Structure of the DNA Repair Enzyme Endonuclease IV and Its DNA Complex

Hosfield, David J.; Guan, Yue; Haas, Brian J.; Cunningham, Richard P.; Tainer, John A.

doi:10.1016/s0092-8674(00)81968-6

Cited by 271 publications

(196 citation statements)

References 58 publications

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“…Indeed, we have shown that truncated Ape1 (N⌬61-Ape1) lacking the first 61 N-terminal amino acids has reduced NIR but normal AP endonuclease activity (3). The crystal structure of Nfo complexed to duplex DNA containing an AP site shows that three Zn 2ϩ ions are bound in the protein by conserved residues that cluster at the center of a crescent-shaped cavity (6). Site-directed mutagenesis of the con- Nfo-H69A Nfo-G149D served amino acid residues support participation of Zn 2ϩ ions in catalysis of phosphodiester bond cleavage (18).…”

Section: Resultsmentioning

confidence: 99%

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Uncoupling of the base excision and nucleotide incision repair pathways reveals their respective biological roles

Ищенко

Deprez

Maksimenko

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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The multifunctional DNA repair enzymes apurinic͞apyrimidinic (AP) endonucleases cleave DNA at AP sites and 3 -blocking moieties generated by DNA glycosylases in the base excision repair pathway. Alternatively, in the nucleotide incision repair (NIR) pathway, the same AP endonucleases incise DNA 5 of a number of oxidatively damaged bases. At present, the physiological relevance of latter function remains unclear. Here, we report genetic dissection of AP endonuclease functions in base excision repair and NIR pathways. Three mutants of Escherichia coli endonuclease IV (Nfo), carrying amino acid substitutions H69A, H109A, and G149D have been isolated. All mutants were proficient in the AP endonuclease and 3 -repair diesterase activities but deficient in the NIR. Analysis of metal content reveals that all three mutant proteins have lost one of their intrinsic zinc atoms. Expression of the nfo mutants in a repair-deficient strain of E. coli complemented its hypersensitivity to alkylation but not to oxidative DNA damage. The differential drug sensitivity of the mutants suggests that the NIR pathway removes lethal DNA lesions generated by oxidizing agents. To address the physiological relevance of the NIR pathway in human cells, we used the fluorescence quenching mechanism of molecular beacons. We show that in living cells a major human AP endonuclease, Ape1, incises DNA containing ␣-anomeric 2 -deoxyadenosine, indicating that the intracellular environment supports NIR activity. Our data establish that NIR is a distinct and separable function of AP endonucleases essential for handling lethal oxidative DNA lesions. apurinic͞apyrimidinic endonuclease ͉ oxidative DNA damage ͉ tert-butyl hydroperoxide ͉ 3Ј-blocking groups ͉ ␣-anomeric 2Ј-deoxyadenosine

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Section: Resultsmentioning

confidence: 99%

“…Nfo is an EDTA-resistant multifunctional enzyme with AP endonuclease, 3Ј-phosphatase and 3Ј-phosphoglycoaldehyde diesterase activities (4). The protein contains three Zn 2ϩ ions in the active site that are critical for its functions (5,6). The nfo gene is under the control of the soxRS system and inducible by O 2 Ϫ and NO⅐ radicals (7).…”

mentioning

confidence: 99%

Uncoupling of the base excision and nucleotide incision repair pathways reveals their respective biological roles

Ищенко

Deprez

Maksimenko

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

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“…Several groups of crystallographers have revealed the structures of DNA protein complexes with the base flipping (31)(32)(33)(34)(35)(36)(37)(38). However, in the case of the CPD photolyase, no structural evidence of CPD-flipping has been shown.…”

Section: Table II Analyses Of the Interactions Between The Cpd Photolmentioning

confidence: 99%

Investigation of the Cyclobutane Pyrimidine Dimer (CPD) Photolyase DNA Recognition Mechanism by NMR Analyses

Torizawa

Ueda

Kuramitsu

et al. 2004

Journal of Biological Chemistry

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The cyclobutane pyrimidine dimer (CPD) is one of the major forms of DNA damage caused by irradiation with ultraviolet (UV) light. CPD photolyases recognize and repair UV-damaged DNA. The DNA recognition mechanism of the CPD photolyase has remained obscure because of a lack of structural information about DNA-CPD photolyase complexes. In order to elucidate the CPD photolyase DNA binding mode, we performed NMR analyses of the DNA-CPD photolyase complex. Based upon results from 31 P NMR measurements, in combination with site-directed mutagenesis, we have demonstrated the orientation of CPD-containing single-stranded DNA (ssDNA) on the CPD photolyase. In addition, chemical shift perturbation analyses, using stable isotope-labeled DNA, revealed that the CPD is buried in a cavity within CPD photolyase. Finally, NMR analyses of a double-stranded DNA (dsDNA)-CPD photolyase complex indicated that the CPD is flipped out of the dsDNA by the enzyme, to gain access to the active site.Irradiation of DNA with ultraviolet (UV) light produces various damaged bases, leading to cellular transformation and cell death (1-3). One of the major products formed by UV irradiation is the cyclobutane pyrimidine dimer (CPD) 1 (Fig. 1), from the photo [2 ϩ 2] cycloaddition of the 5,6-double bond of two adjacent pyrimidine nucleotides. Organisms have a variety of enzymes playing crucial roles in repair-systems for damaged DNA, including nucleoside excision repair systems and photoreactivation (4). CPD photolyases, which function as members of the DNA repair systems, restore the CPD to normal pyrimidines by photoreactivation. To elucidate the mechanism of DNA repair by CPD photolyase, various biological and spectroscopic experiments have been performed. This enzyme has a flavin adenine dinucleotide (FAD) as an essential cofactor (5).The FAD is excited by light, and then transfers one electron to the CPD bases (6 -8). After the electron transfer, the cyclobutane ring splits and then one electron is transferred back to the FAD (9, 10). In order to understand the mechanism based on structural analyses, the crystal structures of the CPD photolyases from Escherichia coli, Anacystis nidulans, and Thermus thermophilus have been solved without the substrates, i.e. CPD-containing DNAs (11-13). The x-ray studies revealed that the enzymes share a similar global fold, which consists of an ␣/␤ domain and a helical domain. The helical domain is composed of clusters I and II, and a cavity is formed between the clusters, where the FAD is deeply buried. It has been suggested that the cavity is used for the CPD binding, because the asymmetric polarity of the cavity fits well with that of the CPD (11-15). Based upon the crystal structures without the substrates, two research groups have proposed computer models of the DNA-CPD photolyase complex, in which the relative orientations of the DNA chain are different from each other (11-15). However, no crystallographic or NMR structure information on the complexes is presently available.Here, we report NMR analyses of...

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“…5D). Following incision, Ape1 and endonuclease IV both remain tightly bound to their abasic DNA products (35,45), so these two proteins may inhibit DNA synthesis by pol ␤ using a similar mechanism. However, the substrate-specific inhibition of pol ␤ by Ape1, and the apparent lack of specificity of endonuclease IV, may reflect different modes of binding employed by these two enzymes (14).…”

Section: Concentration-dependent Inhibition Of Pol ␤ By Ape1 and E Cmentioning

confidence: 99%

“…However, the substrate-specific inhibition of pol ␤ by Ape1, and the apparent lack of specificity of endonuclease IV, may reflect different modes of binding employed by these two enzymes (14). Both proteins induce DNA bends, but Ape1 induces a relatively mild 35°bend compared with the 90°kink caused by endonuclease IV (45,46). Table I for sequence), 1 nM pol ␤, 20 M dCTP, and either no added Ape1 (filled squares), 50 nM wild-type Ape1 (filled circles), or 50 nM D283A/D308A mutant form of Ape1 (filled triangles).…”

Section: Concentration-dependent Inhibition Of Pol ␤ By Ape1 and E Cmentioning

confidence: 99%

Modulation of the 5′-Deoxyribose-5-phosphate Lyase and DNA Synthesis Activities of Mammalian DNA Polymerase β by Apurinic/Apyrimidinic Endonuclease 1

Wong¹,

Demple

2004

Journal of Biological Chemistry

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The Ape1 protein initiates the repair of apurinic/ apyrimidinic sites during mammalian base excision repair (BER) of DNA. Ape1 catalyzes hydrolysis of the 5-phosphodiester bond of abasic DNA to create nicks flanked by 3-hydroxyl and 5-deoxyribose 5-phosphate (dRP) termini. DNA polymerase (pol) ␤ catalyzes both DNA synthesis at the 3-hydroxyl terminus and excision of the 5-dRP moiety prior to completion of BER by DNA ligase. During BER, Ape1 recruits pol ␤ to the incised apurinic/apyrimidinic site and stimulates 5-dRP excision by pol ␤. The activities of these two enzymes are thus coordinated during BER. To examine further the coordination of BER, we investigated the ability of Ape1 to modulate the deoxynucleotidyltransferase and 5-dRP lyase activities of pol ␤. We report here that Ape1 stimulates 5-dRP excision by a mechanism independent of its apurinic/apyrimidinic endonuclease activity. We also demonstrate a second mechanism, independent of Ape1, in which conditions that support DNA synthesis by pol ␤ also enhance 5-dRP excision. Ape1 modulates the gap-filling activity of pol ␤ by specifically inhibiting synthesis on an incised abasic substrate but not on single-nucleotide gapped DNA. In contrast to the wild-type Ape1 protein, a catalytically impaired mutant form of Ape1 did not affect DNA synthesis by pol ␤. However, this mutant protein retained the ability to stimulate 5-dRP excision by pol ␤. Simultaneous monitoring of 5-dRP excision and DNA synthesis by pol ␤ demonstrated that the 5-dRP lyase activity lags behind the polymerase activity despite the coordination of these two steps by Ape1 during BER.The repair of damaged DNA is a critical function in all organisms and is required to maintain the integrity of genetic information. DNA can be damaged by exposure of cells to ionizing radiation, ultraviolet light, and xenobiotic agents as well as by exposure to endogenously generated alkylating agents and reactive oxygen species (1, 2). DNA base lesions are a common result of these genotoxic agents, which contribute to the formation of mutations that lead to cancer (3). In mammalian cells, these DNA lesions are repaired by enzymes of the base excision repair (BER) 1 pathway. BER is initiated by a family of DNA glycosylase enzymes. These proteins recognize and excise modified bases from the sugar-phosphate backbone of DNA (4, 5). The resultant apurinic/apyrimidinic (AP) site is both a repair intermediate and also a highly prevalent primary DNA lesion that is also created via acid-catalyzed hydrolysis of DNA bases (6, 7). An estimated 10,000 spontaneously generated AP sites are formed daily in each human cell (8), in addition to the contributions made by the action of DNA glycosylases. Indeed, the steadystate level of AP sites has been estimated to be quite high in some tissue types, especially following oxidative stress (9 -11).AP sites are processed by AP endonucleases, which are highly conserved in both prokaryotic and eukaryotic organisms. Mammalian Ape1 hydrolyzes the phosphodiester backbone of DNA 5Ј of AP s...

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Structure of the DNA Repair Enzyme Endonuclease IV and Its DNA Complex

Cited by 271 publications

References 58 publications

Uncoupling of the base excision and nucleotide incision repair pathways reveals their respective biological roles

Uncoupling of the base excision and nucleotide incision repair pathways reveals their respective biological roles

Investigation of the Cyclobutane Pyrimidine Dimer (CPD) Photolyase DNA Recognition Mechanism by NMR Analyses

Modulation of the 5′-Deoxyribose-5-phosphate Lyase and DNA Synthesis Activities of Mammalian DNA Polymerase β by Apurinic/Apyrimidinic Endonuclease 1

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