DNA Purine Bases Research Articles

The method of crosslinking histones to DNA partly depurinated at neutral pH

The procedure of either reversible or stable “zero-length” crosslinking of histones through their lysine residues directly to DNA partly depurinated under mild conditions at neutral pH is described. DNA in chromatin was methylated with dimethyl sulfate. Methylated purine bases in DNA can be readily excised under mild conditions at neutral pH. Aldehyde groups formed in the depurinated residues of DNA react with the ϵ-amino groups of lysines in histones and crosslink histones to DNA through the Schiff bases. The Schiff bases catalyze the β-elimination reactions causing quantitative single-stranded scissions of DNA at the points of crosslinking in such a way that only the 5′-terminal fragments of DNA formed upon the breakage become attached to histones. These covalent DNA-protein crosslinks are reversible and can be split at pH 3 or stabilized by reduction of the Schiff bases with NaBH 4.

Preferential methylation of target organ DNA by the oesophageal carcinogen N-nitrosomethylbenzylamine.

Male Wistar rats received a single i.v. injection of the oesophageal carcinogen N-nitroso[methyl-14C]-methylbenzylnitrosamine (2.5 mg/kg body weight). Rapid distribution of the carcinogen occurred, with highest initial concentrations in liver and kidney. Within 10 min after the injection, 14C-labelled metabolites accounted for 50% of the total radioactivity present in the oesophagus, for approximately 25% in liver and forestomach, and for less than 20% in all other organs investigated. Decay of the carcinogen in rat serum followed first-order kinetics with a half-life of 35 min. Of the total radioactivity administered, 49% was exhaled as 14CO2 within 10 h and an additional 5-10% was excreted via urine and faeces. Four hours after a single i.v. injection of N-nitroso-[methyl-14C]benzylnitrosamine methylation of purine bases in DNA was most extensive in the oesophagus, followed by liver, lung and forestomach DNA. In the remaining tissues, DNA methylation was either considerably less (kidney, glandular stomach, spleen) or not at all detectable (ileum, colon, brain). At this time the concentration of the promutagenic base O6-methylguanine in oesophageal DNA was six times higher than in lung and nine times higher than in hepatic DNA. These data suggest that in the rat the selective induction of oesophageal tumours by N-nitrosomethylbenzylamine and related asymmetrical nitrosamines is mediated by a preferential bioactivation of the carcinogen in the target organ.

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.

Excision in vitro of the DNA bound carcinogen, 4-nitroquinoline 1-oxide

It has been shown that 4-hydroxyaminoquinoline 1-oxide, the proximate form of the carcinogen 4-nitroquinoline 1-oxide, binds covalently to the purine bases of DNA. Here we report that carcinogen-bound nucleotides can be excised from DNA by a 5' leads to 3' exonuclease associated with DNA polymerase I of E. coli in the forms of either mononucleotides or oligonucleotides. Beef spleen phosphodiesterase II (5' leads to 3') also split carcinogen-bound nucleotides, while a 3' leads to 5' exonuclease of DNA polymerase I and E. coli exonuclease III (3' leads to 5') could not excise the modified nucleotide.

Chemical carcinogenesis in the nervous system. Preferential accumulation of O6-methylguanine in rat brain deoxyribonucleic acid during repetitive administration of N-methyl-N-nitrosourea.

The alkylation of purine bases in DNA of several rat tissues was determined during weekly injections (10 mg/kg) of N-[3H]methyl-N-nitrosourea, a dose schedule known to selectively induce tumours of the nervous system. Each group of animals was killed 1 week after the final injection, and the DNA hydrolysates were analysed by chromatography on Sephadex G-10. After five weekly applications, O6-methylguanine had accumulated in brain DNA to an extent which greatly exceeded that in kidney, spleen and intestine. In the liver, the final O6-methylguanine concentration was less than 1% of that in brain. Between the first and the fifth injection, the O6-methylguanine/7-methylguanine ratio in cerebral DNA increased from 0.28 to 0.68. In addition, 3-methylguanine was found to accumulate in brain DNA whereas in the other organs no significant quantities of this base were detectable. The results are compatible with the hypothesis that O6-alkylation of guanine in DNA plays a major role in the induction of tumours by N-methyl-N-nitrosourea and related carcinogens. The kinetics of the increase of O6-methylguanine in cerebral DNA suggest that there is no major cell fraction in the brain which is capable of excising chemically methylated bases from DNA. This repair deficiency could be a determining factor in the selective induction of nervous-system tumours by N-methyl-N-nitrosourea and other neuro-oncogenic compounds.

Binding of 14C-labeled 4-nitroquinoline 1-oxide to DNA in vivo

The mode of binding of a carcinogen, 4-nitroquinoline 1-oxide (4NQO) to DNA was investigated by incubating Ehrlich ascites tumor cells growing in the peritoneal cavity of mice with 14C-labeled 4NQO. DNA extracted from incubated cells had radioactivity associated with DNA after neutral and alkaline cesium chloride equilibrium density-gradient centrifugation. The radioactivity was not released from DNA by heat or alkaline denaturation. Digestion of the 14C-associated DNA by deoxyribonuclease and snake venom phosphodiesterase gave a nucleotide compound which was eluted before ordinary mononucleotides on gel filtration. Phosphomonoesterase treatment of this material yielded a compound which was eluted after mononucleotides on gel filtration and which was not adsorbed on DEAE-cellulose column chromatography. Upon depurination of the 14C-associated DNA under mild conditions, almost all the radioactivity was released with purine bases. The data suggest a covalent binding of 4NQO or its derivatives with purine bases of DNA.

Interaction of 2-Amino-1, 4-naphthoquinone Imine with Nucleic Acid

On 2-amino-1, 4-naphthoquinone imine-HCl (ANQI), which had previously been reported to interact with nucleic acid and to inhibit the synthesis of nucleic acid and protein, especially of DNA, in Ehrlich mouse ascites tumor cells in vitro, the details of the interaction with nucleic acid were investigated by the methods of melting experiment for DNA, difference spectrum, equilibrium dialysis, and flow dichroism. The results are as follows : 1. The sites in DNA for attachment of ANQI were the purine base moieties, i.e., both of adenine and guanine. ANQI interacted with a highly polymerized native DNA at the highest degree and hardly interacted with such monomers as bases, nucleosides, and nucleotides. 2. ANQI was found to be able to bind to calf thymus DNA up to the ratio of 2 moles per 4 nucleotides by the method of equilibrium dialysis. This means that ANQI can attach with all the purine bases in DNA. 3. Such bivalent metal ions as Mg2+, Ca2+, Mn2+, Cu2+, Zn2+, and Ba2+ interfered with the interaction between ANQI and DNA. On the other hand, Hg2+ increased the extent of interaction. 4. It was found from the measurement of flow dichroism on DNA-bound ANQI that the orientation of ANQI was nearly parallel with the planes of DNA bases.