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Comparison of the kinetic constants for the excision of HX residues by the four enzymes shows that the E. Since the use of various substrates to monitor the activity of HX-DNA glycosylases has generated conflicting results, the efficacy of the four 3-meAde-DNA glycosylases of different origin was compared using three different substrates. We found up to 2—5-fold difference in the rates of HX excision between the various sequences of the oligonucleotides studied. When the dIMP residue was placed opposite to each of the four bases, a preferential recognition of dI: The rules that govern the recognition of a damaged base by the enzyme in charge of its elimination, in vitro and in vivowould be important for the understanding of the molecular mechanisms involved in DNA repair.
The mutagenic properties of dIMP residues have been ascertained by site-specific mutagenesis in vivo 11 A single dIMP residue, inserted in vitro at a specific locus in a M13mp9 RF molecule, exhibits miscoding properties leading to mutagenesis in E. The presence of hot spots of mutagenesis in E. A significant variation in the rate of repair, depending upon the surrounding sequence context, has been shown in the case of repair of uracil 13AP-sites 14formamidopyrimidine residues 15O 6 -methylguanine 16N-methylated bases 17 and UV-induced photoproducts The slow rate of repair of cyclobutane dimers in the human p53 gene at mutational hotspots in skin cancer is documented, suggesting that hotspots for mutations arise partly as a consequence of heterogeneous repair within specific sequences The aim of the present study was to investigate the structural requirements for the interaction of the 3-meAde-DNA glycosylases of different origin with dIMP residues, when present in different sequence contexts.
The best substrate is a double-stranded oligonucleotide containing a single HX residue.
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The results show that pure preparations of human, rat, yeast and E. Up to 2—5-fold difference in the rates of HX removal between various sequences was measured. Moreover, the bacterial, yeast and mammalian enzymes show definite sequence preference. Escherichia coli BH X:: Seeberg University of Oslo, Norway.
The AlkA protein prepared as described 22 was a gift from Dr B. The MAG protein was purified as described Nucleic acids and nucleotides were purchased from Roche Mannheim, Germany. Radiolabeled reagents were obtained from the following sources: Its specific activity was 51 c. The specific activities of the 3 H-methylated substrates were c. Lescot this laboratory or purchased from Genset Paris, France. The sequence of duplex oligonucleotides that we chose was part of the gpt gene The sequences of the various oligonucleotides used are shown in Table 1.
The gels were subjected to autoradiography. The kinetic parameters of the binding reactions were determined using BIAevaluation 2. When it is treated either with piperidine lane 2 or by an excess of Fpg protein lane 3treatments that will reveal AP sites, it is not incised, showing that it does not contain the AP site. When this double-stranded oligodeoxyribonucleotide is treated either with the ANPG40 protein lane 4 or the AlkA protein lane 10it is only minimally incised due to the instability of the generated abasic site under the experimental conditions used.
Cs-3111d substrates were used in order to compare and to ascertain their relative properties. As shown in Table 2the duplex oligonucleotide containing a dIMP residue is by far the best substrate for all the three enzymes. This observation leads to the use of this latter substrate to establish the kinetic parameters and the influence of the sequence context upon the excision of HX by the various DNA repair proteins.
The quantification of the amount of radioactivity migrating at the position of i-rando, 19mer shows that it increased linearly during the initial part of the reaction Fig.
Similar kinetics were obtained with i-rwndom other proteins and allowed the determination of the i-randon velocities of the enzymatic reactions used to determine the kinetic constants.
These data show a dramatic difference i-randkm the three enzymes, the human one being by far the most efficient. The initial cs-311f of cleavage of various substrates having different sequence context by the mammalian 3-meAde-DNA glycosylases was compared.
Moreover, this study was extended by using, for each oligonucleotide, duplexes containing dC, dG or dA positioned opposite the dIMP residue. The latter substrate is recognized 1. The results show a dramatic change 5. It is clear that the two flanking bases are not the main reason for the good or poor removal of HX compare HXDL and HX irandom, but it should be noted that the stability of the duplex containing inosine flanked by pyrimidines is lower than when inosine is flanked by purines However, the rat enzyme had a lower activity on all the substrates tested.
The analysis of the efficiency of excision of HX when opposite to each of the four different bases by mammalian 3-meAde-DNA glycosylases reveals that dI: The results presented in Table 5 show that the AlkA protein does not exhibit any clear preference for a given substrate, which is at variance with the mammalian enzymes, although in most cases dI: T mismatch was the best repaired.
Interestingly, the HXDL substrate for all the mismatches shows again in the case of the mismatch dI: It is very poorly recognized. Among the substrates tested, the HX was the best.
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The results presented in Table 6 show that as already cs-311x for the AlkA protein, the MAG protein has no clear preference for any of the substrates tested. However, regardless of the sequence context, the excision of HX by the Mag protein present in a dI: Among the substrates tested, the HX oligonucleotide was the best. The distinctive behavior of the yeast 3-meAde-DNA glycosylase compared to the other enzymes tested should be o-random.
The purpose of this study was to investigate in detail the factors that influence the efficacy of four 3-meAde-DNA gly-cosylases of different origin for the same substrate.
Since the glycosidic bond of akylated bases is quite unstable and does not allow such detailed studies, we chose to study the activity of these 3-meAde-DNA glycosylases on substrates containing HX residues. This study was facilitated by the use of duplex oligonucleotides containing dIMP residues at i-randim precise location and the availability of data concerning the structure of duplex oligonucleotides containing mismatched dIMP residue 38 — The evidence from crystal structure and NMR studies i-ranvom that i the HX base l-random form stable hydrogen-bonded mismatches with all four normal bases T, G, A and C, in DNA and ii a duplex oligonucleotide containing mismatches made of dIMP residues mainly takes a B-DNA helix conformation and does not show large perturbations at the local level 38 — Studies of the thermal stability of oligonucleotides containing deoxyinosine have shown that the order of the stability is independent of sequence effects, and is: The comparison of the efficiency of HX excision by the mammalian 3-meAde-DNA glycosylases shows that it correlates with the thermal stability of the mismatch: The bacterial enzyme also preferentially repairs dI: In cs-131d case of the MAG protein, the dI: It should be recalled that NMR studies of the dI: As Syn conformation of dI takes place only in the dI: Since the recognition of a relatively short oligodeoxyribonucleotide by a DNA gly-cosylase could be modified by the termini of such molecules, the activity of the various enzymes was measured by cs311d the same sequence in i-andom core of the oligonucleotide but having blunt or sticky ends.
The results show that oligonucleotides having blunt ends are somehow better recognized. The comparison of the kinetic constants for the excision of HX by the three different enzymes shows that the efficiency of repair by E.
This is in agreement with the previous in vivo data showing that, in E. Taken together these results strongly suggest that, in vivoHX, as a DNA lesion, is not physiologically important for the fast propagating monocellular organisms, perhaps due to the slow rate of adenine deamination in duplex DNA Nevertheless, the difference between the binding constants of E.
This data possibly means that the inefficient repair of dIMP residue in DNA by bacterial and yeast proteins could be due to an extremely slow catalysis. The interaction of the human truncated ANPG protein with oligodeoxyribonucleotides containing either HX or abasic sites has been investigated The region of direct protein—DNA interactions consists only of one turn of helix.
However, the crystal structures of the AlkA protein 4647 and the truncated ANPG protein complexed to a pyrrolidine-containing 13 bp duplex oligonucleotide have been reported AlkA is a globular protein consisting of three equal-sized domains.
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Its active site is located in a large hydrophobic cleft rich in aromatic residues between domains 2 and 3 46 Both enzymes utilize activated water molecules for nucleophilic attack. According to the general acid and general base catalysis, Asp of the AlkA protein and Glu of the ANPG protein act as a general base to abstract a proton from bound water. This hypothesis might explain the difference of kinetic constants for the HX repair between bacterial and human enzymes.
Possible absence in the active site of the AlkA protein of an amino acid residue which could act as a general acid to protonate HX may explain the slow rate of catalysis observed.
Since the kinetic constants for the removal of alkylated bases by the AlkA and ANPG cs-311v are quite similar 750this could be explained by the fact that alkylated bases have in common a positive charge and a labilized glycosidic bond. Therefore one can tentatively propose that for excision of alkylated bases such as 3meAde and 7meGua, nucleophilic attack by water activated by the general base could be sufficient for catalysis. The results show that the bacterial, yeast and mammalian c-311d investigated have different sequence preferences.
Neighboring groups can have a large effect on thermal stability of duplex DNA containing dIMP residues 33therefore affecting the repair of the lesion. The thermal stability and NMR studies of the oligonucleotides used can give insight into cs-31d sequences for good and poor repair i-randlm HX from duplex DNA. However, the sequence effect on the repair of HX was studied in vitro on naked DNA, and it is possible that repair in vivo on coated DNA could be different.
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The generation of 3-meAde-DNA glycosylase deficient APDG ko mice 5152 will allow the assessment of the role of this enzyme in the repair of HX and the physiological relevance of such damage in vivo. Unexpectedly, these APDG ko mice do not exhibit any particular sensitivity to alkylating agents Lanes 6 and 12, with the Fpg i-randlm ng ; lanes 7 and 13, with the Nfo protein ng ; lanes 8 and 14, with the Nth protein ng. Arrow a indicates the 34mer oligonucleotide. For details see Materials i-gandom Methods.
Kinetics of excision, by l-random ANPG40 protein, of HX from an oligonucleotide duplex containing thymine opposite a deoxyinosine residue. Lanes 1—7, incubation time 0, 5, 10, 15, 20, 25 and 30 min, respectively. B The cs-3311d of the cs-311f of the reaction shown in A was quantified and plotted as a function of time. The initial velocities measured for excision of HX were treated as described by Lineweaver and Burk. The kinetic parameters ka and kd were determined using BIAevaluation 2.
Initial velocities of cleavage, by various mammalian 3-methyladenine DNA glycosylases, of oligonucleotide duplexes having different structures and containing different bases opposite to the dIMP residue.
Initial velocities were calculated from the linear part of the curve where the amount of HX released is plotted as a function of time. Initial velocities of cleavage by the E.
Initial velocities of cleavage by the yeast MAG protein of oligonucleotide duplexes containing a different base opposite the dIMP residue. Oxford University Press is a department of the University of Oxford.