Browse
Submit Data
Databases
API
Help

Dataset Information

13 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

A domain in human EXOG converts apoptotic endonuclease to DNA-repair exonuclease.

ABSTRACT: Human EXOG (hEXOG) is a 5'-exonuclease that is crucial for mitochondrial DNA repair; the enzyme belongs to a nonspecific nuclease family that includes the apoptotic endonuclease EndoG. Here we report biochemical and structural studies of hEXOG, including structures in its apo form and in a complex with DNA at 1.81 and 1.85?Å resolution, respectively. A Wing domain, absent in other ???-Me members, suppresses endonuclease activity, but confers on hEXOG a strong 5'-dsDNA exonuclease activity that precisely excises a dinucleotide using an intrinsic 'tape-measure'. The symmetrical apo hEXOG homodimer becomes asymmetrical upon binding to DNA, providing a structural basis for how substrate DNA bound to one active site allosterically regulates the activity of the other. These properties of hEXOG suggest a pathway for mitochondrial BER that provides an optimal substrate for subsequent gap-filling synthesis by DNA polymerase ?.

SUBMITTER: Szymanski MR

PROVIDER: S-EPMC5418593 | biostudies-literature | 2017 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

A domain in human EXOG converts apoptotic endonuclease to DNA-repair exonuclease.

Szymanski Michal R MR Yu Wangsheng W Gmyrek Aleksandra M AM White Mark A MA Molineux Ian J IJ Lee J Ching JC Yin Y Whitney YW

Nature communications 20170503

Human EXOG (hEXOG) is a 5'-exonuclease that is crucial for mitochondrial DNA repair; the enzyme belongs to a nonspecific nuclease family that includes the apoptotic endonuclease EndoG. Here we report biochemical and structural studies of hEXOG, including structures in its apo form and in a complex with DNA at 1.81 and 1.85 Å resolution, respectively. A Wing domain, absent in other ββα-Me members, suppresses endonuclease activity, but confers on hEXOG a strong 5'-dsDNA exonuclease activity that p ...[more]

PMID: 28466855

Similar Datasets

Nuclear localization of human DNA mismatch repair protein exonuclease 1 (hEXO1).

Project description:Human exonuclease 1 (hEXO1) is implicated in DNA mismatch repair (MMR) and mutations in hEXO1 may be associated with hereditary nonpolyposis colorectal cancer (HNPCC). Since the subcellular localization of MMR proteins is essential for proper MMR function, we characterized possible nuclear localization signals (NLSs) in hEXO1. Using fluorescent fusion proteins, we show that the sequence 418KRPR421, which exhibit strong homology to other monopartite NLS sequences, is responsible for correct nuclear localization of hEXO1. This NLS sequence is located in a region that is also required for hEXO1 interaction with hMLH1 and we show that defective nuclear localization of hEXO1 mutant proteins could be rescued by hMLH1 or hMSH2. Both hEXO1 and hMLH1 form complexes with the nuclear import factors importin beta/alpha1,3,7 whereas hMSH2 specifically recognizes importin beta/alpha3. Taken together, we infer that hEXO1, hMLH1 and hMSH2 form complexes and are imported to the nucleus together, and that redundant NLS import signals in the proteins may safeguard nuclear import and thereby MMR activity.

| S-EPMC1885640 | biostudies-literature

Immunodetection of DNA repair endonuclease ERCC1-XPF in human tissue.

Project description:The high incidence of resistance to DNA-damaging chemotherapeutic drugs and severe side effects of chemotherapy have led to a search for biomarkers able to predict which patients are most likely to respond to therapy. ERCC1-XPF nuclease is required for nucleotide excision repair of helix-distorting DNA damage and the repair of DNA interstrand crosslinks. Thus, it is essential for several pathways of repair of DNA damage by cisplatin and related drugs, which are widely used in the treatment of non-small cell lung carcinoma and other late-stage tumors. Consequently, there is tremendous interest in measuring ERCC1-XPF expression in tumor samples. Many immunohistochemistry studies have been done, but the antibodies for ERCC1-XPF were not rigorously tested for antigen specificity. Herein, we survey a battery of antibodies raised against human ERCC1 or XPF for their specificity using ERCC1-XPF-deficient cells as a negative control. Antibodies were tested for the following applications: immunoblotting, immunoprecipitation from cell extracts, immunofluorescence detection in fixed cells, colocalization of ERCC1-XPF with UV radiation-induced DNA damage in fixed cells, and immunohistochemistry in paraffin-embedded samples. Although several commercially available antibodies are suitable for immunodetection of ERCC1-XPF in some applications, only a select subset is appropriate for detection of this repair complex in fixed specimens. The most commonly used antibody, 8F1, is not suitable for immunodetection in tissue. The results with validated antibodies reveal marked differences in ERCC1-XPF protein levels between samples and cell types.

| S-EPMC2739111 | biostudies-literature

Human exonuclease 1 and BLM helicase interact to resect DNA and initiate DNA repair.

Project description:The error-free repair of double-stranded DNA breaks by homologous recombination requires processing of broken ends. These processed ends are substrates for assembly of DNA strand exchange proteins that mediate DNA strand invasion. Here, we establish that human BLM helicase, a member of the RecQ family, stimulates the nucleolytic activity of human exonuclease 1 (hExo1), a 5'-->3' double-stranded DNA exonuclease. The stimulation is specific because other RecQ homologs fail to stimulate hExo1. Stimulation of DNA resection by hExo1 is independent of BLM helicase activity and is, instead, mediated by an interaction between the 2 proteins. Finally, we show that DNA ends resected by hExo1 and BLM are used by human Rad51, but not its yeast or bacterial counterparts, to promote homologous DNA pairing. This in vitro system recapitulates initial steps of homologous recombination and provides biochemical evidence for a role of BLM and Exo1 in the initiation of recombinational DNA repair.

| S-EPMC2579351 | biostudies-literature

Isolation of cDNA clones encoding a human apurinic/apyrimidinic endonuclease that corrects DNA repair and mutagenesis defects in E. coli xth (exonuclease III) mutants.

Project description:Apurinic/apyrimidinic (AP) sites in cellular DNA are considered to be both cytotoxic and mutagenic, and can arise spontaneously or following exposure to DNA damaging agents. We have isolated cDNA clones which encode an endonuclease, designated HAP1 (human AP endonuclease 1), that catalyses the initial step in AP site repair in human cells. The predicted HAP1 protein has an Mr of 35,500 and shows striking sequence similarity (93% identity) to BAP 1, a bovine AP endonuclease enzyme. Significant sequence homology to two bacterial DNA repair enzymes, E. coli exonuclease III and S. pneumoniae ExoA proteins, and to Drosophila Rrp1 protein is also apparent. We have expressed the HAP1 cDNA in E. coli mutants lacking exonuclease III (xth), endonuclease IV (nfo), or both AP endonucleases. The HAP1 protein can substitute for exonuclease III, but not for endonuclease IV, in respect of some, but not all, DNA repair and mutagenesis functions. Moreover, a dut xth (ts) double mutant, which is nonviable at 42 degrees C due to an accumulation of unrepaired AP sites following excision of uracil from DNA, was rescued by expression of the HAP1 cDNA. These results indicate that AP endonucleases show remarkable conservation of both primary sequence and function. We would predict that the HAP1 protein is important in human cells for protection against the toxic and mutagenic effects of DNA damaging agents.

| S-EPMC328951 | biostudies-other

Human AP endonuclease suppresses DNA mismatch repair activity leading to microsatellite instability.

Project description:The multifunctional mammalian apurinic/apyrimidinic (AP) endonuclease (APE) participates in the repair of AP sites in the cellular DNA as well as participating in the redox regulation of the transcription factor function. The function of APE is considered as the rate-limiting step in DNA base excision repair. Paradoxically, an unbalanced increase in APE protein leads to genetic instability. Therefore, we investigated the mechanisms of genetic instability that are induced by APE. Here, we report that the overexpression of APE protein disrupts the repair of DNA mismatches, which results in microsatellite instability (MSI). We found that expression of APE protein led to the suppression of the repair of DNA mismatches in the normal human fibroblast cells. Western blot analysis revealed that hMSH6 protein was markedly reduced in the APE-expressing cells. Moreover, the addition of purified Mutalpha (MSH2 and MSH6 complex) to the extracts from the APE-expressing cells led to the restoration of mismatch repair (MMR) activity. By performing MMR activity assay and MSI analysis, we found that the co-expression of hMSH6 and APE exhibited the microsatellite stability, whereas the expression of APE alone generated the MSI-high phenotype. The APE-mediated decrease in MMR activity described here demonstrates the presence of a new and highly effective APE-mediated mechanism for MSI.

| S-EPMC1201336 | biostudies-literature

Distinct activities of exonuclease 1 and flap endonuclease 1 at telomeric g4 DNA.

Project description:BackgroundExonuclease 1 (EXO1) and Flap endonuclease 1 (FEN1) are members of the RAD2 family of structure-specific nucleases. Genetic analysis has identified roles for EXO1 and FEN1 in replication, recombination, DNA repair and maintenance of telomeres. Telomeres are composed of G-rich repeats that readily form G4 DNA. We recently showed that human EXO1 and FEN1 exhibit distinct activities on G4 DNA substrates representative of intermediates in immunoglobulin class switch recombination.Methodology/principal findingsWe have now compared activities of these enzymes on telomeric substrates bearing G4 DNA, identifying non-overlapping functions that provide mechanistic insight into the distinct telomeric phenotypes caused by their deficiencies. We show that hFEN1 but not hEXO1 cleaves substrates bearing telomeric G4 DNA 5'-flaps, consistent with the requirement for FEN1 in telomeric lagging strand replication. Both hEXO1 and hFEN1 are active on substrates bearing telomeric G4 DNA tails, resembling uncapped telomeres. Notably, hEXO1 but not hFEN1 is active on transcribed telomeric G-loops.Conclusion/significanceOur results suggest that EXO1 may act at transcription-induced telomeric structures to promote telomere recombination while FEN1 has a dominant role in lagging strand replication at telomeres. Both enzymes can create ssDNA at uncapped telomere ends thereby contributing to recombination.

| S-EPMC2811187 | biostudies-literature

Structure and function of TatD exonuclease in DNA repair.

Project description:TatD is an evolutionarily conserved protein with thousands of homologues in all kingdoms of life. It has been suggested that TatD participates in DNA fragmentation during apoptosis in eukaryotic cells. However, the cellular functions and biochemical properties of TatD in bacterial and non-apoptotic eukaryotic cells remain elusive. Here we show that Escherichia coli TatD is a Mg(2+)-dependent 3'-5' exonuclease that prefers to digest single-stranded DNA and RNA. TatD-knockout cells are less resistant to the DNA damaging agent hydrogen peroxide, and TatD can remove damaged deaminated nucleotides from a DNA chain, suggesting that it may play a role in the H2O2-induced DNA repair. The crystal structure of the apo-form TatD and TatD bound to a single-stranded three-nucleotide DNA was determined by X-ray diffraction methods at a resolution of 2.0 and 2.9 Å, respectively. TatD has a TIM-barrel fold and the single-stranded DNA is bound at the loop region on the top of the barrel. Mutational studies further identify important conserved metal ion-binding and catalytic residues in the TatD active site for DNA hydrolysis. We thus conclude that TatD is a new class of TIM-barrel 3'-5' exonuclease that not only degrades chromosomal DNA during apoptosis but also processes single-stranded DNA during DNA repair.

| S-EPMC4176340 | biostudies-literature

Kinetic Features of 3'-5' Exonuclease Activity of Human AP-Endonuclease APE1.

Project description:Human apurinic/apyrimidinic (AP)-endonuclease APE1 is one of the key enzymes taking part in the repair of damage to DNA. The primary role of APE1 is the initiation of the repair of AP-sites by catalyzing the hydrolytic incision of the phosphodiester bond immediately 5' to the damage. In addition to the AP-endonuclease activity, APE1 possesses 3'-5' exonuclease activity, which presumably is responsible for cleaning up nonconventional 3' ends that were generated as a result of DNA damage or as transition intermediates in DNA repair pathways. In this study, the kinetic mechanism of 3'-end nucleotide removal in the 3'-5' exonuclease process catalyzed by APE1 was investigated under pre-steady-state conditions. DNA substrates were duplexes of deoxyribonucleotides with one 5' dangling end and it contained a fluorescent 2-aminopurine residue at the 1st, 2nd, 4th, or 6th position from the 3' end of the short oligonucleotide. The impact of the 3'-end nucleotide, which contained mismatched, undamaged bases or modified bases as well as an abasic site or phosphate group, on the efficiency of 3'-5' exonuclease activity was determined. Kinetic data revealed that the rate-limiting step of 3' nucleotide removal by APE1 in the 3'-5' exonuclease process is the release of the detached nucleotide from the enzyme's active site.

| S-EPMC6225374 | biostudies-literature

The role of DNA repair endonuclease XPF in transcription

Project description:In this experiment we want to explore and compare the bindind of XPF in normal MEFs, in tRA treated MEFs and in UV radiated MEFs

2020-08-19 | E-MTAB-8154 | biostudies-arrayexpress

BapE DNA endonuclease induces an apoptotic-like response to DNA damage in Caulobacter.

Project description:In the presence of extensive DNA damage, eukaryotes activate endonucleases to fragment their chromosomes and induce apoptotic cell death. Apoptotic-like responses have recently been described in bacteria, but primarily in specialized mutant backgrounds, and the factors responsible for DNA damage-induced chromosome fragmentation and death have not been identified. Here we find that wild-type Caulobacter cells induce apoptotic-like cell death in response to extensive DNA damage. The bacterial apoptosis endonuclease (BapE) protein is induced by damage but not involved in DNA repair itself, and mediates this cell fate decision. BapE fragments chromosomes by cleaving supercoiled DNA in a sequence-nonspecific manner, thereby perturbing chromosome integrity both in vivo and in vitro. This damage-induced chromosome fragmentation pathway resembles that of eukaryotic apoptosis. We propose that damage-induced programmed cell death can be a primary stress response for some bacterial species, providing isogenic bacterial communities with advantages similar to those that apoptosis provides to multicellular organisms.

| S-EPMC3497781 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data