Inroads into base excision repair I. The discovery of apurinic/apyrimidinic (AP) endonuclease. "An endonuclease for depurinated DNA in Escherichia coli B," Canadian Journal of Biochemistry, 1972.

DNA treated with alkylating agents is incised at sites of damage by cell extracts. A key component of this DNA repair function was shown by Verly and co-workers to be an endonuclease acting at secondary lesions, apurinic sites, rather than directly at alkylated nucleotide residues.

The use of thioglycollate to demonstrate DNA AP (apurinic/apyrimidinic-site) lyase activities. Biological consequences of thiol addition to the 5' product of a beta-elimination reaction at an AP site in DNA.

Thioglycollate reacts with the 5' product of AP lyase activity on apurinic/apyrimidinic (AP) sites in DNA. The 3'-terminal thioglycollate-unsaturated sugar 5-phosphate adduct can be released by the use of Escherichia coli endonuclease IV or endonuclease VI, and identified by DEAE-Sephadex chromatography. In contrast, the mammalian AP endonuclease is unable to excise a 3'-terminal thiol-unsaturated sugar adduct; this lesion, which must sometimes occur in vivo, might be irreparable and have pathological consequences.

Mechanism of DNA strand nicking at apurinic/apyrimidinic sites by Escherichia coli [formamidopyrimidine]DNA glycosylase.

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.

The use of thioglycolate to distinguish between 3' AP (apurinic/apyrimidinic) endonucleases and AP lyases.

Addition of thioglycolate and DEAE-Sephadex chromatography were used to analyze the cleavage of the C(3')-O-P bond 3' to AP (apurinic/apyrimidinic) sites in DNA and to distinguish between a mechanism of hydrolysis (which would allow the nicking enzyme to be called 3' AP endonuclease) or beta-elimination (so that the nicking enzyme should be called AP lyase). For this purpose, DNA labelled in the AP sites was first cleaved by rat-liver AP endonuclease, then with the 3' nicking catalyst in the presence of thioglycolate and the reaction products were analyzed on DEAE-Sephadex: deoxyribose-5-phosphate (indicating a 3' cleavage by hydrolysis) and the thioglycolate:unsaturated sugar-5-phosphate adduct (indicating a cleavage by beta-elimination) are well separated allowing to eventually easily discard the hypothesis of a hydrolytic process and the appellation of 3' AP endonuclease. We have shown that addition of thioglycolate to the unsaturated sugar resulting from nicking the C(3')-O-P bond 3' to AP sites by beta-elimination is an irreversible reaction.

Delta-elimination in the repair of AP (apurinic/apyrimidinic) sites in DNA.

[5'-32P]pdT8d(-)dT7, containing an AP (apurinic/apyrimidinic) site in the ninth position, and [d(-)-1',2'-3H, 5'-32P]DNA, containing AP sites labelled with 3H in the 1' and 2' positions of the base-free deoxyribose [d(-)] and with 32P 5' to this deoxyribose, were used to investigate the yields of the beta-elimination and delta-elimination reactions catalysed by spermine, and also the yield of hydrolysis, by the 3'-phosphatase activity of T4 polynucleotide kinase, of the 3'-phosphate resulting from the beta delta-elimination. Phage-phi X174 RF (replicative form)-I DNA containing AP (apurinic) sites has been repaired in five steps: beta-elimination, delta-elimination, hydrolysis of 3'-phosphate, DNA polymerization and ligation. Spermine, in one experiment, and Escherichia coli formamidopyrimidine: DNA glycosylase, in another experiment, were used to catalyse the first and second steps (beta-elimination and delta-elimination).

The rat-liver microsomal AP endonuclease. The endoplasmic reticulum is presented as a net thrown into the cytosol to capture newly synthesized karyophilic proteins.

Although most of the rat-liver AP (apurinic/apyrimidinic) endonuclease is in chromatin, some activity is found in microsomes. A quantitative assay of the microsomal AP endonuclease is described. The enzyme is a peripheral membrane protein that is located on the outside surface of microsomes.

The multiple activities of Escherichia coli endonuclease IV and the extreme lability of 5'-terminal base-free deoxyribose 5-phosphates.

Escherichia coli endonuclease IV hydrolyses the C(3')-O-P bond 5' to a 3'-terminal base-free deoxyribose. It also hydrolyses the C(3')-O-P bond 5' to a 3'-terminal base-free 2',3'-unsaturated sugar produced by nicking 3' to an AP (apurinic or apyrimidinic) site by beta-elimination; this explains why the unproductive end produced by beta-elimination is converted by the enzyme into a 3'-OH end able to prime DNA synthesis. The action of E.

Bacteriophage-T4 and Micrococcus luteus UV endonucleases are not endonucleases but beta-elimination and sometimes beta delta-elimination catalysts.

Bacteriophage-T4 UV endonuclease nicks the C(3')-O-P bond 3' to AP (apurinic or apyrimidinic) sites by a beta-elimination reaction. The breakage of this bond is sometimes followed by the nicking of the C(5')-O-P bond 5' to the AP site, leaving a 3'-phosphate end; delta-elimination is proposed as a mechanism to explain this second reaction. The AP site formed when this enzyme acts on a pyrimidine dimer in a polynucleotide chain undergoes the same nicking reactions.

Importance of thiols in the repair mechanisms of DNA containing AP (apurinic or apyrimidinic) sites.

Addition of thiol compounds containing an anionic group to the 3'-terminal unsaturated sugar of the 5' fragment obtained from an oligonucleotide containing an AP site cleaved by beta-elimination, can be followed by gel electrophoresis. The technique enables to distinguish between two mechanisms of cleavage of the C3'-O-P bond 3' to an AP site: hydrolysis or beta-elimination. Addition of thiols to the double-bond of the 3'-terminal sugar resulting from beta-elimination prevents a subsequent delta-elimination.

Pairing properties of bromouracil and repair of bromouracil-containing DNA. Possible utilization of bromodeoxyuridine triphosphate for site-directed mutagenesis.

5-Bromo-2'-deoxyuridine triphosphate (Br-dUTP) and dTTP are used interchangeably for DNA synthesis in vitro by the Klenow fragment of Escherichia coli DNA polymerase I. When DNA containing Br-dUMP instead of dTMP at a few preselected sites is transfected into competent bacteria, no mutation occurs, indicating that in vivo E. coli DNA polymerase always places a dAMP residue in front of any unrepaired Br-dUMP residue.

Possible roles of beta-elimination and delta-elimination reactions in the repair of DNA containing AP (apurinic/apyrimidinic) sites in mammalian cells.

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.

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.

The relative importance of Escherichia coli exonuclease III and endonuclease IV for the hydrolysis of 3'-phosphoglycolate ends in polydeoxynucleotides.

In vitro, in the presence of Mg++, the 3'-phosphoglycolatase activity of endonuclease IV is about 4-times smaller than that of exonuclease III for the same AP endonuclease activity. It thus seems that endonuclease IV has only a minor role in the repair of strand breaks limited by 3'-phosphoglycolate ends in Escherichia coli even after the amount of enzyme has been increased by induction with O2 -generating agents.

Further purification and characterization of the DNA 3'-phosphatase from rat-liver chromatin which is also a polynucleotide 5'-hydroxyl kinase.

The DNA 3'-phosphatase activity of rat-liver chromatin has been purified. A DNA 5'-hydroxyl kinase activity comigrates at each step of purification. Both enzymes have the same molecular mass (79 kDa) and the same isoelectric point (8.

Nicks 3' or 5' to AP sites or to mispaired bases, and one-nucleotide gaps can be sealed by T4 DNA ligase.

Using synthetic oligodeoxynucleotides with 3'-OH ends and 32P-labelled 5'-phosphate ends and the technique of polyacrylamide gel electrophoresis, it is shown that, in the presence of the complementary polynucleotide, an AP (apurinic or apyrimidinic) site at the 3' or the 5' end of the labelled oligodeoxynucleotides does not prevent their ligation by T4 DNA ligase, although the reaction rate is decreased. This decrease is more severe when the AP site is at the 3' end; the activated intermediates accumulate showing that it is the efficiency of the adenyl-5'-phosphate attack by the 3'-OH of the base-free deoxyribose which is mostly perturbed. Using the same technique, it is shown that a mispaired base at the 3' or 5' end of oligodeoxynucleotides does not prevent their ligation.

Escherichia coli endonuclease III is not an endonuclease but a beta-elimination catalyst.

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.

Chromatin 3'-phosphatase/5'-OH kinase cannot transfer phosphate from 3' to 5' across a strand nick in DNA.

Rat liver chromatin contains a 3'-phosphatase/5'-OH kinase which may be involved in the repair of DNA strand breaks limited by 3'-phosphate/5'-OH ends. In order to determine whether the phosphate group can be transferred directly from the 3' to the 5' position, a polynucleotide duplex was synthesized between poly (dA) and oligo (dT) segments which had 3'-[32P]phosphate and 5'-OH ends. The oligo (dT) segments were separated by simple nicks as shown by the ability of T4 DNA ligase to seal the nick after the 3'-phosphate was removed by a phosphatase and the 5' end was phosphorylated with a kinase.

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.

Action of intact AP (apurinic/apyrimidinic) sites and AP sites associated with breaks on the transcription of T7 coliphage DNA by Escherichia coli RNA polymerase.

The effect of apurinic/apyrimidinic (AP) sites in DNA on RNA and protein synthesis was studied in vitro using T7 coliphage DNA. Initiation of RNA synthesis by Escherichia coli RNA polymerase was synchronized and heparin was used to prevent reinitiation. When the T7 DNA contained AP sites, the rate of RNA synthesis was decreased but it remained higher than the values calculated on the assumption that an AP site in the transcribed strand is a complete block to the enzyme progression.

Deoxyribonuclease IV from rat liver chromatin and the excision of apurinic sites from depurinated DNA.

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.

The excision of AP sites by the 3'-5' exonuclease activity of the Klenow fragment of Escherichia coli DNA polymerase I.

The 3' AP endonucleases (class I) are said to hydrolyze the phosphodiester bond 3' to AP sites yielding 3'-OH and 5'-phosphate ends; on the other hand, the resulting 3' terminal AP site is not removed by the 3'-5' exonuclease activity of the Klenow fragment [1]. We show that AP sites in DNA are easily removed by the 3'-5' exonuclease activity of the Klenow fragment and that they are excised as deoxyribose-5-phosphate. It is suggested that the 3' AP endonucleases are perhaps not the hydrolases they are supposed to be.

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.