The oligonucleotide [5'-32P]pdT8d(-)dTn, containing an apurinic/apyrimidinic (AP) site [d(-)], yields three radioactive products when incubated at alkaline pH: two of them, forming a doublet approximately at the level of pdT8dA when analysed by polyacrylamide-gel electrophoresis, are the result of the beta-elimination reaction, whereas the third is pdT8p resulting from beta delta-elimination. The incubation of [5'-32P]pdT8d(-)dTn, hybridized with poly(dA), with E. coli endonuclease III yields two radioactive products which have the same electrophoretic behaviour as the doublet obtained by alkaline beta-elimination. The oligonucleotide pdT8d(-) is degraded by the 3'-5' exonuclease activity of T4 DNA polymerase as well as pdT8dA, showing that a base-free deoxyribose at the 3' end is not an obstacle for this activity. The radioactive products from [5'-32P]pdT8d(-)dTn cleaved by alkaline beta-elimination or by E. coli endonuclease III are not degraded by the 3'-5' exonuclease activity of T4 DNA polymerase. When DNA containing AP sites labelled with 32P 5' to the base-free deoxyribose labelled with 3H in the 1' and 2' positions is degraded by E. coli endonuclease VI (exonuclease III) and snake venom phosphodiesterase, the two radionuclides are found exclusively in deoxyribose 5-phosphate and the 3H/32P ratio in this sugar phosphate is the same as in the substrate DNA. When DNA containing these doubly-labelled AP sites is degraded by alkaline treatment or with Lys-Trp-Lys, followed by E. coli endonuclease VI (exonuclease III), some 3H is found in a volatile compound (probably 3H2O) whereas the 3H/32P ratio is decreased in the resulting sugar phosphate which has a chromatographic behaviour different from that of deoxyribose 5-phosphate. Treatment of the DNA containing doubly-labelled AP sites with E. coli endonuclease III, then with E. coli endonuclease VI (exonuclease III), also results in the loss of 3H and the formation of a sugar phosphate with a lower 3H/32P ratio that behaves chromatographically as the beta-elimination product digested with E. coli endonuclease VI (exonuclease III). From these data, we conclude that E. coli endonuclease III cleaves the phosphodiester bond 3' to the AP site, but that the cleavage is not a hydrolysis leaving a base-free deoxyribose at the 3' end as it has been so far assumed. The cleavage might be the result of a beta-elimination analogous to the one produced by an alkaline pH or Lys-Trp-Lys. Thus it would seem that E. coli 'endonuclease III' is, after all, not an endonuclease.

Escherichia coli [formamidopyrimidine]DNA glycosylase catalyses the nicking of both the phosphodiester bonds 3' and 5' of apurinic or apyrimidinic sites in DNA so that the base-free deoxyribose is replaced by a gap limited by 3'-phosphate and 5'-phosphate ends. The two nickings are not the results of hydrolytic processes; the [formamidopyrimidine]DNA glycosylase rather catalyses a beta-elimination reaction that is immediately followed by a delta-elimination. The enzyme is without action on a 3'-terminal base-free deoxyribose or on a 3'-terminal base-free unsaturated sugar produced by a beta-elimination reaction nicking the DNA strand 3' to an apurinic or apyrimidinic site.

Histones and polyamines nick the phosphodiester bond 3' to AP (apurinic/apyrimidinic) sites in DNA by inducing a beta-elimination reaction, which can be followed by delta-elimination. These beta- and delta-elimination reactions might be important for the repair of AP sites in chromatin DNA in either of two ways. In one pathway, after the phosphodiester bond 5' to the AP site has been hydrolysed with an AP endonuclease, the 5'-terminal base-free sugar 5'-phosphate is released by beta-elimination. The one-nucleotide gap limited by 3'-OH and 5'-phosphate ends is then closed by DNA polymerase-beta and DNA ligase. We have shown in vitro that such a repair is possible. In the other pathway, the nicking 3' to the AP site by beta-elimination occurs first. We have shown that the 3'-terminal base-free sugar so produced cannot be released by the chromatin AP endonuclease from rat liver. But it can be released by delta-elimination, leaving a gap limited by 3'-phosphate and 5'-phosphate. After conversion of the 3'-phosphate into a 3'-OH group by the chromatin 3'-phosphatase, there will be the same one-nucleotide gap, limited by 3'-OH and 5'-phosphate, as that formed by the successive actions of the AP endonuclease and the beta-elimination catalyst in the first pathway.

A method is presented to measure the alkylation of phosphates in DNA after a treatment with an alkylating agent. Using this method, we have shown that phosphate alkylation represents 15O/, of total alkylation when DNA is alkylated with ethyl methanesulfonate and only l o / , of total alkylation when DNA is alkylated with methyl methanesulfonate. Experiments are also presented which show that phosphate triesters resulting from the alkylation of DNA by ethyl methanesulfonate are very stable, most of them remaining intact after heating a t 100 "C for 90 min at pH 7.0.Lett et al. [I] have stated that alkylating agents react primarily with phosphates in DNA and have presented spectrophotometrical evidence for a transalkylation process a t room temperature from triester phosphates to bases in the macromolecule. Epoxides were an exception because they reacted directly with the bases, while methyl methanesulfonate had an intermediate behaviour, alkylating initially both phosphates and bases. On the other hand, the transalkylation process did not occur a t room temperature with ethyl methanesulfonate which, according to these authors, gave onlyethylated DNAphosphates. Lawley and Brookes [Z], using labelled alkylating agents, found that nearly all alkyl groups bound to DNA were liberated by formic acid hydrolysis a t 175 "C as alkylated bases, especially alkylated purines ; they concluded that the alkylation of the phosphates was negligible. As ethyl methanesulfonate was not an exception in their work, it was suggested by Alexader [3] that the formation of ethylated bases was an artifact occurring during acid hydrolysis a t high temperature.Some work has been done using oligonucleotides. Holy and Scheit [4,5], in contrast to Brimacombe et al. [6], have observed phosphate alkylation with diazomethane. Also Rhaese and Freese [7] observed phosphate alkylation with ethyl and methyl methanesulfonates.Because of these contradictions concerning the fundamental question of DNA phosphate alkylation, it is not surprising that little work has been done on the assay of phosphate alkylation in DNA. To achieve this goal, Strauss and Hill [S] used labelled alkylating Abbreviations. PPO, 2,5-diphenyloxazole; dimethyl-POPOP, 1,4-bis-2(4-methyl-5-oxazolyl)-benzene.Enzymes. Alkaline phosphatase (EC 3.1.3.1); phosphodiesterase (EC 3.1.4.1). agents and measured the radioactivity bound to DNA which resisted heating the aqueous soIution to 100°C a t neutral pH. One might except such a treatment to lead to a complete loss of the alkylated purines and, as the alkylation of pyrimidines is very low, it seems that most of the residual radioactivity ought to be due to alkyl groups bound to phosphates. The aim of the first part of our work was to check this assumption using ethyl and methyl methanesulfonates and to develop a reliable method for the estimation of DNA phosphate alkylation.The second part of our work deals with the stability of the phosphate triesters formed by alkylation of DNA. If it seems well established that phosphate alkylation i...

The main endonuclease for apurinic sites of Eschrrichia coli (endonuclease VI) has no action on normal strands, either in double-stranded or single-stranded DNA, or on alkylated sites. The enzyme has an optimum pH at 8.5, is inhibited by EDTA and needs Mgz+ for its activity; it has a half-life of 7 min at 40 "C. A purified preparation of endonuclease VI, free of endonuclease I1 activity, contained exonuclease 111 ; the two activities (endonuclease VI and exonuclease 111) copurified and were inactivated with the same half-lives at 40 "C. Endonuclease VI cuts the DNA strands on the 5' side of the apurinic sites giving a 3'-OH and a 5'-phosphate, and exonuclease 111, working afterwards, leaves the apurinic site in the DNA molecule; this apurinic site can subsequently be removed by DNA polymerase I. The details of the excision of apurinic sites in vitro from DNA by endonuclease VI/exonuclease 111, DNA polymerase I and ligase, are described; it is suggested that exonuclease 111 works as an antiligase to facilitate the DNA repair.An enzyme which hydrolyzes a phosphoester bond near apurinic sites was found in Escherichia c d i by Verly and Paquette [l]. This enzyme, which has no action on normal DNA strands or on alkylated sites [ 2 ] , has been completely purified by Verly and Rassart[3]; it is a monomeric protein of M , = 32000. Depurinated DNA has been repaired in vitro by Verly et al. [4] with three enzymes: the endonuclease specific for apurinic sites from E. coli, DNA polymerase I and the four deoxynucleoside triphosphates, ligase and its coenzyme. Mutants of E. coli lacking DNA polymerase I are very sensitive to methylmethanesulfonate and it is known that the lethal action of this alkylating agent is due mostly to the depurination of DNA [5]. On the other hand, however, mutations in gene sthA, which codes for the endonuclease specific for apurinic sites, only slightly increase the sensitivity of E. coli to methylmethanesulfonate [6]; this is most likely due to the fact that E. coli possesses two enzymes active on apurinic sites: the main enzyme which we have isolated