Walter G. Verly

Appearance and repair of apurinic/apyrimidinic sites in DNA during early germination of Zea mays

Abstract The number of AP (apurinic or apyrimidinic) sites found in DNA of radicle cells of Zea mays quiescent embryos after 2 years of storage is low; the low rate of spontaneous base loss is probably due to the low water content of the seed. But this number increases 4-fold during the first 20 h of germination, to decrease between 20 and 25 h, and increase again afterwards. These variations may well be due to competition between the formation of AP sites and their repair during early germination. Formation must be due to DNA glycosylases removing bases which have been damaged during the storage of the seeds. The first increase in the number of AP sites would be due to DNA glycosylases which have survived the storage period; the second increase might be the result of the synthesis of new DNA glycosylase molecules. The nuclear DNA-repair synthesis monitored by autoradiography closely follows the number of AP sites during early germination, suggesting that the repair of most of the damage which has accumulated in DNA during storage has AP sites as an intermediary step.

A chromatin factor in rat liver which destroys O6‐ethylguanine in DNA

Adapted Escherichia coli cells (i.e. pretreated with a low concentration of N-methyl-N’nitro-N-nitrosoguanidine) contain a factor able to promote the disappearance of U6-methylguanine from DNA [ 1,2]. The 06-methylguanine is not released as a free base and the transformation product remains in DNA [2]; the factor seems to be active at 0°C and to disappear during the reaction [ 11. Renard et al. [3] observed the disappearance of @-ethylguanine from the DNA of isolated rat liver nuclei treated in vitro with ethylnitrosourea and Pegg [4] has found, in the total proteins of a rat liver extract, an activity which induces the disappearance of @-methylguanine from DNA. As in E. co/i, the glycosylic bond is not hydrolyzed to release free @-methylguanine. In this communication, we show that, in rat liver, chromatin has the highest concentration of the factor capable to decrease the @‘-ethylguanine content of an added ethylated DNA. Some properties of the chromatin factor are also presented.

The DNA 3′‐phosphatase and 5′‐hydroxyl kinase of rat liver chromatin

Some nicks, accidently produced in DNA, might be limited by 3 ‘-phosphate and 5 ‘-OH ends and not be suitable for ligase or polymerase activities. A 3 ‘-phosphatase might thus be helpful to the cell whereas access of a 5 ’ -phosphatase to nuclear DNA could only be injurious. We have studied the action of chromatin proteins on double-strand DNA containing nicks limited by 3 ’ -phosphate/5 ’ -OH or 3 ’ -OH/5 ’ -phosphate ends, thus mimicking possible situations of the cell nuclear DNA. In agreement with the above prediction, chromatin proteins had only a 3 ’ -phosphatase activity; this is in contrast with cytoplasmic proteins which hydrolyze both 3 ’ -phosphate and 5 ’ -phosphate ends. T4 polynucleotide 5 ‘-OH kinase has an associated 3 ’ -phosphatase activity [ 11. We have shown that chromatin likely possesses a protein with the same two activities: a 5’-OH kinase activity accompanies the 3 ’ -phosphatase during its purification. This work was completed when the complementary observation was published [2]: the purified DNA kinase from rat liver contains a 3 ’ -phosphatase activity.

A common chromatin factor involved in the repair of O6-methylguanine and O6-ethylguanine lesions in DNA

Repair of DNA containing 06-methylguanine or 06-ethylguanine by rat liver total proteins was observed in [ 1,2]. We have shown that the factor responsible for the disappearance of 06-ethylguanine is mostly located in chromatin [3] and that the ethyl group is transferred onto 2 cysteine residues of acceptor proteins [4]. Using a factor partially purified from mouse liver supernatant repair of DNA containing 06-methylguanine and transfer of the methyl group onto cysteine residues of proteins was shown in [S]. This work shows that chromatin roteins from rat liver contain factors acting on f? 0 -methylguanine as well as 06-ethylguanine in DNA. Competition experiments indicate that a limiting component is common to both repair systems.

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.

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.

Interstrand DNA crosslinks due to AP (apurinic/apyrimidinic) sites

Storage of a solution of DNA containing apurinic sites, even at 4°C leads to the appearance of interstrand crosslinks. Possible biological consequences of these crosslinks, when they appear in cell DNA, are briefly discussed. Formation of interstrand crosslinks in DNA containing tritium‐labelled thymine and kept in an aqueous solution might be due, at least partly, to the loss of bases by the autoirradiated DNA.

Kinetic analysis of O6-ethylguanine disappearance from DNA catalyzed by the chromatin factor of rat liver.

Rat liver chromatin contains a factor which induces the disappearance of O’-ethylguanine from DNA alkylated with ethylnitrosourea f 1,2]. The transformation product has not yet been isolated nor, of course, identified. We noticed that, in contrast to the situation in Escherichia coli [3,4], this factor is present constitutively in chromatin and that it seems to be an enzyme rather than a stoichiometric reagent: it is inactive at 0°C and, when there is an excess of substrate, it is still working after 120 min at 37’C. Here, we give additions details on the chromatin repair factor showing that it behaves as an enzyme which is competitively inhibited by the reaction product.

Action of rat liver AP endodeoxyribonuclease on DNA treated with hydrazine or bleomycin

Endonuclease IV, the minor Escherichia coli endonuclease specific for apurinic sites, has been shown [ 1) to be active on apyrimidinic sites produced by uracil-DNA glycosylase. It was of interest to investigate whether the rat liver enzyme was also active on apyrimidinic sites. However, we have used an Al? endodeoxy~bonuclease (i.e., an endonuclease specific for apu~nic and apyr~idi~ic sites in DNA) to analyze the products of the reaction of DNA with chemical agents such as hydrazine and bleomycin. Bleomycin, a glycopeptide with mol. wt of 1500, is an antibiotic produced by Streptomyces verticillus, isolated [2,3] . The action of the drug on DNA strongly depends on its concentration. At high concentrations, the four bases are removed from DNA [4] and double-strand as well as single-strand breaks are produced [S] . But, at low concentrations, bleomycin splits off mostly thymine bases [6] and the possibly of interc~ation of one of the thiazole rings of the antibiotic in the double-held at the place of the lost pyrimidine has been suggested [ 71. Because bleomycin is currently used in the chemotherapy of neoplastic diseases [&-lo] , the problem of the repair of DNA lesions produced by this agent is important; we wanted particularly to know whether the base-free sites were accessible to a repair endodeoxyribonuclease.

Action of phenobarbital given to rats together with diethylnitrosamine on the O6-ethylguanine content of liver DNA

When rats are fed diethylnitrosamine (10 mg/kg/day), no O6-ethylguanine is found in liver DNA after 2 weeks, but a considerable amount accumulates after 4 weeks. On the other hand, a 2-week feeding of diethylnitrosamine is not sufficient to induce liver cancers, whereas a 4-week treatment leads to hepatocarcinomas in 50% of the animals. Administration of phenobarbital (75 mg/kg/day) together with diethylnitrosamine during 4 weeks prevents the formation of liver cancers. It also prevents accumulation of O6-ethylguanine in liver DNA. Phenobarbital does not change the amount of O6-ethylguanine repair activity present in liver chromatin after 2 or 4 weeks of treatment with diethylnitrosamine. It is thus concluded that, by inducing the development of the endoplasmic reticulum, phenobarbital decreases the equilibrium concentration of the ultimate carcinogen derived from this indirect alkylating agent.