A Barley Endonuclease Specific for Apurinic DNA

Abstract

An endonuclease specific for depurinated native DNA was isolated and partially purified from extracts of barley leaves. The procedure included streptomycin sulphate precipitation, ammonium sulphate fractionation, phosphocellulose, hydroxyapatite and Sephadex G-150 chromatography. Purity of the resulting enzyme was determined by gel electrophoresis and gel chromatography and specificity by testing the activity on intact and depurinated bacterial DNAs. At lower concentrations, the enzyme is specific for DNA containing apurinic sites. At higher concentrations, however, it degrades DNA in a non-specific manner. The nuclease has a pH optimum at 7.6, and a molecular weight of about 18 000.Endonuclease activities which introduce singlestrand breaks into native DNA containing apurinic sites have been found in extracts of bacteria [I -41, mammalian tissues [5], human cells [6,7] and also higher plants [8-lo]. There is good evidence to support the assumption that at least some of these enzymes, called apurases, play a role in the repair of apurinic sites in vivo. Mutants of Escherichia coli with low levels in apurase are sensitive to methyl methanesulphonate [I 1,141. In human fibroblasts derived from patients with xeroderma pigmentosum complementation groups A and D apurase manifested lower activity, different pH dependence and altered K, compared to the apurase of normal human fibroblasts [7]. In cooperation with E. coli polymerase I and T4 ligase, apurase from either E. coli or Micrococcus luteus completely repaired depurinated DNA in vitro [12,13]. The relatively high rate of spontaneous depurination of DNA in vivo is also hardly compatible with a low rate of spontaneous mutagenesis or cell lethality without assuming a repair function of apurases in living cells [15].The enzyme has been purified from E. coli [16,17], rat liver [18], calf thymus [I91 and Phaseolus [20]. The barley apurases have previously been purified from barley embryos [21]. In this paper, their isolation Abbreviations. NaCl/Cit, 0.15 M NaCl in 0.015 M trisodium citrate, pH 7 ; Hepes, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid.Enzymes. Deoxyribonuclease I (EC 3.1.4.5); apurinic-DNAspecific endodeoxyribonuclease or apurase . from barley leaves is described. Part of the data published here has already been presented previously In barley, recovery from genetic damage [23] induced by several alkylating agents, is accompanied by the rejoining of single-strand breaks [23] and repair synthesis [24]. This communication presents additional data concerning the probable steps by which alkylated DNA may be repaired in higher plants.[221. MATERIALS AND METHODSApurase activity was assayed either according to Tomilin [4] or according to Paquette et al. [16] using the modification described by Ljungquist et al. [25]. E. coli B/3 thy-[thy~zine-~H]DNA (about lo3 counts . min-' . pg-') or E. coli [32P]DNA (about lo4 counts . min-' . pg-') were used as substrates.In Tomilin's method 2 ml of labelled DNA (100-250 pg/ml) were mixed with 4 ml of the solutio...

Apurinic acid endonuclease activity from mouse epidermal cells

“…The molecular weights of the apurinic endonucleases from calf thymus (48), calf liver (48), and placenta (49) are also around 30 000 daltons. However, two such enzymes with higher (50,59) and one with lower molecular weight (52) have been found. The present endonuclease resembles the analogous enzymes from calf liver and placenta in its chromatographic behaviour (48,49).…”

Apurinic endonuclease from Saccharomyces cerevisiae

“…Enzymes with similar substrate specificities have been isolated from a number of prokaryotes (Paterson, 1978) as well as from eukaryotic cells, namely rat liver , calf liver (Kuebler & Goldthwait, 1977), calf thymus (Ljungquist & Lindahl, 1974), placenta (Linsley et al, 1977) and seed embryos of the plant Phaseolus multiflorus (Thibodeau & Verly, 1977). Endonucleases active on depurinated DNA have also been shown to exist in barley (Svachulova et al, 1978), xeroderma pigmentosum fibroblasts (Kuhnlein et al, 1978), in malignant mouse epidermal cells grown in culture (Ludwig & Thielmann, 1979), and in extracts of HeLa cells (Teebor & Duker, 1975) and of a mouse cell line (Nes & Nissen-Meyer, 1978). reported on endonuclease activity specific for AP sites in 105 000g supernatants from wild type RAD yeast cells, and from the MeSO2OMe-sensitive mutants rad6, radl8-2, and rad21.…”

Isolation and Characterization of an Apurinic Endodeoxyribonuclease from the Anaerobic Thermophile Desulfotomaculum nigrificans

“…APendodeoxyribonuclease was first discovered in Escherichia coli (Verly & Paquette, 1972). This enzyme has been found in other bacteria (Bibor & Verly, 1978;Clements et al, 1978;Hecht & Thielman, 1978;Inoue & Kada, 1978;Ljungquist, 1977;Pierre & Laval, 1980;Sako et al, 1980a;Verly & Rassart, 1975), in animals (Brent, 1976;Kane & Linn, 1981;Linsley et al, 1977;Ljungquist & Lindahl, 1974;Teebor & Dukor, 1975;Thibodeau et al, 1980) and in a plant (Svachilova et al, 1978). Recently we reported the purification and characterization of a new magnesium-dependent APendodeoxyribonuclease from vegetative cells of Bacillus subtilis (Sako et al, 1980a, b;Sako et al, 1981).…”

“…Moreover, the molecular weights of the APendodeoxyribonuclease from E. coli (Ljungquist, 1977;Verly & Rassart, 1975), B. stearothermophilus (Bibor & Verly, 1978) (Ljungquist & Lindahl, 1974), calf liver (Kuebler & Goldthwait, 1977), placenta (Linsley et al, 1977), and HeLa cells (Kane & Linn, 1981) are approximately 30000. However, the molecular weights of APendodeoxyribonuclease from barley leaves (Svachilova et al, 1978) and rat-liver chromatin (Thibodeau et al, 1980) are 18000 and 12500, respectively. Substrate speciJicity.…”