An endonuclease of Escherichia coli active on a DNA treated with methylmethane sulfonate has been separated from an endonuclease active on depurinated sites. The former enzyme is designated here as endonuclease II, while the latter enzyme is designated as apurinic acid endonuclease. Endonuclease II is also active on DNA treated with-methylnitrosourea, 7-bromomethyl-12-methylbenz[a anthracene, and 'y-irradiation. A third fraction which contains activities for both depurinated and alkylated sites needs further study. Endonuclease II, molecular weight 33,000, has been purified 12,500-fold and does not have exonuclease III activity. Apurinic acid endonuclease, molecular weight 31,500, has been purified 11,000-fold and does not have exonuclease III activity. Exonuclease III, molecular weight 26,000, has been purified 2300-fold and does not have endonucleolytic activity at depurinated reduced sites or at alkylated sites in DNA. Therefore, these are three separate proteins. Exonuclease III can produce, presumably by its exonucleolytic activity, double-strand breaks in heavily alkylated DNA under conditions where it does not make single-strand endonucleolytic breaks at either depurinatedreduced or alkylated sites. The first purpose of this paper is to define endonuclease II of Escherichia coli as an activity different from the apurinic acid endonuclease of E. coli. Strauss and Robbins first described an endonucleolytic activity in extracts of Bacillus subtilis that recognized alkylated DNA (1). In this laboratory, an enzyme in extracts of E. coli, active on heavily alkylated DNA, was partially purified, characterized, and designated endonuclease II of E. coli (2, 3). The substrate used for these experiments was DNA that was entrapped in a polyacrylamide gel and then alkylated with methylmethane sulfonate [MeSO2OMe (MMS)] at an MeSO2OMe-to-nucleotide ratio of 6000 to 1. A partially purified preparation of endonuclease II was also found to have an endonucleolytic activity on depurinated reduced DNA (4), and this activity was thought to be due to the same enzyme that was active on MeSO20Me-treated DNA. However, Verly et al. (5, 6), using the purification procedure originally described in this laboratory, obtained an enzyme that was active on depurinated DNA but not on alkylated DNA. Subsequently, we succeeded in separating the activity on depurinated sites in DNA from the activity on MeSO2OMe-treated DNA (7,8). The former we designate as the apurinic acid endonuclease of E. coli, while the latter we designate as endonuclease II of E. coli. Endonuclease II of E. coli is also active on DNA treated with methylnitrosourea, 7-bromomethyl-12-methvlbenz-[alanthracene, and y-irradiation (7-11).The second purpose of this paper is to demonstrate that endonuclease II, the apurinic acid endonuclease, and exonuclease III are separate proteins. Originally, Yajko and Weiss (12) demonstrated that a number of E. coli mutants deficient in exonuclease III were also deficient in "endonuclease II" and vice versa. The "endonucleas...
Endonuclease II (deoxyribonucleate oligonucleotidohydrolase, EC 3.1.4.30) of Escherichia coli has been shown to break phosphodiester bonds in alkylated DNA and depurinated DNA. The hypothesis that depurination. is a step in the mechanism of the reaction with alkylated DNA is supported by in vitro experiments with DNA reacted with N-methyl-Nl-nitrosourea. Endonuclease I1 releases 08-methylguanine and 3-methyladenine, but not 7-methylguanine, from DNA that has been methylated by the carcinogen N-methyl-N-nitrosourea.Endonuclease II (deoxyribonucleate oligonucleotidohydrolase, EC 3.1.4.30) of Escherichia coli is an enzyme capable of breaking p)hosphodiester bonds in DNA which has been reacted with the alkylating agent methyl methanesulfonate (MAIS) (1-3). MMTIS-treated DNA contain.s 7-methylguanine and 3-methyladenine. Endonuclease IT also recognizes dep)urinated and depurinated-reduced sites in DNA (4) and at high concentrations makes a limited iiumber of single-strand breaks in native DNA from T-4 and T-7 bacterioplhages (5). The enzyme hydrolyses the phosphodiester bond.s in native D)NA to yield 5'-phosphomonoesters (5), and indirect evidence has been presented to support the proposal that enzymeinduced chain breaks are on the 5'-phosphate side of the depurinated reduced sites (5). Because the enzyme recognizes both alkylated and depurinated sites in the DNA, it was l)ostillated that the process of depurination was an intermediate step prior to phosphodiester bond cleavage of alkylated DNA. Enzymatic depurination by the endonuclease II preparation has now been demonstrated. This has allowed us to observe a specificity of the enzyme for some but not all of the methylated bases. This paper describes the ability of the enzyme to release 00-methylguanine and 3-methyladenine, and the inability of the enzyvme to release 7-methylguanine from DNA 'that has been methylated by the carcinogen ,Nmethyl-N-nitrosourea (MNU).
Irradiation of DNA in a nitrogen atmosphere with 60Co gamma-radiation produces at least two types of damage. The first type leads to single strand breaks in the DNA observed after exposure to alkali. This type of alkali-labile bond will be designated a spontaneous break. The second type of damage to DNA is an alteration which makes the DNA susceptible to phosphodiester bond hydrolysis by a 1600-fold purified preparation of endonuclease II of Escherichia coli and is designated an enzyme-sensitive site. This site is not alkali-labile. After irradiation, preincubation of the DNA either for days at 0 degrees or for 4 hr at 37 degrees increases both the spontaneous breaks and the enzyme sensitive sites. There is a greater increase of spontaneous breaks when the preincubation is in O2 compared to N2. The increase of enzyme sensitive sites due to the preincubation is not altered significantly by O2. The increase of spontaneous breaks during the preincubation is almost completely prevented by addition of either NaBH4 or NH2OH after the irradiation. The treatment can be before or after the preincubation. This effect indicates that these breaks are due to alkali-labile bonds possibly produced by depurination or depyrimidination reactions. That the spontaneous breaks are due primarily to alkali-labile bonds is supported by an experiment in which formamide gradients were used. Neither NaBH4 nor NH2OH has any effect on the enzyme sensitive sites. Addition of beta-mercaptoethanol (0.5 M) at the start of the preincubation prevents in part the appearance of both spontaneous breaks and enzyme-sensitive sites. It has no effect when added at the end of the preincubation. Catalase added before the preincubation has no effect on either type of damage. It is postulated that the spontaneous breaks occur because purine or pyrimidine radicals are formed (possibly hydroxyl radicals) which can then interact with oxygen to produce unstable intermediates. The intermediates then undergo either depurination or depyrimidination. The subsequent alkali catalyzed beta-elimination reaction of depurinated or depyriminidinated DNA is prevented by NaBH4 or NH2OH. An alternative hypothesis would involve damage to the sugar rather than to bases. The enzyme-sensitive sites represent another form of base damage which is not oxygen dependent. The chemical nature of either form of primary damage is not known.
A nonradioactive, colorimetric microplate hybridization procedure was used to assay human immunodeficiency virus (HIV) DNA, amplified by the polymerase chain reaction (PCR). Under the PCR conditions used, four proviral copies per 150,000 cells were detected by amplifying a series of DNA mixtures that contained various copy numbers of HIV. Assays of PCR-amplified DNA from peripheral blood mononuclear cells of seronegative individuals yielded negative results (104 of 104), whereas samples from seropositive individuals yielded > 99% positive results (141 of 142). Similar results were obtained in a chemiluminescent assay with an acridinium ester-labeled probe and in a solution hybridization assay in which a 32P-labeled probe was used.
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