Further characterization of a depurinated DNA-purine base insertion activity from cultured human fibroblasts.

The purification from cultured human fibroblasts of a protein that binds specifically to partially depurinated DNA and inserts purines into those sites is described. The purine insertion, but not the binding, requires K+. The DNA binding can be saturated with increasing apurinic sites and is weakened by the presence of adenine or guanine. Base insertion into depurinated DNA is specific for adenine or guanine; none is observed with dATP or dGTP. When the depurinated DNA substrate is specifically cleaved with apurinic endonuclease, no purine insertion occurs. Guanine insertion does not occur into tRNA or depyrimidinated DNA, and thymine is not inserted into either depyrimidinated DNA or depurinated DNA. Purine insertion activity follows Michaelis-Menten kinetics with respect to purintes; the apparent Km values for both adenine and guanine are 5 microM. The enzyme binds the purine bases very tightly. Adenine binding saturates at less than 1 microM adenine, perhaps reflecting the low intracellular adenine concentration. The binding protein specific for UV-irradiated DNA (Feldberg, R.S., and Grossman, L. (1976) Biochemistry 15, 2402-2408) had no detectable purine or pyrimidine base insertion activity with depurinated or depyrimidinated DNAs.     

Characterization of a depurinated-DNA purine-base-insertion activity from Drosophila.

An activity that binds preferentially to depurinated DNA and inserts purines into those sites was partially purified from Drosophila melanogaster embryos. The protein has a sedimentation coefficient of 4.9 S and is devoid of AP (apurinic/apyrimidinic) endonuclease activity. Upon incorporation of purines into apurinic DNA, the number of alkali-labile sites decreases, thus establishing the conversion of depurinated sites into normal nucleotides. The activity requires K+, and is totally inhibited by caffeine or EDTA. Guanine is specifically incorporated into partially depurinated poly(dG-dC) and adenine is specifically incorporated into poly(dA-dT), thus demonstrating the apparent template specificity of the enzyme.     

Survival of apurinic SV40 DNA in the d-complementation group of xeroderma pigmentosum.

The survival of depurinated Form I SV40 DNA was studied in normal human fibroblasts and in D-complementation Xeroderma pigmentosum (XP) fibroblasts. Survival was measured with an infective center assay. Heat-acid and methyl methanesulfonate (MMS) were used as depurinating agents. After 3 hrs of depurination by heat--acid treatment, infectivity in normal cells was less than 15% of the controls compared to more than 50% for the XP D cell strains. Similar results were obtained with MMS-treated DNA. These results are contrary to expectation since apurinic endonuclease activity, which is presumed to be involved in the repair of apurinic sites, is much lower in XP D cell strains than in normal cell strains. Our results indicate that another mechanism for the repair of apurinic sites could exist.     

Comparison of human plasma low- and high-density lipoproteins as substrates for lecithin: cholesterol acyltransferase

The interstrand crosslinks that appear in stored depurinated DNA interfere with the counting of apurinic sites and strand breaks by sucrose gradient analysis. They could not be cleaved at acid or alkaline pH, or by treatment with methoxyamine.     

Apurinic sites cause mutations in simian virus 40

SV40 has been used as a molecular probe to study the mutagenicity of apurinic sites (Ap) in mammalian cells. Untreated or UV-irradiated monkey kidney cells were transfected with depurinated DNA from the temperature-sensitive tsB201 SV40 late mutant which grows normally at the permissive temperature of 33 degrees C but which is unable to grow at 41 degrees C. Phenotypic revertants were screened at 41 degrees C for their ability to grow at the restrictive temperature and the mutation frequency was calculated in the viral progeny. Ap sites were introduced into DNA by heating at 70 degrees C under acid conditions (pH 4.8). This treatment induces one Ap site per SV40 genome per 15 min of heating as measured by alkaline denaturation or by treatment with the T4-encoded UV-specific endonuclease which possesses Ap-endonuclease activity. The experiments reported here show that Ap sites strongly decrease virus survival with a lethal hit corresponding roughly to 3 Ap lesions per SV40 genome, and indicate for the first time that apurinic sites produced by heating are highly mutagenic in animal cells. UV irradiation of the host cells 24 h prior to transfection with depurinated DNA did not modify the mutation frequency in the virus progeny.     

Some properties of the interstrand crosslinks in depurinated DNA

The interstrand crosslinks that appear in stored depurinated DNA interfere with the counting of apurinic sites and strand breaks by sucrose gradient analysis. They could not be cleaved at acid or alkaline pH, or by treatment with methoxyamine.     

Purification and properties of a Bacillus subtilis endonuclease specific for apurinic sites in DNA.

An endonuclease which hydrolyzes depurinated DNA has been purified from extracts of Bacillus subtilis cells. The endonuclease is a monomeric protein and has a molecular weight of around 56,000. The enzyme is specific for apurinic sites in double-stranded DNA, has a pH optimum at 8.0, and is slightly stimulated with 50 mM NaCl but completely inhibited with 500 mM NaCl. It requires no divalent cations and is insensitive to EDTA; it has no associated exonuclease. These properties are very similar to those of Escherichia coli endonuclease IV, which is also insensitive to EDTA and has no exonuclease activity, and very different from those of the main endonuclease for apurinic sites (endonuclease IV) of the same bacterium.     

Purification and properties of a plant endonuclease specific for apurinic sites.

An endonuclease which hydrolyzes depurinated DNA has been isolated from Phaseolus multiflorus enbryos; it has a molecular weight around 40,000. The enzyme is specific for apurinic sites; it has no action on normal DNA strands or on alkylated sites, and is without exonulcease activity. The rate of phosphoester bond hydrolysis near apurinic sites is far greater in native than in denatured DNA. The endonuclease is not inactivated by 10 mM EDTA, but is activity is however stimulated by Mg2+ or Mn2+. Its optimum pH is 7.5 to 8.0, and its optimum temperature 40degrees although, at this temperature, it is rapidly denatured; even low NaCl concentrations inhibit the enzyme activity. The endonuclease for apurinic sites of P. multiflorus is a non-histone protein of chromatin; the properties (like thermosensitivity of susceptibility to ionic strength) of the enzyme in situ, working on chromatin DNA, might be different from those described for the isolated endonuclease in homogenous aqueous solution.     

Response of phage T4 polynucleotide kinase toward dinucleotides containing apurinic sites: design of a 32P-postlabeling assay for apurinic sites in DNA

We have examined the capacity of bacteriophage T4 polynucleotide kinase (EC 2.7.1.78) to phosphorylate the partially depurinated products of d-ApA, namely, d-SpA and d-ApS (where S represents an apurinic deoxyribose group). It was observed that the enzyme acted only on the latter isomer. Since molecules of this type (d-NpS) are the sole apurinic site containing products resulting from the combined digestion of lightly depurinated DNA by snake venom phosphodiesterase and calf alkaline phosphatase [Weinfeld, M., Liuzzi, M., & Paterson, M. C. (1989) Nucleic Acids Res. 17, 3735-3745], we were able to devise a postlabeling assay for these biologically important DNA lesions. The method offers several advantages, including (a) elimination of the need for prelabeled DNA, (b) high (femtomole range) sensitivity, and (c) nearest-neighbor analysis of bases 5' to apurinic/apyrimidinic sites. Using this assay, we obtained a value for the rate of depurination of form I pRSVneo plasmid DNA, incubated at pH 5.2 at 70 degrees C, of approximately 3.3 apurinic sites per plasmid molecule per hour. This value compares favorably with previously published data of others, acquired by alternative approaches. The rate of depurination of poly(dA), treated in a similar fashion, was found to be approximately 1 base per 10(3) nucleotides per hour.     

den V gene of bacteriophage T4 codes for both pyrimidine dimer-DNA glycosylase and apyrimidinic endonuclease activities.

Recent studies have shown purified preparations of phage T4 UV DNA-incising activity (T4 UV endonuclease or endonuclease V of phage T4) contain a pyrimidine dimer-DNA glycosylase activity that catalyzes hydrolysis of the 5' glycosyl bond of dimerized pyrimidines in UV-irradiated DNA. Such enzyme preparations have also been shown to catalyze the hydrolysis of phosphodiester bonds in UV-irradiated DNA at a neutral pH, presumably reflecting the action of an apurinic/apyrimidinic endonuclease at the apyrimidinic sites created by the pyrimidine dimer-DNA glycosylase. In this study we found that preparations of T4 UV DNA-incising activity contained apurinic/apyrimidinic endonuclease activity that nicked depurinated form I simian virus 40 DNA. Apurinic/apyrimidinic endonuclease activity was also found in extracts of Escherichia coli infected with T4 denV+ phage. Extracts of cells infected with T4 denV mutants contained significantly lower levels of apurinic/apyrimidinic endonuclease activity; these levels were no greater than the levels present in extracts of uninfected cells. Furthermore, the addition of DNA containing apurinic or apyrimidinic sites to reactions containing UV-irradiated DNA and T4 enzyme resulted in competition for pyrimidine dimer-DNA glycosylase activity against the UV-irradiated DNA. On the basis of these results, we concluded that apurinic/apyrimidinic endonuclease activity is encoded by the denV gene of phage T4, the same gene that codes for pyrimidine dimer-DNA glycosylase activity.     

A barley endonuclease specific for apurinic DNA. Isolation and partial characterization

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 18000.     

Depurination-induced infidelity of deoxyribonucleic acid synthesis with purified deoxyribonucleic acid replication proteins in vitro

Removal of purine bases from phi X174 single-stranded DNA leads to increased reversion frequency of amber mutations when this DNA is copied in vitro with purified DNA polymerases. This depurination-induced mutagenesis is observed at three different genetic loci and with several different purified enzymes, including Escherichia coli DNA polymerases I and III, avian myeloblastosis virus DNA polymerase, and eukaryotic DNA polymerases alpha, beta, and gamma. The extent of mutagenesis correlates with the estimated frequency of bypass of the lesion and is greatest with inherently inaccurate DNA polymerases which lack proofreading capacity. With E. coli DNA polymerase I, conditions which diminish proofreading result in a 3-5-fold increase in depurination-induced mutagenesis, suggesting a role for proofreading in determining the frequency of bypass of apurinic sites. The addition of E. coli single-stranded DNA-binding protein to polymerase I catalyzed reactions with depurinated DNA had no effect on the extent of mutagenesis. Analysis of wild-type revertants produced during in vitro DNA synthesis by polymerase I or avian myeloblastosis virus DNA polymerase on depurinated phi X174 amber 3 DNA indicates a preference for insertion of dAMP opposite the putative apurinic site at position 587. These results are discussed in relation both to the mutagenic potential of apurinic sites in higher organisms and to studies on error-prone DNA synthesis.     

Relationship between DNA acid-solubility and frequency of single-strand breaks near apurinic sites

Using [32P]DNA alkylated with [3H]methyl methanesulfonate, depurinated by heating at 50 degrees C for various periods, then treated with sodium hydroxide, a table was constructed giving the DNA fraction soluble in 5% perchloric acid at 0 degree C as a function of the frequency of strand breaks. The alkaline treatment placed a break near each apurinic site; the apurinic sites were counted in two ways which gave consonant results: by the loss of [3H]methyl groups and by reaction with [14C]methoxyamine. The 32P label of DNA was used to measure the acid-solubility.     

Isolation from barley embryo of endonuclease specific for apurinic sites in DNA

The endonuclease activity specific for apurinic sites in DNA was detected in barley embryos. The enzyme was partially purified. It reveals high activity on partially depurinated DNA but low or nil activity on intact and alkylated DNA. The method used for the detection of enzyme activity was based on the changes in the sedimentation velocity of substrate DNA in neutral sucrose gradients with 80 % formamide.     

The phosphonomethyl analogue of 3-phosphoglycerate is a potent competitive inhibitor of phosphoglycerate mutases.

Deoxyribonuclease IV, a 5'-3' exonuclease degrading double-stranded DNA from intra-strand nicks, has been purified from the chromatin of rat liver cells. The enzyme, which has an Mr of 58000, excises the apurinic (AP) sites from a depurinated DNA nicked 5' to these AP sites with the chromatin AP endonuclease. The excision is not the result of hydrolysis of the phosphodiester bond 3' to the AP sites since the excision product does not behave as deoxyribose 5-phosphate but as its 2,3-unsaturated derivative. This result suggests that, to remove the AP sites from the DNA nicked by an AP endonuclease, the chromatin deoxyribonuclease IV rather acts as a catalyst of beta-elimination.     

Apurinic DNA endonucleases from Drosophila melanogaster embryos

Summary Apurinic DNA endonuclease activity from Drosophila melanogaster embryos was resolved into two separable forms by phosphocellulose chromatography, one which flowed through the column (Fraction I) and the other which was retained and eluted at approximately 200 mM potassium phosphate (Fraction II). Both fractions, purified further by glycerol gradient sedimentation, were found to introduce nicks into DNA that were specific for and equal in number to the alkali-labile sites in depurinated DNA. They had similar apparent K_m values for apurinic sites (0.7 nM apurinic sites for Fraction I and 0.8 nM for Fraction II), but differed with respect to optimal pH, Mg⁺⁺ requirement and sensitivity to EDTA.     

Properties of 3-methyladenine-DNA glycosylase from Escherichia coli.

An Escherichia coli enzyme that releases 3-methyladenine and 3-ethyladenine in free form from alkylated DNA has been purified 2800-fold in 7% yield. The enzyme does not liberate several other alkylation products from DNA, including 7-methylguanine,O6-methylguanine, 7-methyladenine, N6-methyladenine, 7-ethylguanine, O6-ethylguanine, and the arylalkylated purine derivatives obtained by treatment of DNA with 7-bromomethyl-12-methylbenz[a]anthracene. The reaction of the enzyme with alkylated DNA leads to the introduction of apurinic sites but no chain breaks (less than one incision per ten apurinic sites), and there is no detectable nuclease activity with native DNA, depurinated DNA, ultraviolet-irradiated DNA, or X-irradiated DNA as potential substrates. The enzyme is termed 3-methyladenine-DNA glycosylase. It is a small protein, Mr = 19 000, that does not require divalent metal ions, phosphate, or other cofactors in order to cleave base-sugar bonds in alkylated DNA.     

Partial purification and characterization of a human 3-methyladenine-DNA glycosylase.

A DNA glycosylase was purified about 30-fold from cultured human lymphoblasts (CCRF-CEM line) and was found to cleave 3-methyladenine from DNA alkylated with methyl methanesulfonate. The enzyme did not promote the release of 1-methyladenine, 7-methyladenine, or 7-methylguanine from DNA nor did it act on denatured methylated DNA. It produced apurinic sites in DNA alkylated with N-methyl-N-nitrosourea and ethyl methane-sulfonate as well as methyl methanesulfonate but not in untreated DNA or in DNA alkylated with nitrogen mustard or irradiated with ultraviolet light or X-rays. The glycosylase was free of detectable endonuclease activity in experiments with untreated DNA or DNA exposed to ultraviolet light; low levels of endonuclease activity, obtained when X-irradiated, alkylated, or depurinated DNA was the substrate, were attributed to contaminant apurinic endonuclease activity. This 3-methyladenine-DNA glycosylase has an estimated molecular weight of 34,000, is not dependent on divalent metal ions, and shows optimal activity at pH 7.5--8.5.     

Apurinic endonuclease from Saccharomyces cerevisiae.

An endonuclease cleaving depurinated and alkylated double-stranded DNA has been purified 500-fold from Saccharomyces cerevisiae, strain MB 1052. The enzyme has an Mr of 31 000 +/- 2000, a sedimentation value of 3.2S and a diffusion coefficient of 9.5 X 10-7 cm2/s. The enzyme was active only at apurinic/apyridiminic sites, regardless of whether they were produced by heating the DNA at acidic pH or by alkylation with the ultimate carcinogen methyl methanesulphonate. Native DNA was not acted upon. U.v.-irradiated DNA and DNA treated with the ultimate carcinogen N-acetoxy-2-acetylaminofluorene were cleaved to an extent related to the extent of apurinic/apyridiminic sites. Enzymic activity was not dependent upon Mg2+, but was stimulated approx. 3-fold by 4mM-Mg2+. The enzyme did not bind to DEAE-cellulose or CM-cellulose at KCl concentrations greater than 160 mM. The endonuclease was obtained free of exonuclease and 3-methyladenine-DNA glycosylase activity in five chromatographic steps.     

Single-strand-specific nuclease of pea seeds: glycoprotein nature and associated nucleotidase activity

A single-strand-specific nuclease from germinating pea seeds has been purified to homogeneity. The purification procedure includes affinity chromatography on concanavalin A-Sepharose and gel filtration. The nuclease exhibits its activity at neutral pH and does not have an absolute requirement for a divalent cation. The purified nuclease also possesses a 3'-nucleotidase activity and is a glycoprotein containing about 20% carbohydrate. On native polyacrylamide gels the nuclease activity comigrates with the nucleotidase. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of two subunits in the native enzyme. The nuclease and nucleotidase activities show differential rates of thermal inactivation, the latter following simple first order kinetics and the former exhibiting a more complex reaction. The nucleotidase was also found to be stimulated by DNA, the increase being greater with native DNA than with denatured DNA. These properties are possibly accounted for by the dimeric structure of the enzyme where the nucleotidase catalytic site resides in one subunit while the nuclease site is formed by interaction of both subunits. The enzyme also hydrolyzes double-stranded alkylated DNA and depurinated DNA at a higher rate than native DNA. Experimental evidence suggests that depurinated DNA is hydrolyzed in the region of apurinic sites.