Enzymatic insertion of purine bases into depurinated DNA in vitro.

Livneh, Zvi; Elad, D.; Sperling, Joseph

doi:10.1073/pnas.76.3.1089

Cited by 54 publications

(22 citation statements)

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“…Base insertion enzymes (insertases) have been detected in several organisms (5,6,15 13 and DNase V can form a complex (21), suggesting a concerted action of two enzymes in DNA repair. An alternative pathway is suggested by the recent report by Franklin and Lindahl (8).…”

Section: Resultsmentioning

confidence: 99%

Repair of a synthetic abasic site in DNA in a Xenopus laevis oocyte extract.

Matsumoto¹,

Bogenhagen²

1989

Mol. Cell. Biol.

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Covalently closed circular DNA containing a synthetic analog of an abasic site at a unique position was used as a substrate to study DNA repair. Incubation of this DNA in Xenopus laevis oocyte extracts resulted in rapid cleavage of the DNA at the abasic site by a class II apurinic-apyrimidinic endonuclease, followed by complete repair within 40 min. Nicked circular DNAs persisted for several minutes before repair by an ATP-dependent DNA synthesis reaction. The repair-related DNA synthesis was localized within 3 or 4 nucleotides surrounding the abasic site. These results are consistent with the short-patch repair reported for DNA damage at heterogeneous sites in human cells (J. D. Regan and R. B. Setlow, Cancer Res. 34:3318-3325, 1974).DNA repair follows a variety of pathways in response to various kinds of DNA damage (reviewed in references 9 and 26). Genetic and biochemical analyses have identified sequential reactions in these pathways that involve a host of repair-related enzymes. Nevertheless, our knowledge of repair mechanisms, especially in eucaryotes, remains fragmentary. In most studies on repair, DNA substrates are prepared by treatment of whole cells or DNA molecules with DNA-damaging agents. The resulting randomly distributed adducts are poorly suited for detailed analyses throughout the repair process.Recent technical progress in chemical DNA synthesis has made it possible to introduce a specific adduct into DNA in a site-directed manner (reviewed in reference 3). Grollman and Johnson and their colleagues have succeeded in synthesizing oligonucleotides containing an analog of one of the most common products of DNA damage, an abasic site (28). The apurinic-apyrimidinic (AP) sites are not only generated spontaneously or by direct action of damaging agents but are also common intermediate products in base excision repair. The 3-hydroxy-2-hydroxymethyltetrahydrofuran residue is a structural analog of the sugar residue in the natural AP site. This alkali-stable tetrahydrofuran moiety can be introduced into oligonucleotides during automated DNA synthesis. When inserted into double-stranded DNA, this synthetic AP site is recognized and cleaved by bacterial and mammalian AP endonucleases (26a, 28).We have used DNA substrates bearing tetrahydrofuran residues at defined positions to develop accurate and sensitive assays for reactions involved in repair of this lesion. In this paper, we describe the vigorous repair of an abasic site in a cell-free system derived from Xenopus laevis oocytes. Similar oocyte extracts have been used in studies of transcription and chromatin assembly (10,18 ase activity were detected in some commercially supplied enzymes. We used only restriction enzymes free from contamination by AP endonucleases as determined by our preliminary tests. T3 RNA polymerase and avian myeloblastosis virus reverse transcriptase were from Stratagene and Life Sciences, Inc., respectively. 32P-labeled nucleotides were obtained from ICN Radiochemicals. Adult X. laevis females were obtained from Xenopus I, An...

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Section: Resultsmentioning

confidence: 99%

Repair of a synthetic abasic site in DNA in a Xenopus laevis oocyte extract.

Matsumoto¹,

Bogenhagen²

1989

Mol. Cell. Biol.

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“…was extracted twice with equal volumes of phenol, and 40 Ml of the DNA solution was electrophoresed on agarose gels to separate the superhelical circular DNA (fastmoving band) from the nicked circular DNA (slow-moving band). Details of the procedure have been described (31).…”

Section: Methodsmentioning

confidence: 99%

Endonucleolytic activity directed towards 8-(2-hydroxy-2-propyl) purines in double-stranded DNA.

Livneh¹,

Elad²,

Sperling³

1979

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

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Photoalkylation of circular covalently closed DNA from phage PM2 with isopropyl alcohol by using a free radical photoinitiator and UV light of X > 305 nm led to the specific 8-substitution of purine moieties in the DNA, yielding 842-hydroxv-2-propyl)adenine and 8(2-hydroxy-2-propyl)guanine as the only detectable damage in the DNA. Using this specifically photoalkylated DNA as a substrate, we discovered in extracts of Micrococcus luteus an endonucleolytic activity that is directed towards 8(2-hydroxy-2-propyl) purines in DNA. The activity is not a combination of a DNA-glycosylase and an apurinic site endonuclease. It is not inhibited by single-stranded DNA, by UV-or y-irradiated single-stranded DNA, or by normal or depurinated double-stranded DNA; however, 'y-or UV-(254 nm) irradiated double-stranded DNAs do inhibit the activity, hinting at the possibility of a common type of lesion in these damaged DNAs. Divalent cations are not required for the incising activity, and it is fully active in 1 mM EDTA, whereas caffeine and ATP cause inhibition. Extracts of mutant M. luteus lacking pyrimidine-dimer-directed endonucleases were found to contain the endonucleolytic activity in levels comparable to those present in the wild type. After the incision, we could demonstrate the specific excision of the 8-alkylated purines from the damaged DNA. The special conformational consequences of the 8-a kylation of purines, at the nucleotide level, namely their nonregular syn conformation, suggest that it is the distortion in the DNA that is recognized by the endonuclease. Nucleotide excision repair is one of the main repair mechanisms that acts to restore the structural integrity of damaged DNA. It is initiated by an endonuclease that incises the damaged DNA strand in the neighborhood of the damage, followved by excision of the damaged sequences by an exonuclease, repolymerization by a DNA polymerase, and finally covalent sealing by DNA ligase to restore the DNA molecule (1-3).The nucleotide excision repair is initiated by specific endonucleases that recognize chemically modified regions, or consequent conformational changes in the DNA, and incise the DNA at the vicinity of such lesions. So far only two endonucleases that are directed towards known chemically defined lesions have been described. These are the pyrimidine-dimer endonuclease (4-6) and the apurinic/apyrimidinic site endonuclease (7-13). Other endonuicleases have been found to incise UV-, 'y-ray-, or chemically damaged DNA, but their specificities are not yet known (14)(15)(16)(17)(18)(19)(20)(21)(22). This is mainly due to the fact that the damaged DNA substrates usually contain a multiplicity of products, not all of them chemically characterized, thus preventing an unambiguous assignment of a specific enzyme to a defined lesion. This difficulty interferes with the characterization of repair endontucleases and with the elucidation of the molecular basis of the damage to enzyme interactions, which play a key role in the initiation of the repair process.We have a...

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“…There are several reports of an enzyme that can directly reinsert purines at apurinic sites in DNA, both in E. coli (78) and in human fibroblasts (79,80). Such a proposed activity would be reminiscent of certain tRNA modifying enzymes such as the one that removes an unsubstituted guanine residue within the anticodon region by cleavage of a glycosyl bond and inserts a hypermodified guanine at the same site (81).…”

Section: Do Purine Insert Ases Exist?mentioning

confidence: 99%