The E.coii gene ogt encodes the DNA repair protein C^-alkylguanine-DNA-alkyltransferase (C^-AlkG ATase). The protein coding region of the gene was cloned into a multicopy expression vector to obtain high yields of the enzyme (~ 0.2% of total protein) which was purified to apparent homogeneity by affinity, molecular exclusion and reverse-phase chromatography. Good correlation was found between the determined and predicted amino acid compositions. The ability of the purified protein to act on C^-methylguanine (C^-MeG), C^-ethylguanine (C^-EtG) and (?*-methylthymine (O'-MeT) in self-complementary dodecadeoxyribonucleotides was compared to that of 19 kDa fragment of the related ada-protein. With both proteins the rate order was C/'-MeG > C^-EtG > <7*-MeT, however, the ogt protein was found to repair (/-MeG, C^-EtG and (T'-MeT, 1.1, 173 and 84 times, respectively, faster than the ada protein. INTRODUCTION The reaction of alkylating agents with DNA results in the formation of a number of base and phosphate modifications (1,2). Some of these have been shown to be toxic and mutagenic in bacteria and mammalian cells (3,4) which contain repair enzymes capable of correcting certain types of damage (1,3-5). The repair of the lesion (/-MeG was first demonstrated in E.coii following administration of A^-methyl-N-nitro-N-nitrosoguanidine (MNNG) (6). Under such conditions repair is most likely to be mediated predominantly by the C^-AlkG-ATase that is the product of the ogt gene (7). This 19 kDa protein transfers the methyl group from the Opposition of guanine to a cysteine residue within the protein itself (7,8) a process which is stoicheiometric and autoinactivating. Exposure of E. coli to low doses of MNNG results in an increase in their resistance to the toxic and mutagenic effects of a subsequent higher dose of MNNG, a phenomenon known as the adaptive response (9,10). This response is controlled by the ada gene (11) the product of which was later shown to be a 39 kDa protein that contained 2 ATase functions (8,12,13). One of these repairs C -̂AlkG and O*-AlkT (15) and the other repairs the Sstereoisomer of alkylphosphotriesters (AlkP) (16). This ATase is very susceptible to proteolytic cleavage at amino acid residue 178 and gives rise to two subfragments of 19 and 20 kDa. The latter of these is responsible for the repair of AlkP and, in its alkylated form the 39 kDa protein has been shown to be a positive regulator of the adaptive response. This involves its binding to a region known as the ada box within the ada promoter and the subsequent upregulation and transcription of the gene (17). The 19 kDa protein encoded by ogt does not repair AlkP (7,8) however the amino acid sequence demonstrates 29% homology to the C-terminal domain of the ada protein, the © IRL Press 8475 at U niersity C olege L odon on N ovem er 5, 2013 http://narrdjournals.org/ D ow nladed from Nucleic Acids Research region responsible for the repair of C^-AlkG (7). Analysis of the ogt promoter does not reveal any homology to the ada box sequences suggesting that ogt would not be upregulated during the adaptive response (7) and no evidence of upregulation has been found (Potter et al, unpublished results). A number of experiments in which the characteristics of the ogt ATase are being compared to those of the ada ATases are in hand. In the present report we describe the purification of the ogt encoded C^-AlkG-ATase to apparent homogeneity from extracts of bacteria harbouring ogt in a multicopy expression vector. The amino acid composition of the homogeneous protein has been determined and the protein has been used to measure its rate of action, in comparison with the ada encoded C^-AlkG-ATase, on selfcomplementary synthetic oligonucleotides containing C/'-MeG, C^-EtG or <7*-MeT. MATERIALS AND METHODS Bacteria and Plasmids E.coli JM83 (18) harbouring the plasmid pO61M was used throughout this study. pO61M consists of a 1304 bp insert isolated from pO61SPl (7) (see Figure 1) containing the complete promoter region and protein coding sequence of the ogt gene inserted in the unique Sma\ site of pUC9 (19). pO62SX (20) contains 793 bp of the 3' region of the protein coding sequence of the ada gene inserted into the multiple cloning site of pUC9 and encodes protein that only repairs C'-AlkG. pO62HSR (Potter et al, unpublished results) contains all of the ada gene except for 157 bp of the 3' end which encodes the active site for the repair of C^-AlkG and (^-AlkT. Alkyltransferase Assay Extracts of E.coli harbouring various plasmids, fractions collected during chromatography or pooled activity peaks were assayed for ATase activity as described elsewhere (7). Assays for AlkP ATase and (^-AlkG-ATase activities individually involved use of modified [H]-methylnitrosourea treated DNA substrates from which a) C^-MeG (and C^-MeT) were removed by prior incubation with the C'-AlkG ATase fragment of the ada gene encoded by the plasmid pO62SX (20) or b) AlkP were removed by incubation with the AlkP ATase fragment of the ada gene encoded by the plasmid pO62HSR. The preparation and verification of these substrates will be described more completely elsewhere. Polyacrylamide Gel Electrophoresis (PAGE) Protein extracts were analysed using an SDS discontinuous buffer system (20) adapted by Bury (22) and visualised with Coomassie Blue or by silver-staining (23). Purification of the ogt Gene Product Eight litres of E.coli JM83 harbouring pO61M were grown to stationary phase in LB medium and centrifuged to obtain approximately 15 g of cells. These were disrupted by sonication (three pulses of 2 minutes at 30 n peak to peak with cooling on ice) in 100 ml of buffer I (50 mM Tris-HCl, 1 mM EDTA, 3 mM dithiothreitol DTT pH 8.3). Phenylmethylsulphonyl fluoride was added to 0.5 mM immediately after the last sonication. Following centrifugation at 30,000 g for 10 minutes, the sample was applied to a doublestranded DNA-cellulose column (15 cmx2.5 cm) equilibrated in buffer I. After washing extensively with the same buffer, proteins were eluted into 4 ml fractions with buffer I containing 0.1, 0.25 or 0.5 M NaCl. Monitoring of the eluant was performed at 280 nm. Fractions containing the ATase were pooled and concentrated to a final volume of 1.2 ml using an Amicon ultrafiltration system employing a YM10 membrane. The sample was then applied to a Sephacryl S200HR column (85 x 1.5 cm i.d.) and proteins eluted 8476 at U niersity C olege L odon on N ovem er 5, 2013 http://narrdjournals.org/ D ow nladed from Nucleic Acids Research P M H I I pO61SP1 I k^£

RT-PCR was used to find whether cytochromes P450 of the 2A, 2B and 2E sub-families are expressed in the rat oesophagus. This showed that this tissue expresses a previously unknown member of the CYP2B sub-family, now designated CYP2B21. Using a combination of 5'- and 3'-RACE (rapid amplification of cDNA ends) and library screening, the cDNA was amplified and sequenced. The cDNA sequence (GenBank accession no. AF159245) covers the whole of the coding region and the whole of the 3'-untranslated region (UTR), but only 17 nt of the 5'-UTR. The DNA sequence has strong similarity to those of CYP2B1 and CYP2B2, with the derived amino acid sequence being 84 and 83% identical, respectively. The ease with which this cDNA was found in the cDNA library suggests that CYP2B21 is a major P450 of the oesophagus. The catalytic activity of this new CYP2B is not yet known, but as previous authors have reported that other members of this sub-family (CYP2B1 or 2B2) metabolize the selective oesophageal carcinogen N:-nitrosomethylbutylamine with the chemical selectivity necessary for carcinogenesis, i.e. they preferentially hydroxylate the alpha-carbon of the butyl chain, this new CYP2B may be the nitrosamine-activating enzyme of the oesophagus.

In vitro, following the removal of thymine from a G·T mismatch, thymine DNA glycosylase binds tightly to the apurinic site it has formed. It can also bind to an apurinic site opposite S 6-methylthioguanine (SMeG) or opposite any of the remaining natural DNA bases. It will therefore bind to apurinic sites formed by spontaneous depurination, chemical attack, or other glycosylases. In the absence of magnesium, the rate of dissociation of the glycosylase from such complexes is so slow (k off 1.8 − 3.6 × 10−5 s−1; i.e. half-life between 5 and 10 h) that each molecule of glycosylase removes essentially only one molecule of thymine. In the presence of magnesium, the dissociation rates of the complexes with C·AP andSMeG·AP are increased more than 20-fold, allowing each thymine DNA glycosylase to remove more than one uracil or thymine from C·U and SMeG·T mismatches in DNA. In contrast, magnesium does not increase the dissociation of thymine DNA glycosylase from G·AP sites sufficiently to allow it to remove more than one thymine from G·T mismatches. The bound thymine DNA glycosylase prevents human apurinic endonuclease 1 (HAP1) cutting the apurinic site, so unless the glycosylase was displaced, the repair of apurinic sites would be very slow. However, HAP1 significantly increases the rate of dissociation of thymine DNA glycosylase from apurinic sites, presumably through direct interaction with the bound glycosylase. This effect is concentration-dependent and at the probable normal concentration of HAP1 in cells the dissociation would be fast. This interaction couples the first step in base excision repair, the glycosylase, to the second step, the apurinic endonuclease. The other proteins involved in base excision repair, polymerase β, XRCC1, and DNA ligase III, do not affect the dissociation of thymine DNA glycosylase from the apurinic site.

The time course of removal of thymine by thymine DNA glycosylase has been measured in vitro. Each molecule of thymine DNA glycosylase removes only one molecule of thymine from DNA containing a G.T mismatch because it binds tightly to the apurinic DNA site left after removal of thymine. The 5'-flanking base pair to G.T mismatches influences the rate of removal of thymine: k(cat) values with C.G, T.A, G.C, and A.T as the 5'-base pair were 0.91, 0.023, 0.0046, and 0.0013 min(-l), respectively. Thymine DNA glycosylase can also remove thymine from mismatches with S(6)-methylthioguanine, but, unlike G.T mismatches, a 5'-C.G does not have a striking effect on the rate: k(cat) values for removal of thymine from (SMe)G.T With C.G, T.A, G.C, and A.T as the 5'-base pair were 0.026, 0.018, 0.0017, and 0.0010 min(-l), respectively. Thymine removal is fastest when it is from a G.T mismatch with a 5'-flanking C.G pair, suggesting that the rapid reaction of this substrate involves contacts between the enzyme and oxygen 6 or the N-l hydrogen of the mismatched guanine as well as the 5'-flanking C.G pair. Disrupting either of these sets of contacts (i,e. replacing the 5'-flanking C.G base pair with a T.A or replacing the G.T mismatch with (SMe)G.T) has essentially the same effect on rate as disrupting both sets (i,e. replacing CpG.T with Tp(Sme)G.T), and so these contacts are probably cooperative.The glycosylase removes uracil from G.U, C.U, and T.U base pairs faster than it removes thymine from G.T. It can even remove uracil from A.U base pairs, although at a very much lower rate. Thus, thymine DNA glycosylase may play a backup role to the more efficient general uracil DNA glycosylase.

Oligonucleotides containing 4-thiothymine or 6-thioguanine cross-link to lambda-Cro repressor upon irradiation with long wavelength UV light. The results are consistent with the Cro-DNA interaction model based on its crystal structure.

Postsynthetic functionization of bases in oligonucleotides provides a useful approach to the preparation of DNA and RNA containing modified bases. In this communication is presented our recent work on this subject.

It has been suggested that the cytotoxicity of 6-thioguanine depends upon (1) incorporation of 6-thioguanine into DNA, (2) methylation by S-adenosylmethionine (SAM) of the thio group to give S6-methylthioguanine, (3) miscoding during DNA replication to give [SMeG] x T base pairs, and (4) recognition of these base pairs by proteins of the postreplicative mismatch repair system. Here we have investigated systematically the ability of proteins present in human cell extracts to bind to DNA containing S6-methylthioguanine. We found that [SMeG] x T base mismatches were bound by the mismatch binding complex, hMutS alpha, and that the level of binding was dependent upon the base 5' to the S6-methylthioguanine in the order G > C = A > T. Extracts from cells that lack either hMSH2 (LoVo cells) or GTBP (HCT-15 cells), two components of the hMutS alpha complex, were unable to bind the [SMeG] x T base pair. We also found that hMutS alpha was able to bind to [SMeG] x C base pairs when the S6-methylthioguanine was in the sequence 5'-Cp[SMeG]. This suggests that miscoding by S6-methylthioguanine residues in DNA during DNA synthesis may not be an absolutely required step in the mechanism of cytotoxicity. Also, since CpG sequences are so important in gene regulation, this result may be of considerable significance.

It is proposed here that the delayed cytotoxicity of thioguanine involves the postreplicative DNA mismatch repair system. After incorporation into DNA, the thioguanine is chemically methylated by S -adenosylmethionine to form S 6 -methylthioguanine. During DNA replication, the S 6 -methylthioguanine directs incorporation of either thymine or cytosine into the growing DNA strand, and the resultant S 6 -methylthioguanine-thymine pairs are recognized by the postreplicative mismatch repair system. Azathioprine, an immunosuppressant used in organ transplantation, is partly converted to thioguanine. Because the carcinogenicity of N -nitrosamines depends on formation of O 6 -alkylguanine in DNA, the formation of the analog S 6 -methylthioguanine during azathioprine treatment may partly explain the high incidence of cancer after transplantation.

a simple and efficient protocol is described for site-specific 15N-labelling of adenine in synthetic oligodeoxynucleotides by post-synthetic substitution with 15N-labelled ammonia of the oxidized methylthio group on purine. A labelled DNA of 20-mer was characterized by NMR spectroscopy.

A simple procedure is described for the preparation of a versatile oligodeoxynucleotide which contains 4-phenylthiothymidine. This versatile oligomer has been successfully used for synthesis of oligonucleotides containing labile 5-methyl-N-4,N-4-ethanocytosine (7) or 4-azido-5-methyl-2-pyrimidinone-1-beta-(2'-deoxyriboside) (8).

Two methods (chemical synthesis and specific methylation) are described for the preparation of oligodeoxynucleotides containing 6-methylthiopurine residues. 6-Methylthiopurine phosphoramidite (6) is prepared and incorporated into oligomers. Methylation with methyl iodide of 6-thiopurine (or 6-thioguanine) in oligonucleotides also leads to exclusive production of 6-methylthiopurine (or 6-methylthioguanine) oligomers.

simple procedures for preparation of O-2-alkylthymidines and of their phosphoramidite monomers (IV) are described. These monomers have been successfully incorporated into DNA oligomers. The measurements of the melting temperature (Tm) of DNA duplexes show that O-2-methylthymine preferentially pairs with guanine rather than with adenine.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

The first-pass clearance of dietary N-nitrosodimethylamine (NDMA) by the liver is the most important factor in the pharmacokinetics of this carcinogen in the rat, but is less important in the pharmacokinetics of N-nitrosodiethylamine (NDEA). The reason for the difference in clearance of these two nitrosamines is not known. These experiments were carried out to see whether the general characteristics of the clearance of these two carcinogens in vivo could be reproduced in the perfused liver, and whether the clearance could be correlated with the Michaelis-Menten parameters Km and Vmax for their metabolism. If this could be done one would be able to predict the possible extent of first-pass clearance of nitrosamines in man from measurement of Km and Vmax for nitrosamine metabolism by the human liver. The Km (22 microM) and Vmax (10.2 and 13.4 nmol/g liver/min) for the metabolism of NDMA by slices from two human livers, the inhibition of that metabolism by ethanol (Ki 0.5 microM), and the rate of N-7 methylation of DNA when slices are incubated with NDMA, were measured. These results are similar to those reported previously with rat liver. The Km (27 microM) for the metabolism of NDEA by rat liver slices and the inhibition of that metabolism by ethanol (Ki 1 microM) were estimated from the rate of ethylation of the DNA of the slices. The clearance of both these nitrosamines by the perfused rat liver was measured, and the results appeared to parallel those in vivo with a striking difference between the clearance of NDMA and NDEA. The maximal rate of clearance of NDMA was 11.2 nmol/g liver/min and of NDEA 8.9 nmol/g liver/min, similar to the Vmax for metabolism of NDMA by liver slices and to the estimated maximal rate of liver metabolism of both nitrosamines in the living rat. However, although the Km for metabolism of these two nitrosamines by liver slices is similar (about 25 microM), the logarithmic mean sinusoidal concentration [see Bass and Keiding, Biochem Pharmacol 37: 1425-1431, 1988] giving half maximal clearance during perfusion (the equivalent to Km) was 2.3 microM for NDMA and 10.6 microM for NDEA. The almost 5-fold difference between these two values is the basis for the difference between the clearance of the two nitrosamines.(ABSTRACT TRUNCATED AT 400 WORDS)

6-(2,4-dinitrophenyl)thioguanine phosphoramidite monomers with the 2-amino group of the base protected by either the isobutyryl or phenylacetyl group were incorporated into oligodeoxynucleotides with an automatic DNA synthesizer. N2-protection with the isobutyryl group was unsatisfactory because of the difficulty of removing it after synthesis of the oligemer. However, post-synthetic conversion of the N2-phenylacetyl protected 6-(2,4-dinitrophenyl)thioguanine gives oligomers containing 6-thioguanine, 2,6-diaminopurine, 2-amino-6-methylaminopurine, O6-methylguanine or guanine in high yield and purity. Potentially oligemers containing other labile functional groups at the 6-position could be produced by the procedure. DNA duplexes containing 6-thioguanine paired to cytosine had a markedly lower melting temperature (Tm) than counterparts containing G:C. However a DNA duplex containing 4-thiothymine paired to A had a Tm similar to that of a DNA duplex containing T:A. The distortion in DNA structure caused by 6-thioguanine may play a role in the biological effect of this compound.

The effect of methylation of the 04 atom of thymine in two oligonucleotide sequences is investigated by molecular dynamics simulations. Three types of environments are considered including: (i) in vacuo calculation, with a distance-dependent dielectric function and unhydrated counter-ions; (ii) in vacuo calculation, with a distance-dependent dielectric constant and hydrated counter-ions; and (iii) with a 9 angstrom thick explicit water layer and counter-ions. In all environments, the oligonucleotide sequence containing the chemically modified thymine paired with guanine is more stable than the oligonucleotide sequence in which the modified thymine is paired with adenine. The methyl group attached to the O4 atom of thymine is found in a syn configuration with respect to the N3 atom. The best fit between the experimental NMR results and the molecular dynamics simulations is obtained using the environment with hydrated counter-ions.

A general procedure is described for separation and purification of oligodeoxynucleotides of identical length but different base composition, in particular, of oligomers containing modified bases such as 4-substituted thymines and 6-substituted guanines, using an anion-exchange column (either Mono Q or NucleoPac). The modified oligomers can be well separated from the analogous oligomers containing unmodified thymine or guanine under the basic conditions of the chromatography. The effects of oligomer length, base composition, and lipophilicity on the separation are discussed. A general rule which can be used for prediction of the order of elution of different oligomers and for estimation of tautomeric form of a modified base in the oligomer is presented.

A strategy is described for synthesis of oligomers modified in the 4-position of thymine by postsynthetic substitution. 4-Triazolothymine phosphoramidite monomer has been prepared in one step from thymine amidite monomer and incorporated into a 12 mer AGCGAAXTCGCT using a DNA-synthesizer. The fully protected oligomer containing 4-triazolothymine, while still bound to CPG-support, was treated at 25-degrees-C with either alcohol/DBU, dilute aqueous NaOH, concentrated aqueous ammonia, 1,1-dimethylhydrazine, or thiolacetic acid, to produce essentially pure oligodeoxynucleotides containing O4-alkylthymine, thymine, 5-methylcytosine, N4-(dimethylamino)-5-methylcytosine [i.e., 4-(2,2-dimethylhydrazino)-5-methylpyrimid-2-one (T(DH))], or 4-thiothymine respectively. This first and efficient synthesis of T(DH) oligomers indicates that this may be a general route to the synthesis of oligomers containing thymine with a reactive group at the 4-position. The melting temperature (Tm) of a DNA duplex containing T(DH):G or T(DH):A pairs was similar to that of a duplex with A:C mismatch.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.