O(6)-Alkylguanine-DNA alkyltransferase (ATase) confers resistance to many of the biological effects of certain classes of alkylating agents by repairing the DNA lesions responsible. The role of ATase in the mutagenic and toxic effects of the carcinogenic and antitumour alkylating agents are of interest in relation to the prevention and treatment of cancer in man. In this commentary we specifically focus on the variation in ATase levels and our current understanding of the factors involved in the regulation of ATase expression.

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Variability and regulation of O6-alkylguanine–DNA alkyltransferase

Some evolutionary consequences of different rates and trends in DNA damage and repair are explained. Different types of DNA damaging agents cause nonrandom lesions along the DNA. The type of DNA sequence motifs to be preferentially attacked depends upon the chemical or physical nature of the assaulting agent and the DNA base composition. Higher-order chromatin structure, the nonrandom nucleosome positioning along the DNA, the absence of nucleosomes from the promoter regions of active genes, curved DNA, the presence of sequence-specific binding proteins, and the torsional strain on the DNA induced by an increased transcriptional activity all are expected to affect rates of damage of individual genes. Furthermore, potential Z-DNA, H-DNA, slippage, and cruciform structures in the regulatory region of some genes or in other genomic loci induced by torsional strain on the DNA are more prone to modification by genotoxic agents. A specific actively transcribed gene may be preferentially damaged over nontranscribed genes only in specific cell types that maintain this gene in active chromatin fractions because of (1) its decondensed chromatin structure, (2) torsional strain in its DNA, (3) absence of nucleosomes from its regulatory region, and (4) altered nucleosome structure in its coding sequence due to the presence of modified histones and HMG proteins. The situation in this regard of germ cell lineages is, of course, the only one to intervene in evolution. Most lesions in DNA such as those caused by UV or DNA alkylating agents tend to diminish the GC content of genomes. Thus, DNA sequences not bound by selective constraints, such as pseudogenes, will show an increase in their AT content during evolution as evidenced by experimental observations. On the other hand, transcriptionally active parts may be repaired at rates higher than inactive parts of the genome, and proliferating cells may display higher repair activities than quiescent cells. This might arise from a tight coupling of the repair process with both transcription and replication, all these processes taking place on the nuclear matrix. Repair activities differ greatly among species, and there is a good correlation between life span and repair among mammals. It is predicted that genes that are transcriptionally active in germ-cell lineages have a lower mutation rate than bulk DNA, a circumstance that is expected to be reflected in evolution. Exception to this rule might be genes containing potential Z-DNA, H-DNA, or cruciform structures in their coding or regulatory regions that appear to be refractory to repair. This study supports the molecular clock hypothesis when applied to one gene within a group of related species and contends that evolutionary rates might vary between genes and gene segments not only as a result of differences in selective constraints but also as a result of differences in the rate of damage minus rate of repair among different segments of chromatin DNA.

Evolutionary consequences of nonrandom damage and repair of chromatin domains

O6-Methylguanine was measured by a competitive repair assay in blood leukocyte DNA of seven patients with Hodgkin's or non-Hodgkin's lymphoma during therapeutic exposure to procarbazine involving three daily p.o. doses (50 mg each) for 10 days (corresponding to 2.1 mg/kg/day for a 70-kg human). Adduct accumulation was observed in all seven cases, reaching levels up to 0.28 fmol/microgram of DNA (0.45 mumol/mol of guanine). In one individual, maximal levels of adduct were reached after 7 days of exposure, followed by a steady decline, whereas in all other individuals continuous accumulation was observed throughout the exposure period. In four individuals for which data were available for Day 11 (12 to 16 h after the final intake of procarbazine), decreased amounts of O6-methylguanine were observed relative to the last previous measurements. The accumulation of O6-methylguanine was linearly correlated (P less than 0.01) with the cumulative dose of procarbazine, with a slope of 0.011 fmol of O6-methylguanine/microgram of DNA per mg/kg of body weight or 2.68 x 10(-4) fmol of O6 methylguanine DNA per mg/m2. (Two h after the administration of single p.o. doses of 1 to 10 mg/kg of procarbazine to rats, O6-methylguanine formation in leukocyte DNA was just under half that in liver DNA and showed a linear relationship with dose with a slope of 0.017 fmol/microgram of DNA per mg/kg of body weight or 5.67 x 10(-4) fmol of O6-methylguanine/microgram of DNA per mg/m2. A negative correlation (P less than 0.05) between the rate of accumulation of O6-methylguanine in different individuals and lymphocyte O6-alkylguanine-DNA alkyltransferase (AGT) was observed, demonstrating a probable protective effect of AGT against the accumulation of O6-methylguanine during exposure to methylating agents. This observation supports the suggestion of a possible role of procarbazine-induced O6-methylguanine in the pathogenesis of acute nonlymphocytic leukemia appearing after treatment with chemotherapeutic protocols which include procarbazine, based on the finding of low lymphocyte AGT levels in patients with such therapy-related neoplastic disease (Sagher et al., Cancer Res., 48: 3084-3089, 1988). Lymphocyte AGT levels were mainly in the range of 5 to 10 fmol/micrograms of DNA and showed no consistent variation during procarbazine exposure.

Accumulation of O6-methylguanine in human blood leukocyte DNA during exposure to procarbazine and its relationships with dose and repair.

Properties of mammalian O6-alkylguanine-DNA transferases.

The DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) repairs the promutagenic O6-methylguanine lesion by transferring the methyl group to a cysteine residue on the protein. A mechanism in which AGT activates the guanyl moiety as a leaving group by protonation of a heteroatom on guanine was probed by reacting AGT with analogues of O6-methylguanine in which the heteroatoms were changed. The initial rates of reaction were measured at various substrate concentrations in 50 mM Hepes, 1 mM EDTA, 1 mM DTT, and 10% glycerol, pH 7.8 at 37 degrees C. The kinact (h-1) and Kin (mM) were determined for O6-methylguanine (1.66 +/- 0.19, 1.51 +/- 0.32), 6-methoxypurine (1.07 +/- 0.25, 10.6 +/- 4.2), S6-methyl-6-thioguanine (0.63 +/- 0.04, 1.17 +/- 0.18), 6-methylthiopurine (no reaction), Se6-methyl-6-selenoguanine (1.76 +/- 0.28, 10.6 +/- 5.0), 6-methylselenopurine (2.51 +/- 0.62, 15.7 +/- 6.3), O6-methyl-1-deazaguanine (1.71 +/- 0.34, 14.8 +/- 4.4), O6-methyl-3-deazaguanine (1.90 +/- 0.24, 2.54 +/- 0.59), and O6-methyl-7-deazaguanine (1.97 +/- 0.26, 2.56 +/- 0.72). These results indicate that replacement of the nitrogens does not affect the kinact parameter but the Kin is increased upon removal of the exocyclic amino group and the nitrogen at the 1-position. Replacement of the oxygen with sulfur decreases the kinact, and replacement with selenium increases the Kin. The results are consistent with a mechanism in which O6-methylguanine binds to the active site of AGT with hydrogen bonds to the oxygen, the exocyclic amino group, and the nitrogen at the 1-position of the substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Reaction of O6-alkylguanine-DNA alkyltransferase with O6-methylguanine analogues: evidence that the oxygen of O6-methylguanine is protonated by the protein to effect methyl transfer.

A simplified and highly sensitive assay for the determination of O6-alkylguanine-DNA-alkyltransferase has been developed and validated by the analysis of extracts of human urinary bladder mucosa. The new assay involves the use of a synthetic dodecanucleotide containing a single O6-methylguanine residue as substrate for the enzyme. This substrate is 5'-end-labelled with [35S]PO3 and separation of repaired and unrepaired oligonucleotide is achived by immuno-precipitation with polyclonal antibodies specific for O6-methyldeoxyguanosine. Kinetic analysis of the repair of the oligonucleotide by extracts of Escherichia coli and rat liver showed that the reaction is first-order in substrate and enzyme and gave the molecular rate constants 7.5 x 10(6) mol-1 1-1 sec-1 and 8.0 x 10(6) mol-1 1-1 sec-1, respectively. The rate constants for the repair of the corresponding O6-ethylguanine-containing oligonucleotide were 3.0 x 10(5) mol-1 l-1 sec-1 and 3.6 x 10(6) mol-1 l-1 sec-1, respectively. Analysis of extracts of 48 samples of normal or neoplastic human urinary bladder mucosa obtained by transurethral biopsy or at surgery, by the new method or by a method involving use of [3H]-methylated DNA as substrate and HPLC, indicated excellent agreement between the two methods. The mean AGT content of normal urinary bladder mucosa obtained from individuals without diagnosed bladder cancer was 0.181 +/- 0.081 (mean +/- SD) fmol/microgram protein, that of neoplastic samples 0.323 +/- 0.177 fmol/microgram protein and that of normal tissue obtained from patients with bladder cancer 0.183 +/- 0.068 fmol/microgram protein. The new method is convenient, rapid and extremely sensitive (it can readily measure femtomole quantities of enzyme) and should prove useful for studies of the biochemical epidemiology of DNA repair.

Development and validation of a new assay for O6-alkylguanine-DNA-alkyltransferase based on the use of an oligonucleotide substrate, and its application to the measurement of DNA repair activity in extracts of biopsy samples of human urinary bladder mucosa.

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Development and validation of a new assay for O6-alkylguanine-DNA-alkyltransferase based on the use of an oligonucleotide substrate, and its application to the measurement of DNA repair activity in extracts of biopsy samples of human urinary bladder mucosa.