Mutagenesis identifies the critical amino acid residues of human endonuclease G involved in catalysis, magnesium coordination, and substrate specificity

Wu, Shih-Lu; Li, Chia-Cheng; Chen, Jaw-Chyun; Chen, Yi-Jin; Lin, Chin‐Yi; Ho, Tin-Yun; Hsiang, Chien-Yun

doi:10.1186/1423-0127-16-6

Cited by 14 publications

(15 citation statements)

References 46 publications

(73 reference statements)

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“…Amino acid conservation is particularly relevant for the catalytic domain of proteins belonging to the sugar non-specific nuclease family [ 48 ]. Indeed, the motif Hx 22 Nx 4 Qx 5 Nx 8 E, where “x” represents any amino acid, is characteristic to this nuclease family and specifies the key residues directly involved in metal binding and catalysis [ 48 , 60 , 62 , 63 ] ( Fig 1A ). Moreover, motif prediction showed that all three nucleases have a high probability of carrying a putative secretory signal peptide (SP) (cutoff >90) ( Fig 1A ).…”

Section: Resultsmentioning

confidence: 99%

Nucleases as a barrier to gene silencing in the cotton boll weevil, Anthonomus grandis

et al. 2017

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RNA interference (RNAi) approaches have been applied as a biotechnological tool for controlling plant insect pests via selective gene down regulation. However, the inefficiency of RNAi mechanism in insects is associated with several barriers, including dsRNA delivery and uptake by the cell, dsRNA interaction with the cellular membrane receptor and dsRNA exposure to insect gut nucleases during feeding. The cotton boll weevil (Anthonomus grandis) is a coleopteran in which RNAi-mediated gene silencing does not function efficiently through dsRNA feeding, and the factors involved in the mechanism remain unknown. Herein, we identified three nucleases in the cotton boll weevil transcriptome denoted AgraNuc1, AgraNuc2, and AgraNuc3, and the influences of these nucleases on the gene silencing of A. grandis chitin synthase II (AgraChSII) were evaluated through oral dsRNA feeding trials. A phylogenetic analysis showed that all three nucleases share high similarity with the DNA/RNA non-specific endonuclease family of other insects. These nucleases were found to be mainly expressed in the posterior midgut region of the insect. Two days after nuclease RNAi-mediated gene silencing, dsRNA degradation by the gut juice was substantially reduced. Notably, after nucleases gene silencing, the orally delivered dsRNA against the AgraChSII gene resulted in improved gene silencing efficiency when compared to the control (non-silenced nucleases). The data presented here demonstrates that A. grandis midgut nucleases are effectively one of the main barriers to dsRNA delivery and emphasize the need to develop novel RNAi delivery strategies focusing on protecting the dsRNA from gut nucleases and enhancing its oral delivery and uptake to crop insect pests.

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Section: Resultsmentioning

confidence: 99%

Nucleases as a barrier to gene silencing in the cotton boll weevil, Anthonomus grandis

et al. 2017

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“…The active site of EndoG displays a ββα-metal finger topology, harboring conserved residues involved in metal ion binding and nuclease activity ( 2 , 23 ). Based on sequence similarity with related bacterial nucleases, homology models have been proposed for EndoG.…”

Section: Introductionmentioning

confidence: 99%

Crystal structure of the EndoG/EndoGI complex: mechanism of EndoG inhibition

Loll

Gebhardt

Wahle

et al. 2009

Nucleic Acids Research

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EndoG is a ubiquitous nuclease that is translocated into the nucleus during apoptosis to participate in DNA degradation. The enzyme cleaves double- and single-stranded DNA and RNA. Related nucleases are found in eukaryotes and prokaryotes, which have evolved sophisticated mechanisms for genome protection against self-antagonizing nuclease activity. Common mechanisms of inhibition are secretion, sequestration into a separate cellular compartment or by binding to protein inhibitors. Although EndoG is silenced by compartmentalization into the mitochondrial intermembrane space, a nucleus-localized protein inhibitor protects cellular polynucleotides from degradation by stray EndoG under non-apoptotic conditions in Drosophila. Here, we report the first three-dimensional structure of EndoG in complex with its inhibitor EndoGI. Although the mechanism of inhibition is reminiscent of bacterial protein inhibitors, EndoGI has evolved independently from a generic protein-protein interaction module. EndoGI is a two-domain protein that binds the active sites of two monomers of EndoG, with EndoG being sandwiched between EndoGI. Since the amino acid sequences of eukaryotic EndoG homologues are highly conserved, this model is valid for eukaryotic dimeric EndoG in general. The structure indicates that the two active sites of EndoG occupy the most remote spatial position possible at the molecular surface and a concerted substrate processing is unlikely.

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“…In fact, EndoG was originally identified as a nuclear protein in cells that were not undergoing apoptosis (Ruiz-Carrillo and Renaud, 1987; Gerschenson et al, 1995; Ishihara and Shimamoto, 2006; Yu et al, 2006). It is a homodimer that cleaves both single- and double-strand DNA, with an effective R-loop cutting activity (Widlak et al, 2001; Ohsato et al, 2002; Irvine et al, 2005; Huang et al, 2006b; Wu et al, 2009). EndoG preferentially cleaves DNA at dG/dC residues (Widlak et al, 2001), which are also the preferential sites of DSBs in S regions (Rush et al, 2004; Schrader et al, 2005; Stavnezer and Schrader, 2006).…”

Section: Introductionmentioning

confidence: 99%

Endonuclease G plays a role in immunoglobulin class switch DNA recombination by introducing double-strand breaks in switch regions

Zan

Zhang

Al‐Qahtani

et al. 2011

Molecular Immunology

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Immunoglobulin (Ig) class switch DNA recombination (CSR) is the crucial mechanism diversifying the biological effector functions of antibodies. Generation of double-strand DNA breaks (DSBs), particularly staggered DSBs, in switch (S) regions of the upstream and downstream CH genes involved in the specific recombination process is an absolute requirement for CSR. Staggered DSBs would be generated through deamination of dCs on opposite DNA strands by activation-induced cytidine deaminase (AID), subsequent dU deglycosylation by uracil DNA glycosylase (Ung) and abasic site nicking by apurinic/apyrimidic endonuclease. However, consistent with the findings that significant amounts of DSBs can be detected in the IgH locus in the absence of AID or Ung, we have shown in human and mouse B cells that AID generates staggered DSBs not only by cleaving intact double-strand DNA, but also by processing blunt DSB ends generated in an AID-independent fashion. How these AID-independent DSBs are generated is still unclear. It is possible that S region DNA may undergo AID-independent cleavage by structure-specific nucleases, such as endonuclease G (EndoG). EndoG is an abundant nuclease in eukaryotic cells. It cleaves single- and double-strand DNA, primarily at dG/dC residues, the preferential sites of DSBs in S region DNA. We show here that EndoG can localize to the nucleus of B cells undergoing CSR and binds to S region DNA, as shown by specific chromatin immunoprecipitation assays. Using knockout EndoG−/− mice and EndoG−/− B cells, we found that EndoG deficiency resulted in defective CSR in vivo and in vitro, as demonstrated by reduced cell surface IgG1, IgG2a, IgG3 and IgA, reduced secreted IgG1, reduced circle Iγ1-Cμ, Iγ3-Cμ, Iε-Cμ, Iα-Cμ transcripts, post-recombination Iμ-Cγ1, Iμ-Cγ3, Iμ-Cε and Iμ-Cα transcripts. In addition to reduced CSR, EndoG−/− mice showed a significantly altered spectrum of mutations in IgH JH-iEμ DNA. Impaired CSR in EndoG−/− B cells did not stem from altered B cell proliferation or apoptosis. Rather, it was associated with significantly reduced frequency of DSBs. Thus, our findings determine a role for EndoG in the generation of S region DSBs and CSR.

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Mutagenesis identifies the critical amino acid residues of human endonuclease G involved in catalysis, magnesium coordination, and substrate specificity

Abstract: Background: Endonuclease G (EndoG), a member of DNA/RNA nonspecific ββα-Me-finger nucleases, is involved in apoptosis and normal cellular proliferation. In this study, we analyzed the critical amino acid residues of EndoG and proposed the catalytic mechanism of EndoG.

Cited by 14 publications

References 46 publications

Nucleases as a barrier to gene silencing in the cotton boll weevil, Anthonomus grandis

Nucleases as a barrier to gene silencing in the cotton boll weevil, Anthonomus grandis

Crystal structure of the EndoG/EndoGI complex: mechanism of EndoG inhibition

Endonuclease G plays a role in immunoglobulin class switch DNA recombination by introducing double-strand breaks in switch regions

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