Damage repertoire of the Escherichia coli UvrABC nuclease complex includes abasic sites, base-damage analogues, and lesions containing adjacent 5' or 3' nicks

Using oligonucleotide synthesis, we demonstrate a rapid and efficient method for the construction of DNA duplexes containing defined DNA lesions at specific positions. These DNA lesions include apyrimidinic sites, reduced apyrimidinic sites, and base-damage analogues consisting of O-methyl- or O-benzylhydroxylamine-modified apyrimidinic sites. A 49 base pair DNA duplex containing these lesions was specifically incised by the UvrABC nuclease complex. The incision sites occurred predominantly at the eighth phosphodiester bond 5' and the fifth phosphodiester bond 3' to the lesion. Multiple incisions were observed 3' to the lesion. The extent of DNA incisions was base-damage analogues greater than reduced apyrimidinic sites greater than apyrimidinic sites. Introduction of 3' or 5' nicks at the site of a base-damage analogue by treatment of these substrates with either endonuclease III or endonuclease IV reduced, but did not abolish, subsequent incision by the UvrABC complex, whereas introduction of a 3' nick at an abasic site increased the incision efficiency of the UvrABC complex. These data demonstrate a convergence of base and nucleotide excision repair pathways in the removal of specific base damages.     

Poly(deoxyadenylic-deoxythymidylic acid) damage by radiolytically activated neocarzinostatin

The anaerobic reaction of poly(deoxyadenylic-deoxythymidylic acid) with neocarzinostatin activated by the carboxyl radical CO2-, an electron donor generated from gamma-ray radiolysis of nitrous oxide saturated formate buffer, has been characterized. DNA damage includes base release and strand breaks. Few strand breaks are formed prior to alkaline treatment; they bear 3'-phosphoryl termini. In contrast, most (66%) of the base release occurs spontaneously. DNA damage is highly (95%) specific for thymidine sites. Neither DNA-drug covalent adduct nor nucleoside 5'-aldehyde, which are major products in the DNA-nicking reaction initiated by mercaptans and oxygen, is formed in this reaction. Data are presented to show that the CO2(-)-activated neocarzinostatin intermediate is a short-lived free radical able to abstract hydrogen atoms from the C-1' and C-5' positions of deoxyribose. Attack occurs mostly (68%) at the C-1' position, producing a lesion whose properties are consistent with those of (oxidized) apyrimidinic sites.     

Atypical abasic sites generated by neocarzinostatin at sequence-specific cytidylate residues in oligodeoxynucleotides

Neocarzinostatin chromophore produces alkali-labile, abasic sites at cytidylate residues in AGC sequences in oligonucleotides in their duplex form. Glutathione is the preferred thiol activator of the drug in the formation of these lesions. The phosphodiester linkages on each side of the abasic site are intact, but when treated with alkali, breaks are formed with phosphate moieties at each end. Similar properties are exhibited by the abasic lesions produced at the purine residue to which the C in AGC is base-paired on the complementary strand. The abasic sites at C residues differ from those produced by acid-induced depurination in the much greater lability of the phosphodiester linkages on both sides of the deoxyribose, in the inability of NaBH4 to prevent alkali-induced cleavage, and in the relative resistance to apurinic/apyrimidinic endonucleases. The importance of DNA microstructure in determining attack site specificity in abasic site formation at C residues is shown not only by the requirement for the sequence AGC but also by the findings that substitution of G by I 5' to the C decreases the attack at C, whereas placement of an I opposite the C markedly enhances the reaction. Quantitation of the abstraction of 3H into the drug from C residues in AGC specifically labeled in the deoxyribose at C-5' or C-1',2' suggests that, in contrast to the attack at C-5' in the induction of direct strand breaks at T residues, abasic site formation at C residues may involve attack at C-1'. Each type of lesion may exist on the complementary strands of the same DNA molecule, forming a double-stranded lesion.     

9-amino-ellipticine inhibits the apurinic site-dependent base excision-repair pathway.

The aromatic amine 9-amino-ellipticine is a synthetic DNA intercalating compound derived from the antitumor agent ellipticine, which cleaves at very low doses DNA containing apurinic sites by beta-elimination through formation of a Schiff base. This compound has been shown to potentiate the cytotoxic effect of alkylating drugs, such as dimethyl sulfate, in E. coli through a mechanism involving apurinic sites. We have studied the ability of 9-amino-ellipticine to inhibit an enzymatic repair system mimicking base-excision repair, in which E. coli exonuclease III only presents an endonuclease for apurinic/apyrimidinic site activity. 10 microM of 9-amino-ellipticine inhibits 70% of apurinic site repair. Other intercalating agents with similar affinities for DNA do not induce any inhibition. In another system designed for the direct assay of the exonuclease III-induced incisions 5' to AP sites 10 microM of 9-amino-ellipticine inhibits 65% of the endonuclease for apurinic/apyrimidinic site activity of E. coli exonuclease III. The 9-amino-ellipticine-induced formation of a 2',3'-unsaturated deoxyribose and cleavage at the 3' side of the apurinic site, and possible creation of an adduct, as suggested by Bertrand and coworkers (1989), on the 3' position of the deoxyribose seem to strongly inhibit the endonuclease for apurinic/apyrimidinic site activity. 9-Amino-ellipticine appears therefore to be the first small ligand which can inhibit, by an irreversible modification of the substrate, the repair of apurinic sites through the base excision-repair pathway at a pharmacological concentration.     

A model for the recA-dependent repair of excision gaps in UV-irradiated Escherichia coli

DNA isolated from lambda phage was treated with bleomycin A2 plus Fe2+. The bleomycin-damaged DNA was added to lambda packaging extracts and the resulting phage were grown in SOS-induced E. coli. Under these conditions, treatment of the DNA with 0.8 microM bleomycin reduced the viability of the repackaged phage to 3% and increased the frequency of clear-plaque mutants in the progeny by a factor of 16. Bleomycin-induced mutations which mapped to the DNA-binding domain of the cI gene were subjected to DNA-sequence analysis. The most frequent events were single-base substitutions at G:C base pairs, nearly all of which occurred at cytosines in the sequence Py-G-C. Cytosines in the third position of the sequence C-G-C-C were particularly susceptible to mutation. At A:T base pairs, mutations were less frequent and were a mixture of single-base substitutions and -1 frameshifts, occurring primarily at G-T and A-T sequences. Thus, the overall specificity of bleomycin-induced mutations matches that of bleomycin-induced DNA lesions (strand breaks and apyrimidinic sites), which are formed at G-C (particularly Py-G-C), G-T and, to a lesser extent, A-T sequences. Furthermore, the frequency of various types of substitutions was consistent with selective incorporation of A and T residues opposite apyrimidinic sites at these sequences. The highly selective nature of bleomycin-induced mutations may explain the lack of mutagenesis by this compound in a number of reversion assays.     

C-1' hydrogen abstraction of deoxyribose in DNA strand scission by dynemicin A.

Dynemicin A, which is a hybrid antitumor antibiotic containing anthraquinone and enediyne cores, abstracts the C-1' hydrogen of DNA deoxyribose and then the damaged DNA leads to strand breaks with the formation of 5'- and 3'-phosphate termini. The lesions of C-4' hydrogen also occur at 3' side of G.C base pairs (i. e., 5'-CT and 5'-GA), leading to 5'-phosphate and 3'-phosphoglycolate termini or 4'-hydroxylated abasic sites. The C-1' hydrogen abstraction by dynemicin A is distinct from the preferential C-5' hydrogen abstraction of calicheamicin and neocarzinostatin.     

Yeast DNA 3'-repair diesterase is the major cellular apurinic/apyrimidinic endonuclease: substrate specificity and kinetics

DNA strand breaks with damaged 3' termini are potentially toxic lesions caused by free radicals. The purified yeast diesterase that removes small nucleotide fragments from such 3' termini in oxidized DNA has been further characterized with respect to its substrate specificity. In addition to the 3'-phosphoglycolaldehyde esters used to monitor the activity during purification, the enzyme efficiently hydrolyzed a variety of other 3'-esters in DNA. These included 3'-phosphates, 3'-(2,3-didehydro-2,3-dideoxyribose phosphates), and the 3'-blocking damages formed in vivo in Escherichia coli by H2O2 or in vitro by DNA treatment with bleomycin. This same transition metal-dependent enzyme also constitutes the major yeast endonuclease for apurinic/apyrimidinic sites in DNA, hydrolyzing these damages to yield normal 3'-hydroxyl nucleotides and 5'-phosphoryl base-free sugar termini (a Type II apurinic/apyrimidinic endonuclease). Yeast 3'-phosphoglycolaldehyde diesterase therefore appears to be involved in two distinct pathways of DNA repair: initiation of the repair of oxidative strand breaks in DNA and the restoration of sites of base loss caused by many types of DNA-damaging agents.     

Specificity of ionizing radiation-induced mutagenesis in the lac region of single-stranded phage M13 mp10 DNA.

M13 mp10 single-stranded phage DNA was irradiated with 60 Co gamma-rays, and transfected into Escherichia coli. One hundred and sixteen mutant clones having lesions in the lac insert were selected, and mutational sites were examined by DNA sequence analysis. Fourteen out of the 15 nucleotide changes thus detected were base substitutions, and the rest was a base addition. Transitions and transversions were almost equal in number. Mutational events were observed at cytosine residues more frequently than at other residues, and the predominant base change was a C ---- T transition. Possible roles in gamma-ray-induced mutagenesis played by the misincorporation of dAMP owing to radiolytic derivatives of cytosine residues and/or formation of apurinic/apyrimidinic sites are discussed.     

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.     

Abasic site recognition by two apurinic/apyrimidinic endonuclease families in DNA base excision repair: the 3' ends justify the means

DNA damage occurs unceasingly in all cells. Spontaneous DNA base loss, as well as the removal of damaged DNA bases by specific enzymes targeted to distinct base lesions, creates non-coding and lethal apurinic/apyrimidinic (AP) sites. AP sites are the central intermediate in DNA base excision repair (BER) and must be processed by 5' AP endonucleases. These pivotal enzymes detect, recognize, and cleave the DNA phosphodiester backbone 5' of, AP sites to create a free 3'-OH end for DNA polymerase repair synthesis. In humans, AP sites are processed by APE1, whereas in yeast the primary AP endonuclease is termed APN1, and these enzymes are the major constitutively expressed AP endonucleases in these organisms and are homologous to the Escherichia coli enzymes Exonuclease III (Exo III) and Endonuclease IV (Endo IV), respectively. These enzymes represent both of the conserved 5' AP endonuclease enzyme families that exist in biology. Crystal structures of APE1 and Endo IV, both bound to AP site-containing DNA reveal how abasic sites are recognized and the DNA phosphodiester backbone cleaved by these two structurally unrelated enzymes with distinct chemical mechanisms. Both enzymes orient the AP-DNA via positively charged complementary surfaces and insert loops into the DNA base stack, bending and kinking the DNA to promote flipping of the AP site into a sequestered enzyme pocket that excludes undamaged nucleotides. Each enzyme-DNA complex exhibits distinctly different DNA conformations, which may impact upon the biological functions of each enzyme within BER signal-transduction pathways.     

T4 DNA ligase can seal a nick in double-stranded DNA limited by a 5''-phosphorylated base-free deoxyribose residue.

The 5' AP endodeoxyribonucleases hydrolyze the phosphodiester bond 5' to AP (apurinic or apyrimidinic) sites in double-stranded DNA leaving 3'-OH and 5'-phosphate ends. These nicks are sealed by T4 DNA ligase although the 5'-phosphate end belongs to a base-free deoxyribose.     

Efficiency of exonucleolytic action of apurinic/apyrimidinic endonuclease 1 towards matched and mismatched dNMP at the 3' terminus of different oligomeric DNA structures correlates with thermal stability of DNA duplexes

Human DNA apurinic/apyrimidinic endonuclease 1 (APE1) is involved in the DNA base excision repair process. In addition to its AP (apurinic/apyrimidinic) endonucleolytic function, APE1 possesses 3' phosphodiesterase and 3'-5' exonuclease activities. The 3'-5' exonuclease activity is considered important in proofreading of DNA synthesis catalyzed by DNA polymerase beta. Here, we examine the removal of matched and mismatched dNMP from the 3' terminus of the 3'-recessed and nicked DNA by the APE1 activity using two different reaction buffers. To investigate whether the ability of APE1 to excise nucleotides from the 3' terminus depends on the thermal stability of the DNA duplex, we studied this characteristic of the DNAs that were used in the exonuclease assays in these two buffers. Our data confirm that APE1 removes mismatched nucleotides from the 3' terminus of DNA more efficiently than matched pairs. Both the efficiency of the 3'-5' exonuclease activity of APE1 and the thermal stability of DNA duplexes varied depending on the nature of the flanking group at the 5' margin of the nick. The 3'-5' exonuclease activity of APE1 shows a preference for substrates with a hydroxyl group at the 5' margin of the nick as well as for flapped and recessed DNAs.     

Cleavage of DNA at apyrimidinic sites by lysyl-tryptophyl-α-lysine

Incubation of partially depyrimidinated superhelical or linear DNA with the peptide lysyl-tryptophyl-α-lysine results in specific cleavage of the DNA at the base loss sites. No differences were found between the rates of breakage of apyrimidinic sites and apurinic sites in superhelical DNA.     

DNA deoxyribophosphodiesterase.

A previously unrecognized enzyme acting on damaged termini in DNA is present in Escherichia coli. The enzyme catalyses the hydrolytic release of 2-deoxyribose-5-phosphate from single-strand interruptions in DNA with a base-free residue on the 5' side. The partly purified protein appears to be free from endonuclease activity for apurinic/apyrimidinic sites, exonuclease activity and DNA 5'-phosphatase activity. The enzyme has a mol. wt of approximately 50,000-55,000 and has been termed DNA deoxyribophosphodiesterase (dRpase). The protein presumably is active in DNA excision repair to remove a sugar-phosphate residue from an endonucleolytically incised apurinic/apyrimidinic site, prior to gap filling and ligation.     

The yeast 8-oxoguanine DNA glycosylase (Ogg1) contains a DNA deoxyribophosphodiesterase (dRpase) activity.

The yeast OGG1 gene was recently cloned and shown to encode a protein that possesses N-glycosylase/AP lyase activities for the repair of oxidatively damaged DNA at sites of 7,8-dihydro-8-oxoguanine (8-oxoguanine). Similar activities have been identified for Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) and Drosophila ribosomal protein S3. Both Fpg and S3 also contain a deoxyribophosphodiesterase (dRpase) activity that removes 2-deoxyribose-5-phosphate at an incised 5' apurinic/apyrimidinic (AP) sites via a beta-elimination reaction. Drosophila S3 also has an additional activity that removes trans-4-hydroxy-2-pentenal-5-phosphate at a 3' incised AP site by a Mg2+-dependent hydrolytic mechanism. In view of the substrate similarities between Ogg1, Fpg and S3 at the level of base excision repair, we examined whether Ogg1 also contains dRpase activities. A glutathione S-transferase fusion protein of Ogg1 was purified and subsequently found to efficiently remove sugar-phosphate residues at incised 5' AP sites. Activity was also detected for the Mg2+-dependent removal of trans -4-hydroxy-2-pentenal-5-phosphate at 3' incised AP sites and from intact AP sites. Previous studies have shown that DNA repair proteins that possess AP lyase activity leave an inefficient DNA terminus for subsequent DNA synthesis steps associated with base excision repair. However, the results presented here suggest that in the presence of MgCl2, Ogg1 can efficiently process 8-oxoguanine so as to leave a one nucleotide gap that can be readily filled in by a DNA polymerase, and importantly, does not therefore require additional enzymes to process trans -4-hydroxy-2-pentenal-5-phosphate left at a 3' terminus created by a beta-elimination catalyst.     

Product analyses in DNA strand scission by antitumor antibiotic elsamicin A.

Elsamicin A is an antitumor antibiotic with fascinating chemical structure and a good candidate for pharmaceutical development. Molecular mechanism of DNA backbone cleavage mediated by Fe(II)-elsamicin A has been examined. Product analysis using DNA sequencing gels and HPLC reveals the production of damaged DNA fragments bearing 3'-/5'-phosphate and 3'-phosphoglycolate termini associated with formation of free base. In addition, hydrazine-trapping experiments indicate that C-4' hydroxylated abasic sites are formed concomitant with DNA degradation by Fe(II)-elsamicin A. The results lead to the conclusion that the hydroxyl radical formed in Fe(II)-elsamicin A plus dithiothreitol system oxidizes the deoxyribose moiety via hydrogen abstraction predominantly at the C-4' carbon of the deoxyribose backbone and ultimately produces strand breakage of DNA.     

Quantitative parameters of the 3'-5'-exonuclease reaction of human apurinic/apyrimidinic endonuclease 1 and DNA with single-strand breaks containing dYMP or their modified analogues

Human apurinic/apyrimidinic (AP) endonuclease 1 (APE1) is a multifunctional enzyme. In addition to its main AP endonuclease activity, the cleavage of DNA 5' to the AP site, it displays other weak enzymatic activities. One of them is 3'-5' exonuclease activity, which is most effectively pronounced for DNA duplexes containing modified or mismatched nucleotides at the 3' end of the primer chain. There is a presumption that APE1 can correct the DNA synthesis catalyzed by DNA polymerase beta during the base excision repair process. We determined the quantitative parameters of the 3'-5' exonuclease reaction in dependence on the reaction conditions to reveal the detailed mechanism of this process. The kinetic parameters of APE1 exonuclease excision of mismatched dCMP and dTMP from the 3' terminus of single-strand DNA and from photoreactive dCMP analogues applied for photoaffinity modification of proteins and DNA in recombinant systems and cell/nuclear extracts were determined. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.     

den V gene of bacteriophage T4 codes for both pyrimidine dimer-DNA glycosylase and apyrimidinic endonuclease activities.

Recent studies have shown purified preparations of phage T4 UV DNA-incising activity (T4 UV endonuclease or endonuclease V of phage T4) contain a pyrimidine dimer-DNA glycosylase activity that catalyzes hydrolysis of the 5' glycosyl bond of dimerized pyrimidines in UV-irradiated DNA. Such enzyme preparations have also been shown to catalyze the hydrolysis of phosphodiester bonds in UV-irradiated DNA at a neutral pH, presumably reflecting the action of an apurinic/apyrimidinic endonuclease at the apyrimidinic sites created by the pyrimidine dimer-DNA glycosylase. In this study we found that preparations of T4 UV DNA-incising activity contained apurinic/apyrimidinic endonuclease activity that nicked depurinated form I simian virus 40 DNA. Apurinic/apyrimidinic endonuclease activity was also found in extracts of Escherichia coli infected with T4 denV+ phage. Extracts of cells infected with T4 denV mutants contained significantly lower levels of apurinic/apyrimidinic endonuclease activity; these levels were no greater than the levels present in extracts of uninfected cells. Furthermore, the addition of DNA containing apurinic or apyrimidinic sites to reactions containing UV-irradiated DNA and T4 enzyme resulted in competition for pyrimidine dimer-DNA glycosylase activity against the UV-irradiated DNA. On the basis of these results, we concluded that apurinic/apyrimidinic endonuclease activity is encoded by the denV gene of phage T4, the same gene that codes for pyrimidine dimer-DNA glycosylase activity.     

Structure of the DNA repair enzyme endonuclease IV and its DNA complex: double-nucleotide flipping at abasic sites and three-metal-ion catalysis.

Endonuclease IV is the archetype for a conserved apurinic/apyrimidinic (AP) endonuclease family that primes DNA repair synthesis by cleaving the DNA backbone 5' of AP sites. The crystal structures of Endonuclease IV and its AP-DNA complex at 1.02 and 1.55 A resolution reveal how an alpha8beta8 TIM barrel fold can bind dsDNA. Enzyme loops intercalate side chains at the abasic site, compress the DNA backbone, bend the DNA approximately 90 degrees, and promote double-nucleotide flipping to sequester the extrahelical AP site in an enzyme pocket that excludes undamaged nucleotides. These structures suggest three Zn2+ ions directly participate in phosphodiester bond cleavage and prompt hypotheses that double-nucleotide flipping and sharp bending by AP endonucleases provide exquisite damage specificity while aiding subsequent base excision repair pathway progression.     

Reaction of apurinic/apyrimidinic sites with [14C]methoxyamine. A method for the quantitative assay of AP sites in DNA

A simple and rapid method is described for the determination of AP (apurinic/apyrimidinic) sites in DNA. The method involves the reaction of [14C]methoxyamine with the aldehyde group present in the deoxyribose moiety after a base loss. Studies with alkylated-depurinated DNA and with uracil-containing polydeoxyribonucleotides depyrimidinated by uracil-DNA glycosylase show that methoxyamine reacts with both apurinic and apyrimidinic sites in a rapid and exhaustive way. Under standard conditions (30-min incubation with 5 mM methoxyamine at 37 degrees C, pH 7.2) untreated DNA is almost unreactive and the [14C]methoxyamine incorporation in DNA is proportional to the number of AP sites. Since the methoxyamine reaction is free from any degradative effect on DNA, AP sites may be estimated from a simple determination of the acid-insoluble radioactivity.