Analysis of class II (hydrolytic) and class I (β-lyase) apurinic/apyrimidinic endonucleases with a synthetic DNA substrate

Levin, Joshua D.; Demple, Bruce

doi:10.1093/nar/18.17.5069

Cited by 90 publications

(78 citation statements)

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“…The most frequent DNA lesions detected in MMS-treated cells include AP sites and transient single-strand breaks containing 3Ј-hydroxyl termini (62). In mammalian cells, the majority of these strand breaks are thought to result from class II AP endonucleasemediated cleavage of AP sites (45). As shown in the top right panel of Fig.…”

Section: Resultsmentioning

confidence: 99%

Clusters of S1 Nuclease-Hypersensitive Sites Induced In Vivo by DNA Damage

Legault

Tremblay

Ramotar

et al. 1997

Molecular and Cellular Biology

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DNA end-labeling procedures were used to analyze both the frequency and distribution of DNA strand breaks in mammalian cells exposed or not to different types of DNA-damaging agents. The 3 ends were labeled by T4 DNA polymerase-catalyzed nucleotide exchange carried out in the absence or presence of Escherichia coli endonuclease IV to cleave abasic sites and remove 3 blocking groups. Using this sensitive assay, we show that DNA isolated from human cells or mouse tissues contains variable basal levels of DNA strand interruptions which are associated with normal bioprocesses, including DNA replication and repair. On the other hand, distinct dose-dependent patterns of DNA damage were assessed quantitatively in cultured human cells exposed briefly to menadione, methylmethane sulfonate, topoisomerase II inhibitors, or gamma rays. In vivo induction of single-strand breaks and abasic sites by methylmethane sulfonate was also measured in several mouse tissues. The genomic distribution of these lesions was investigated by DNA cleavage with the single-strandspecific S1 nuclease. Strikingly similar cleavage patterns were obtained with all DNA-damaging agents tested, indicating that the majority of S1-hypersensitive sites detected were not randomly distributed over the genome but apparently were clustered in damage-sensitive regions. The parallel disappearance of 3 ends and loss of S1-hypersensitive sites during post-gamma-irradiation repair periods indicates that these sites were rapidly repaired single-strand breaks or gaps (2-to 3-min half-life). Comparison of S1 cleavage patterns obtained with gamma-irradiated DNA and gamma-irradiated cells shows that chromatin structure was the primary determinant of the distribution of the DNA damage detected.DNA is intrinsically unstable: it undergoes spontaneous hydrolysis of labile N-glycosyl bonds, as well as oxidation and nonenzymatic methylation at significant rates in vivo (47). These processes are thought to contribute to mutagenesis, carcinogenesis, and aging (2, 3) and are suspected to be amplified by a plethora of environmental pollutants. Reactive oxygen species (ROS) such as superoxide radicals (O 2 Ϫ ) and H 2 O 2 are generated in all aerobic cells (19). Excess production of these ROS by endogenous sources, for example, mitochondria and activated leukocytes, or by exogenous sources such as redox-cycling quinones (75) will exacerbate oxidative damage to cellular DNA (33, 54). The toxicity of these ROS is thought to result at least in part from their conversion to the highly reactive hydroxyl radical by transition metal-catalyzed Fentontype reactions (4,25). This radical is also formed by the interaction of ionizing radiation with water, e.g., within cells (78), and is known to induce a broad spectrum of DNA lesions, including base and sugar modifications, base loss, and DNA strand breaks (8, 9). The most frequent lesions detected in cells exposed to ionizing radiation are oxidized apurinic/apyrimidinic (AP) (abasic) sites (27) and single-strand breaks, most of which are te...

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Section: Resultsmentioning

confidence: 99%

Clusters of S1 Nuclease-Hypersensitive Sites Induced In Vivo by DNA Damage

Legault

Tremblay

Ramotar

et al. 1997

Molecular and Cellular Biology

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“…Thus, a nonincised AP site in DNA may never be a reaction intermediate in these cases, since the repair enzymes both release a free base by hydrolysis and cleave a phosphodiester bond. The 3'-terminal sugar-phosphate residue occurring in these cases can be excised by an AP endonuclease, since such enzymes can cleave at the 5' side of the lesion independent of the existence of a strand break on the 3' side (24,43). The Fpg protein, a DNA glycosylase-AP lyase which liberates free 8-hydroxyguanine and formamidopyrimidine residues from DNA, and the mammalian enzyme removing ring-saturated pyrimidine residues have an intrinsic ability to cleave first on the 3' side and then on the 5' side of the lesion (3,10).…”

Section: Discussionmentioning

confidence: 99%

“…Moreover, they do so at a reduced rate compared with excision of undamaged 5' nucleotide residues at nicks in DNA (35, 39). E. coli endonuclease III, which is a pyrimidine hydrate-DNA glycosylase with an associated AP lyase activity, promotes chain cleavage by n-elimination at the 3' side of an AP site (2), but such cleavage does not occur at sites already hydrolytically incised on the 5' side by an AP endonuclease (15,24). In a search for alternative excision functions, the major activity detected in cell extracts of E. coli (15) and human cells (39) was a protein of approximately 50 kDa which could hydrolytically excise free dRp from AP sites preincised by an AP endonuclease.…”

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confidence: 99%

Generation of single-nucleotide repair patches following excision of uracil residues from DNA.

Dianov¹,

Price²,

Lindahl³

1992

Mol. Cell. Biol.

274

187

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The extent and location of DNA repair synthesis in a double-stranded oligonucleotide containing a single dUMP residue have been determined. Gently prepared Escherichia coli and mammalian cell extracts were employed for excision repair in vitro. The size of the resynthesized patch was estimated by restriction enzyme analysis of the repaired oligonucleotide. Following enzymatic digestion and denaturing gel electrophoresis, the extent of incorporation of radioactively labeled nucleotides in the vicinity of the lesion was determined by autoradiography. Cell extracts of E. coli and of human cell lines were shown to carry out repair mainly by replacing a single nucleotide. No significant repair replication on the 5' side of the lesion was observed. The data indicate that, after cleavage of the dUMP residue by uracil-DNA glycosylase and incision of the resultant apurinic-apyrimidinic site by an apurinic-apyrimidinic endonuclease activity, the excision step is catalyzed usually by a DNA deoxyribophosphodiesterase rather than by an exonuclease. Gap-filling and ligation complete the repair reaction. Experiments with enzyme inhibitors in mammalian cell extracts suggest that the repair replication step is catalyzed by DNA polymerase 13.DNA bases altered by hydrolytic deamination, oxygen radical-induced damage, and alkylation are often excised by DNA glycosylases (44,54). Certain mismatched residues, such as adenine or thymine opposite guanine, may also be removed by specific DNA glycosylases (1, 56, 57). Thus, a common intermediate in the repair of several different types of DNA damage is an apurinic-apyrimidinic (AP) site. Such sites are also generated by nonenzymatic hydrolysis of base-sugar bonds, in particular by depurination (26), and as a consequence of exposure to ionizing radiation (12,27,34). AP endonucleases, for example, Escherichia coli exonuclease III and endonuclease IV, rapidly incise DNA at the 5' side of an AP site (9). Subsequently, an excision step occurs to generate a small gap, which is filled in by repair replication. A DNA ligase completes the repair process. These consecutive events take place rapidly and efficiently in vivo. It has been shown that AP sites occur transiently in DNA after X-irradiation of mammalian cells but disappear in 2 to 4 min without any detectable accumulation of DNA containing single-strand interruptions (34).The excision of a depurinated-depyrimidinated site has remained the least understood of these events in the base excision repair process. Early work by Painter and Young (37) and Regan and Setlow (40) showed that repair replication of X-irradiated and alkylated DNA in vivo involved the filling-in of much smaller gaps than those observed after UV light-induced DNA damage. Subsequent studies have also indicated that patches only 1 to 4 nucleotides long may be generated during repair of AP sites (8,32). The excision of a 5'-terminal base-free sugar-phosphate residue, possibly accompanied by the removal of one or a few adjacent residues, has been ascribed to exonuclease acti...

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“…The resultant abasic sites are then recognized by AP endonucleases that bind and catalyze a magnesium-dependent 5′ cleavage of the phosphodiester backbone, producing a 3′-OH. Other bifunctional DNA glycosylases cleave the resulting abasic site via an AP lyase mechanism, which leaves a 3′ unsaturated aldehyde (5). In either case, DNA backbone cleavage marks the AP site for subsequent repair by DNA polymerases and ligases.…”

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confidence: 99%

Structural basis for the recognition and cleavage of abasic DNA in Neisseria meningitidis

Lü

Šilhán

MacDonald

et al. 2012

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

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Base excision repair (BER) is a highly conserved DNA repair pathway throughout all kingdoms from bacteria to humans. Whereas several enzymes are required to complete the multistep repair process of damaged bases, apurinic-apyrimidic (AP) endonucleases play an essential role in enabling the repair process by recognizing intermediary abasic sites cleaving the phosphodiester backbone 5′ to the abasic site. Despite extensive study, there is no structure of a bacterial AP endonuclease bound to substrate DNA. Furthermore, the structural mechanism for AP-site cleavage is incomplete. Here we report a detailed structural and biochemical study of the AP endonuclease from Neisseria meningitidis that has allowed us to capture structural intermediates providing more complete snapshots of the catalytic mechanism. Our data reveal subtle differences in AP-site recognition and kinetics between the human and bacterial enzymes that may reflect different evolutionary pressures.X-ray crystallography | genome stability | prokaryote A basic (apurinic-apyrimidic, AP) sites within DNA pose a serious threat to all living organisms; if unrepaired, they lead to stalled replication, increased mutations, and an overall loss of genomic integrity. For example human cells defective in AP-site processing are nonviable (1). AP sites occur through either the spontaneous loss of nucleotide bases via chemical breakage of the N-glycosylic bond or as intermediates in the repair of DNA lesions such as oxidatively damaged bases, spontaneously deaminated cytosines, or alkylation of bases (2). It is estimated that 10 4 abasic sites are produced per day per cell in eukaryotes and if left unrepaired will result in significant mutagenic loading on the cell (3, 4). All living organisms have therefore evolved specific enzymes that recognize and repair damaged or missing DNA bases known as the base excision repair (BER) pathway (2).In BER, DNA glycosylases recognize and remove damaged bases to produce abasic sites. The resultant abasic sites are then recognized by AP endonucleases that bind and catalyze a magnesium-dependent 5′ cleavage of the phosphodiester backbone, producing a 3′-OH. Other bifunctional DNA glycosylases cleave the resulting abasic site via an AP lyase mechanism, which leaves a 3′ unsaturated aldehyde (5). In either case, DNA backbone cleavage marks the AP site for subsequent repair by DNA polymerases and ligases. Two main families of AP endonucleases have been identified: the major human AP endonuclease (APEX1) and Escherichia coli homolog ExoIII belong to the ExoIII family of AP endonucleases and are highly conserved (∼51% sequence homology; ∼29% sequence identity). The other is the EndoIV family, named after the E. coli enzyme, and they cleave abasic sites in a Zn 2+ -dependent reaction. Both E. coli and Saccharomyces cerevisiae possess both ExoIII and EndoIV type enzymes, whereas humans have two ExoIII family enzymes.Molecular and biochemical details of abasic DNA recognition and backbone cleavage have mainly focused on the human APEX1 enz...

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Analysis of class II (hydrolytic) and class I (β-lyase) apurinic/apyrimidinic endonucleases with a synthetic DNA substrate

Cited by 90 publications

References 32 publications

Clusters of S1 Nuclease-Hypersensitive Sites Induced In Vivo by DNA Damage

Clusters of S1 Nuclease-Hypersensitive Sites Induced In Vivo by DNA Damage

Generation of single-nucleotide repair patches following excision of uracil residues from DNA.

Structural basis for the recognition and cleavage of abasic DNA in Neisseria meningitidis

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