-F. Shaw), (NSC-103-3113-P-008-001, and NSC-101-2319-B-001-003 (to A. H.-J. Wang). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. (AHJW) . These authors contributed equally to this work.
    Full-text · Article · Aug 2014 · PLoS ONE
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    • "APE1 is part of the exonuclease III (Xth) family, has a two-layered ␤-sheet core flanked by helices, and has a single Mg 2+ ion in its active site [73]. Nfo has a TIM ␤ barrel core, surrounded by helices with not one but three metal ions—either three Zn 2+ or two Zn 2+ and one Mn 2+ [62] [63]. Additionally, although APE1 and Nfo flip out the abasic site, Nfo flips out the base on the opposite strand as well. "
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    ABSTRACT: To avoid genome instability, DNA repair nucleases must precisely target the correct damaged substrate before they are licensed to incise. Damage identification is a challenge for all DNA damage response proteins, but especially for nucleases that cut the DNA and necessarily create a cleaved DNA repair intermediate, likely more toxic than the initial damage. How do these enzymes achieve exquisite specificity without specific sequence recognition or, in some cases, without a non-canonical DNA nucleotide? Combined structural, biochemical, and biological analyses of repair nucleases are revealing their molecular tools for damage verification and safeguarding against inadvertent incision. Surprisingly, these enzymes also often act on RNA, which deserves more attention. Here, we review protein-DNA structures for nucleases involved in replication, base excision repair, mismatch repair, double strand break repair (DSBR), and telomere maintenance: apurinic/apyrimidinic endonuclease 1 (APE1), Endonuclease IV (Nfo), tyrosyl DNA phosphodiesterase (TDP2), UV Damage endonuclease (UVDE), very short patch repair endonuclease (Vsr), Endonuclease V (Nfi), Flap endonuclease 1 (FEN1), exonuclease 1 (Exo1), RNase T and Meiotic recombination 11 (Mre11). DNA and RNA structure-sensing nucleases are essential to life with roles in DNA replication, repair, and transcription. Increasingly these enzymes are employed as advanced tools for synthetic biology and as targets for cancer prognosis and interventions. Currently their structural biology is most fully illuminated for DNA repair, which is also essential to life. How DNA repair enzymes maintain genome fidelity is one of the DNA double helix secrets missed by James Watson and Francis Crick, that is only now being illuminated though structural biology and mutational analyses. Structures reveal motifs for repair nucleases and mechanisms whereby these enzymes follow the old carpenter adage: measure twice, cut once. Furthermore, to measure twice these nucleases act as molecular level transformers that typically reshape the DNA and sometimes themselves to achieve extraordinary specificity and efficiency.
    Full-text · Article · Apr 2014 · DNA repair
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    • "The Nfo Eco counterparts of these highly conserved residues have been demonstrated to be involved in the interaction with divalent cations in the active site of the protein (Garcin et al., 2008; Hosfield et al., 1999). "
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    ABSTRACT: The DNA recombination and repair machineries of Mycoplasma pneumoniae and Mycoplasma genitalium were predicted to consist of a set of ~11 proteins. The function of one of these proteins was inferred from its homology with proteins belonging to the Endo IV enzyme family. The members of this family function in the repair of apyrimidinic/apurinic (AP) sites in DNA. As such activity may be crucial in the mycoplasmal life cycle, we set out to study the Endo IV-like proteins encoded by M. pneumoniae and M. genitalium. Both proteins, termed NfoMpn and NfoMge, respectively, were assessed for their ability to interact with damaged and undamaged DNA. In the absence of divalent cations, both proteins exhibited specific cleavage of AP sites. Surprisingly, the proteins also recognized and cleaved cholesteryl-bound deoxyribose moieties in DNA, showing that these Nfo proteins may also function in repair of large DNA adducts. In the presence of Mg(2+), NfoMpn and NfoMge also showed 3'>5' exonucleolytic activity. By introduction of 13 single point mutations at highly conserved positions within NfoMpn, two major types of mutants could be distinghuished: (i) mutants that showed no, or limited, AP cleavage activity in the presence of EDTA, but displayed significant levels of AP cleavage activity in the presence of Mg(2+); these mutants displayed no, or very low, exonucleolytic activity, and (ii) mutants that only demonstrated marginal levels of AP site cleavage activity in the presence of Mg(2+), and did not show exonucleolytic activity. Together, these results indicate that the AP endonucleolytic activity of the NfoMpn protein can be uncoupled from its 3'>5' exonucleolytic activity.
    Preview · Article · Apr 2014 · Microbiology
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