The Caenorhabditis elegans gene CeAPN1 encodes a homolog of Escherichia coli and yeast apurinic/apyrimidinic endonuclease

Masson, Jean‐Yves; Tremblay, Stephane; Ramotar, Dindial

doi:10.1016/s0378-1119(96)00375-7

Cited by 24 publications

(17 citation statements)

References 7 publications

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“…However, in the former case the SpApn1 gene is apparently not expressed, and no AP-endonuclease activity has been detected in S. pombe extracts (19). On the other hand, a CeApn recombinant gene did not express a functional protein in E. coli; thus, the activities of this homolog are currently unknown (13). Therefore, YqfS of B. subtilis not only represents the second homolog of bacterial origin but also the fourth member of the family IV of AP-endonucleases with a demonstrated biochemical function.…”

Section: Discussionmentioning

confidence: 96%

YqfS from Bacillus subtilis Is a Spore Protein and a New Functional Member of the Type IV Apurinic/Apyrimidinic-Endonuclease Family

et al. 2003

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The enzymatic properties and the physiological function of the type IV apurinic/apyrimidinic (AP)-endonuclease homolog of Bacillus subtilis, encoded by yqfS, a gene specifically expressed in spores, were studied here. To this end, a recombinant YqfS protein containing an N-terminal His 6 tag was synthesized in Escherichia coli and purified to homogeneity. An anti-His 6 -YqfS polyclonal antibody exclusively localized YqfS in cell extracts prepared from B. subtilis spores. The His 6 -YqfS protein demonstrated enzymatic properties characteristic of the type IV family of DNA repair enzymes, such as AP-endonucleases and 3-phosphatases. However, the purified protein lacked both 5-phosphatase and exonuclease III activities. YqfS showed not only a high level of amino acid identity with E. coli Nfo but also a high resistance to inactivation by EDTA, in the presence of DNA containing AP sites (AP-DNA). These results suggest that YqfS possesses a trinuclear Zn center in which the three metal atoms are intimately coordinated by nine conserved basic residues and two water molecules. Electrophoretic mobility shift assays demonstrated that YqfS possesses structural properties that permit it to bind and scan undamaged DNA as well as to strongly interact with AP-DNA. The ability of yqfS to genetically complement the DNA repair deficiency of an E. coli mutant lacking the major AP-endonucleases Nfo and exonuclease III strongly suggests that its product confers protection to cells against the deleterious effects of oxidative promoters and alkylating agents. Thus, we conclude that YqfS of B. subtilis is a spore-specific protein that has structural and enzymatic properties required to participate in the repair of AP sites and 3 blocking groups of DNA generated during both spore dormancy and germination.During unpredicted periods of dormancy Bacillus subtilis spores are constantly exposed to environmental conditions that have the potential to cause several types of DNA damage. Therefore, the existence of spore-specific protecting mechanisms would seem to be fundamental for spore survival. One of the factors intricately involved in protecting spore DNA from several types of damage, such as oxidative stress, UV-C irradiation, and desiccation, is the presence of ␣/␤ type small acid-soluble proteins (reviewed in references 16, 28, and 27). Although ␣/␤ type small acid-soluble proteins protect spore DNA from several stresses, they confer protection neither to base alkylation (29) nor to UV-induced DNA strand break formation (30). Thus, while the physiological state of the B. subtilis spores prevents or dramatically slows DNA damage during the long periods of dormancy, it is clear that spores do accumulate potentially lethal and mutagenic DNA lesions such as the spore photoproduct, strand breaks, cyclobutane pyrimidine dimers, chemically altered bases and apurinic/apyridiminic (AP) sites which could affect transcription and replication processes during germination (16,26,29). To remove these potentially deleterious DNA damages and alteratio...

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Section: Discussionmentioning

confidence: 96%

YqfS from Bacillus subtilis Is a Spore Protein and a New Functional Member of the Type IV Apurinic/Apyrimidinic-Endonuclease Family

et al. 2003

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“…The first enzyme family is typified by exonuclease (Exo) III from Escherichia coli (7,8) and the homologous APE-1 enzyme in humans (9), which are major AP endonucleases in these organisms. The second conserved AP endonuclease family is typified by E. coli endonuclease (Endo) IV (10,11) and includes the APN-1 protein from Saccharomyces cerevisiae (12) and Schizosaccharomyces pombe (13) and the CeAPN1 gene from the nematode Caenorhabditis elegans (14). In E. coli, Endo IV expression is induced by superoxide anion generators (15), but in S. cerevisiae, APN-1 is the predominant constitutive AP endonuclease.…”

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confidence: 99%

Characterization of an Endonuclease IV 3′-5′ Exonuclease Activity

Kerins

Collins

McCarthy

2003

Journal of Biological Chemistry

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Previous characterization of Escherichia coli endonuclease IV has shown that the enzyme specifically cleaves the DNA backbone at apurinic/apyrimidinic sites and removes 3 DNA blocking groups. By contrast, and unlike the major apurinic/apyrimidinic endonuclease exonuclease III, negligible exonuclease activity has been associated with endonuclease IV. Here we report that endonuclease IV does possess an intrinsic 3-5 exonuclease activity. The activity was detected in purified preparations of the endonuclease IV protein from E. coli and from the distantly related thermophile Thermotoga maritima; it co-eluted with both enzymes under different chromatographic conditions. Induction of either endonuclease IV in an E. coli overexpression system resulted in induction of the exonuclease activity, and the E. coli exonuclease activity had similar heat stability to the endonuclease IV AP endonuclease activity. Characterization of the exonuclease activity showed that its progression on substrate is sensitive to ionic strength, metal ions, EDTA, and reducing conditions. Substrates with 3 recessed ends were preferred substrates for the 3-5 exonuclease activity. Comparison of the relative apurinic/apyrimidinic endonuclease and exonuclease activity of endonuclease IV shows that the relative exonuclease activity is high and is likely to be significant in vivo. Apurinic and apyrimidinic (AP)1 sites threaten genetic stability because they block replication and are mutagenic (1, 2). They arise in DNA through the spontaneous loss of normal or damaged bases or through the release of modified or mismatched bases from DNA by DNA glycosylases (3, 4). The first general step of base excision repair following base loss is the recognition and cleavage of DNA AP sites by an AP endonuclease. This is conserved from bacteria to humans.There are two characterized conserved AP endonuclease families. These enzymes cleave the DNA backbone immediately 5Ј of an AP site, generating a 5Ј-deoxyribose phosphate group and a 3Ј deoxyribose-hydroxyl group that primes DNA repair synthesis (4). Removal of the 5Ј-deoxyribose phosphate group by a 5Ј-deoxyribose phosphodiesterase is considered to create a single-nucleotide gap, and repair is completed by DNA polymerase-mediated DNA resynthesis and rejoining via DNA ligase (5, 6). The first enzyme family is typified by exonuclease (Exo) III from Escherichia coli (7,8) and the homologous APE-1 enzyme in humans (9), which are major AP endonucleases in these organisms. The second conserved AP endonuclease family is typified by E. coli endonuclease (Endo) IV (10, 11) and includes the APN-1 protein from Saccharomyces cerevisiae (12) and Schizosaccharomyces pombe (13) and the CeAPN1 gene from the nematode Caenorhabditis elegans (14). In E. coli, Endo IV expression is induced by superoxide anion generators (15), but in S. cerevisiae, APN-1 is the predominant constitutive AP endonuclease.Genetic studies indicate that Exo III and Endo IV have overlapping but distinctive repair specificities in vivo. In E. coli Exo III is enc...

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“…Exo III also possesses a potent 3Ј-5Ј exonuclease activity. Endo IV belongs to a second group of AP endonucleases that includes a variety of homologs thus far only identified in bacteria and simple eukaryotic organisms such as APN-1 in yeast (36,37) and CeAPN-1 in the nematode Caenorhabditis elegans (38). The in vitro reconstitution studies reported for the bacterial BER pathway utilized Endo IV as the AP endonuclease, suggesting that this protein may be responsible for the step following base removal by DNA glycosylases in vivo (7,10).…”

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confidence: 99%

Escherichia coli Apurinic-Apyrimidinic Endonucleases Enhance the Turnover of the Adenine Glycosylase MutY with G:A Substrates

Pope¹,

Porello

David

2002

Journal of Biological Chemistry

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The DNA repair enzyme MutY plays an important role in the prevention of DNA mutations resulting from the presence of the oxidatively damaged lesion 7,8-dihydro-8-oxo-2-deoxyguanosine (OG). MutY is a base excision repair (BER) glycosylase that removes misincorporated adenine residues from OG:A mispairs, as well as G:A and C:A mispairs. We have previously shown that, under conditions of low MutY concentrations relative to an OG:A or G:A substrate, the time course of the adenine glycosylase reaction exhibits biphasic kinetic behavior due to slow release of the DNA product by MutY. The dissociation of MutY from its product may require the recruitment of other proteins from the BER pathway, such as an apurinic-apyrimidinic (AP) endonuclease, as turnover-enhancing cofactors. The effect of the AP endonucleases endonuclease IV (Endo IV), exonuclease III (Exo III), and Ape1 on the reaction kinetics of MutY with G:A-and OG:A-containing substrates was investigated. The effect of the glycosylases UDG and MutM and the DNA polymerase pol I was also characterized. Endo IV and Exo III, unlike Ape1, UDG, and pol I, greatly enhance the rate of product release with a G:A substrate, whereas the rate constant for the adenine removal step remains unchanged. Furthermore, the turnover rate with a truncated form of MutY, Stop 225, which lacks 125 amino acids of the C terminus, is unaffected by the presence of Endo IV or Exo III. These results constitute the first evidence of an interaction between the MutY-product DNA complex and Endo IV or Exo III. Furthermore, they suggest a role for the C-terminal domain of MutY in mediating this interaction. The base excision repair (BER)1 pathway is the primary cellular mechanism charged with the task of removing DNA bases modified via hydrolysis, oxidation, and alkylation (1). BER relies on the action of damage-specific DNA glycosylases that recognize various types of modified or inappropriate bases within the context of normal Watson-Crick DNA (2). The repair process is initiated by hydrolytic removal of the target base by the relevant DNA glycosylase and proceeds by incision at the abasic site, generation of a gap, reparative DNA synthesis, and ligation of the nicked DNA (1, 3). The BER pathway has been reconstituted in vitro with cell-free extracts or purified protein components, and these experiments have established the minimal requirements for restoration of the damaged DNA (4 -11). For example, repair of uracil in DNA was achieved by use of five Escherichia coli proteins: uracil-DNA glycosylase (UDG), endonuclease IV (an AP endonuclease), RecJ protein, DNA polymerase I, and DNA ligase (7). Both "short patch" and "long patch" BER pathways have been observed, which differ in the number of nucleotides and the various protein components involved (12, 13). The type of BER pathway utilized depends on the organism and the type of DNA damage; however, minimally the BER pathway requires a damage-specific glycosylase, an AP endonuclease, DNA polymerase, and DNA ligase.The DNA glycosylases of BER may ...

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The Caenorhabditis elegans gene CeAPN1 encodes a homolog of Escherichia coli and yeast apurinic/apyrimidinic endonuclease

Cited by 24 publications

References 7 publications

YqfS from Bacillus subtilis Is a Spore Protein and a New Functional Member of the Type IV Apurinic/Apyrimidinic-Endonuclease Family

YqfS from Bacillus subtilis Is a Spore Protein and a New Functional Member of the Type IV Apurinic/Apyrimidinic-Endonuclease Family

Characterization of an Endonuclease IV 3′-5′ Exonuclease Activity

Escherichia coli Apurinic-Apyrimidinic Endonucleases Enhance the Turnover of the Adenine Glycosylase MutY with G:A Substrates

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