Biomedical subjects
Publications and source records attributed to W G Verly.
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.
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.
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.
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. We have also shown that the thioglycolate must be present from the beginning of the reaction with the nicking catalyst to prevent the primary 5' product of the beta-elimination reaction from undergoing other modifications that complicate the interpretation of the results.
[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). These repair pathways, involving a delta-elimination step, may be operational not only in E. coli repairing its DNA containing a formamido-pyrimidine lesion, but also in mammalian cells repairing their nuclear DNA containing AP sites.
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. All the binding sites on the microsomes appear to have the same affinity for the AP endonuclease, suggesting the presence of receptors for the enzyme. The AP endonuclease is displaced from its membrane attachment by submicromolar concentrations of the karyophilic signal of SV-40 T antigen. The AP endonuclease receptors are likely to be on the cytosolic side of the endoplasmic reticulum. It is suggested that binding of the protein to these receptors might be the first step of the transport mechanism that enables the AP endonuclease to penetrate into the nucleus. The same mechanism utilizing the same receptors might be used by other karyophilic proteins, including SV-40 T antigen.
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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. Micrococcus luteus UV endonuclease also nicks the C(3')-O-P bond 3' to AP sites by a beta-elimination reaction. No subsequent delta-elimination was observed, but this might be due to the presence of 2-mercaptoethanol in the enzyme preparation.
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. coli endonuclease IV on an internal AP site is more complex: in a first step the C(3')-O-P bond 5' to the AP site is hydrolysed, but in a second step the 5'-terminal base-free deoxyribose 5'-phosphate is lost. This loss is due to a spontaneous beta-elimination reaction in which the enzyme plays no role. The extreme lability of the C(3')-O-P bond 3' to a 5'-terminal AP site contrasts with the relative stability of the same bond 3' to an internal AP site; in the absence of beta-elimination catalysts, at 37 degrees C the half-life of the former is about 2 h and that of the latter 200 h. The extreme lability of a 5'-terminal AP site means that, after nicking 5' to an AP site with an AP endonuclease, in principle no 5'----3' exonuclease is needed to excise the AP site: it falls off spontaneously. We have repaired DNA containing AP sites with an AP endonuclease (E. coli endonuclease IV or the chromatin AP endonuclease from rat liver), a DNA polymerase devoid of 5'----3' exonuclease activity (Klenow polymerase or rat liver DNA polymerase beta) and a DNA ligase. Catalysts of beta-elimination, such as spermine, can drastically shorten the already brief half-life of a 5'-terminal AP site; it is what very probably happens in the chromatin of eukaryotic cells. E. coli endonuclease IV also probably participates in the repair of strand breaks produced by ionizing radiations: as E. coli endonuclease VI/exonuclease III, it is a 3'-phosphoglycollatase and also a 3'-phosphatase. The 3'-phosphatase activity of E. coli endonuclease VI/exonuclease III and E. coli endonuclease IV can also be useful when the AP site has been excised by a beta delta-elimination reaction.
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. The interpretation of the action of enzymes that start by nicking 3' to AP sites must take into account the presence or absence of thiols in the reaction medium. In living cells, thiols might influence the pathways followed by the repair processes of AP site-containing DNA.
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. On the other hand, in vitro Br-dUTP can also replace dCTP, but only with difficulty: when dCTP is absent, Br-dUMP can be forced in front of a dGMP residue, but the Klenow polymerase pauses before and after addition of Br-dUMP. Transfection into E. coli of the substituted DNA leads to the expected G----A transitions. These mutations can easily be targeted by using a suitable primer and the correctly chosen mix of deoxynucleoside triphosphates containing Br-dUTP. When Br-dUMP has been placed in front of a dGMP residue, the mutation yield is not 100%, showing a partial repair of the transfected DNA before it is replicated. Advantage can be taken of this partial repair to prepare a set of different mutations within a target region in a single experiment.
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.
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.
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 (8.6). It thus seems that the two activities are born by the same protein just as with the phage T4 enzyme which is, at the same time, a 5'-hydroxyl kinase and a 3'-phosphatase. An additional argument is that ATP, which does not influence the rate of the 3'-phosphatase reaction but which is a cosubstrate of the 5'-hydroxyl kinase, protects the 3'-phosphatase activity against thermal denaturation and trypsin digestion. The two active sites must, however, be largely independent within a common support: the thermal denaturation and trypsin inactivation rates are very different for the two activities; increasing the ionic strength activates the kinase and inhibits the phosphatase; polyvalent anions inhibit the phosphatase and have little effect on the kinase. The two active sites might belong to different domains of the protein; they could not however be separated by a partial trypsin digestion. The rates of 3'-dephosphorylation and 5'-phosphorylation by the chromatin enzyme are the same in native and denatured DNA. The 3'-phosphatase has no action on 3'-monodeoxynucleotide, but it hydrolyzes the 3'-phosphate in dinucleotides. The Km of the 3'-phosphatase is 0.548 microM. The Km (5'-OH) and Km (ATP) of the 5'-hydroxyl kinase are about 3.9 microM and 0.69 microM respectively. The chromatin enzyme is unable to hydrolyze 3'-phosphoglycolate ends in 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.
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. A one-nucleotide gap, limited by 3'-OH and 5'-phosphate, can also be closed by T4 DNA ligase although with difficulty; here again the activation of the 5'-phosphate end does not seem to be slowed down, but rather the 3'-OH attack of the adenyl-5'-phosphate. All these anomalous ligations take place with the nick or the gap in front of a continuous complementary strand. Blunt ends ligation of correct duplexes occurs readily; however an AP site or a mispaired base at the 3' or 5' end of one strand of the duplexes prevents ligation between these strands. But a missing nucleotide (responsible for one unpaired nucleotide protruding at the 3' or 5' end of the complementary strand) does not stop ligation of the shorter oligodeoxynucleotides between independent duplexes.
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.
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. The chromatin 3'-phosphatase/5'-OH kinase was unable to transfer phosphate directly from the 3' to the 5' end of the oligo (dT) segments in the original duplex; ATP was needed to phosphorylate the 5'-OH end. It is concluded that the chromatin 3'-phosphatase/5'-OH kinase is unable to convert a 3'-phosphate/5'-OH nick which cannot be repaired by DNA ligase directly into a 3'-OH/5'-phosphate nick which can be repaired by DNA ligase; the chromatin enzyme rather acts in two steps: hydrolysis of the 3'-phosphate followed by ATP-mediated phosphorylation of the 5'-OH end.