Purification to apparent homogeneity and partial amino acid sequence of rat liver O6-alkylguanine-DNA-alkyltransferase.

O6-alkylguanine-DNA-alkyltransferase (ATase) activity was increased in rat liver from 80 to 320 fmoles/mg total protein 48 h after administration of 2-acetylaminofluorene at 60 mg/kg body weight. This tissue was used as a source of ATase which was purified by ammonium sulphate precipitation and DNA-cellulose, molecular exclusion and ion exchange chromatography (IEC). IEC purified material showed a major 24 kDa band after polyacrylamide gel electrophoresis (PAGE) with silver staining. Fluorography of purified ATase following incubation with [3H]-methylated substrate DNA and PAGE showed a single band at 24 kDa suggesting that, as with bacterial ATases, the protein itself accepts the alkyl group from O6-alkylguanine in substrate DNA during the repair reaction. Further purification of the protein using reverse phase HPLC resulted in a single peak representing approximately 125,000 fold purification. This was subjected to amino-terminal sequencing and it was found that the protein was blocked at the amino-terminal end: it was cleaved using trypsin or cyanogen bromide and the amino acid sequence of several reverse phase HPLC purified fragments was determined.     

O6-methylguanine mutation and repair is nonuniform. Selection for DNA most interactive with O6-methylguanine

Mutations were induced in the ampicillinase gene of a bacteriophage f1/pBR322 chimera both by incorporation of O6-methyl-dGTP opposite T during DNA replication in vitro and by site-directed mutagenesis using O6-methylguanine-containing oligonucleotides. After passage of the DNA through Escherichia coli, analysis of 151 O6-methyl-dGTP-induced mutations indicated a significantly greater number of unmutated mutation sites than expected, whereas the mutated sites generally fit a Poisson distribution. The unmutated sites are assumed to be caused by the inability of some sequences to tolerate the presence of a tetrahedral methyl group within the confines of a Watson-Crick helix (Toorchen, D., and Topal, M.D. (1983) Carcinogenesis 4, 1591-1597). A consensus of the DNA sequences surrounding unmutated mutation sites was derived. The consensus sequence had significant similarity to the region of the rat Harvey ras oncogene containing the N-methyl-N-nitrosourea activated site for transformation (Zarbl, H., Sukumar, S., Arthur, A. V., Dionisio, M.-Z., and Barbacid, M. (1985) Nature 315, 382-385). We propose that direct alkylation at O6 of a guanine present within the consensus sequence may produce a DNA conformation less subject to repair. Mutation by O6-methylguanine-containing oligonucleotides demonstrated that repair of the O6-methylguanine lesions varied at least 3-4-fold with position of the lesion.     

Purification of homogeneity and amino acid sequence analysis of a receptor protein for interleukin 1

The interleukin 1 (IL-1) receptor from mouse EL-4 thymoma cells was purified to homogeneity by a method which utilized ligand affinity chromatography and classical chromatographic techniques. After solubilization of the receptor from intact cells with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, the IL-1 binding activity was purified greater than 23,000-fold. Analysis of the purified protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot, and ligand blot demonstrated that a single protein of molecular mass of approximately 80 kDa is the IL-1 binding polypeptide. The purified protein bound IL-1 with a dissociation constant of approximately 1.1 X 10(-10) M, which is indistinguishable from the affinity of the cell-bound receptor. The amino acid composition of this protein is strikingly similar to the composition deduced from the sequence of a cDNA coding for an IL-1 receptor from EL-4 cells. Protein sequence analysis of Staphylococcus aureus V-8 protease-derived peptides yields data consistent with the sequence proposed from cloned cDNA. These studies have demonstrated that the high affinity IL-1 receptor on EL-4 cells is the 80-kDa protein.     

Levels of O6-methylguanine acceptor protein in extracts of human breast tumor tissues.

We have measured the abilities of extracts of tissues from human breast tumors to demethylate adducts of O6-meG in exogenous DNA by transfer of the methyl group to an acceptor protein. The results have shown that all 21 specimens examined (including 5 non-neoplastic, 11 malignant tumors and 5 benign growth) contained significant amounts of O6-meG acceptor activity, removing on average 221.1 +/- 2.1 (SEM) fmol O6-meG per mg protein or 10.07 +/- 0.98 (SEM) fmol O6-megG per microgram DNA in the extracts. There were also wide interindividual variations, which were not age-dependent, and there were no significant differences between the non-neoplastic and neoplastic tissues obtained from individuals with benign or with malignant disease. It was estimated that the average number of O6-meG acceptor molecules per cell in normal human breast tissues was calculated as 46,000 +/- 7000 (SEM).     

DNA-mediated transfer and expression of a human DNA repair gene that demethylates O6-methylguanine.

Human liver DNA was transfected into CHO cells (mex-) along with pSV2gpt and colonies were selected first for resistance to mycophenolic acid and then to chloroethylnitrosourea. Transformants were obtained that contained approximately 10,000 molecules of O6-alkylguanine alkyltransferase (mex+) per cell. Their genome contained at least three copies of the human Alu sequence.     

Active site amino acid sequence of the bovine O6-methylguanine-DNA methyltransferase.

An O6-methylguanine-DNA methyltransferase has been partially purified from calf thymus by conventional biochemical techniques. The enzyme was specifically radioactively labelled at the cysteine residue of the active site and further purified by attachment to a solid support. Following digestion with trypsin, a radioactive peptide containing the active site region of the protein was purified by size fractionation, ion exchange chromatography and reverse phase HPLC. The technique yielded an essentially homogeneous oligopeptide which was subjected to amino acid sequencing. The sequence adjacent to the acceptor cysteine residue of the bovine protein exhibits striking homology to the C-terminal methyl acceptor site of the E. coli Ada protein and the proposed acceptor sites of the E. coli Ogt and the B. subtilis Dat1 proteins.     

Novel synthesis of O6-alkylguanine containing oligodeoxyribonucleotides as substrates for the human DNA repair protein, O6-methylguanine DNA methyltransferase (MGMT)

The human DNA repair protein O⁶-methylguanine DNA methyltransferase (MGMT) dealkylates mutagenic O⁶-alkylguanine lesions within DNA in an irreversible reaction which results in inactivation of the protein. MGMT also provides resistance of tumours to alkylating agents used in cancer chemotherapy and its inactivation is therefore of particular clinical importance. We describe a post-DNA synthesis strategy which exploits the novel, modified base 2-amino-6-methylsulfonylpurine and allows access for the first time to a wide variety of oligodeoxyribonucleotides (ODNs) containing O⁶-alkylguanines. One such ODN containing O⁶-(4-bromothenyl)guanine is the most potent inactivator described to date with an IC₅₀ of 0.1 nM.     

Cloning and characterization of human guanine deaminase. Purification and partial amino acid sequence of the mouse protein

Mouse erythrocyte guanine deaminase has been purified to homogeneity. The native enzyme was dimeric, being comprised of two identical subunits of approximately 50,000 Da. The protein sequence was obtained from five cyanogen bromide cleavage products giving sequences ranging from 12 to 25 amino acids in length and corresponding to 99 residues. Basic Local Alignment Search Tool (BLAST) analysis of expressed sequence databases enabled the retrieval of a human expressed sequence tag cDNA clone highly homologous to one of the mouse peptide sequences. The presumed coding region of this clone was used to screen a human kidney cDNA library and secondarily to polymerase chain reaction-amplify the full-length coding sequence of the human brain cDNA corresponding to an open reading frame of 1365 nucleotides and encoding a protein of 51,040 Da. Comparison of the mouse peptide sequences with the inferred human protein sequence revealed 88 of 99 residues to be identical. The human coding sequence of the putative enzyme was subcloned into the bacterial expression vector pMAL-c2, expressed, purified, and characterized as having guanine deaminase activity with a Km for guanine of 9.5 +/- 1.7 microM. The protein shares a 9-residue motif with other aminohydrolases and amidohydrolases (PGX[VI]DXH[TVI]H) that has been shown to be ligated with heavy metal ions, commonly zinc. The purified recombinant guanine deaminase was found to contain approximately 1 atom of zinc per 51-kDa monomer.     

The nitrosated bile acid DNA lesion O6-carboxymethylguanine is a substrate for the human DNA repair protein O6-methylguanine-DNA methyltransferase

The consumption of red meat is a risk factor in human colorectal cancer (CRC). One hypothesis is that red meat facilitates the nitrosation of bile acid conjugates and amino acids, which rapidly convert to DNA-damaging carcinogens. Indeed, the toxic and mutagenic DNA adduct O⁶-carboxymethylguanine (O⁶-CMG) is frequently present in human DNA, increases in abundance in people with high levels of dietary red meat and may therefore be a causative factor in CRC. Previous reports suggested that O⁶-CMG is not a substrate for the human version of the DNA damage reversal protein O⁶-methylguanine-DNA methyltransferase (MGMT), which protects against the genotoxic effects of other O⁶-alkylguanine lesions by removing alkyl groups from the O⁶-position. We now show that synthetic oligodeoxyribonucleotides containing the known MGMT substrate O⁶-methylguanine (O⁶-MeG) or O⁶-CMG effectively inactivate MGMT in vitro (IC₅₀ 0.93 and 1.8 nM, respectively). Inactivation involves the removal of the O⁶-alkyl group and its transfer to the active-site cysteine residue of MGMT. O⁶-CMG is therefore an MGMT substrate, and hence MGMT is likely to be a protective factor in CRC under conditions where O⁶-CMG is a potential causative agent.     

A common mechanism for repair of O6-methylguanine and O6-ethylguanine in DNA

The mutagenic effects of several ethylating and methylating agents were assessed in Encherichia coli strains that are defective in the adaptive response to alkylating agents. These mutants were either deficient in the response or expressed it constitutively. When expressed, the repair pathway removed the major mutagenic lesion produced by either methylating or ethylating agents. This lesion was almost certainly O⁶-alkylguanine produced by alkylation of DNA, and the mechanism for its removal was characterized in vitro. E. coli cells expressing the adaptive response contain relatively large amounts of a protein that transfers the methyl group from O⁶-methylguanine to one of its own cysteine residues (Olsson & Lindahl, 1980). This methyltransferase was shown to act in an analogous fashion on O⁶-ethylguanine. Incubation of ethylated DNA with purified transferase led to disappearance of the O⁶-ethylguanine residues, and S-ethylcysteine was simultaneously generated in the protein. The greater sensitivity of E. coli wild-type to ethylating than methylating agents may be explained by a slower repair of O⁶-ethylguanine than O⁶-methylguanine and also a weaker ability of ethylating agents to induce the adaptive response.     

Purification and partial amino acid sequence of osteogenin, a protein initiating bone differentiation

Osteogenin was purified from bovine bone matrix and its activity monitored by an in vivo bone induction assay. The purification method utilized extraction of the bone-inducing activity with 6 M urea, followed by chromatography on heparin-Sepharose, hydroxyapatite, and Sephacryl S-200. Active fractions were further purified by preparative sodium dodecyl sulfate gel electrophoresis without reduction. Osteogenin activity was localized in a zone between 30 and 40 kDa. The amino acid sequences of a number of tryptic peptides of the gel-eluted material were determined. Reduction and alkylation of purified osteogenin in 7 M guanidine hydrochloride resulted in the total loss of biological activity. Sodium dodecyl sulfate gel electrophoresis under reducing conditions revealed a broad band with an apparent molecular mass of 22 kDa.     

Reaction of O6-alkylguanine-DNA alkyltransferase with O6-methylguanine analogues: evidence that the oxygen of O6-methylguanine is protonated by the protein to effect methyl transfer.

The DNA repair protein O6-alkylguanine-DNA alkyltransferase (AGT) repairs the promutagenic O6-methylguanine lesion by transferring the methyl group to a cysteine residue on the protein. A mechanism in which AGT activates the guanyl moiety as a leaving group by protonation of a heteroatom on guanine was probed by reacting AGT with analogues of O6-methylguanine in which the heteroatoms were changed. The initial rates of reaction were measured at various substrate concentrations in 50 mM Hepes, 1 mM EDTA, 1 mM DTT, and 10% glycerol, pH 7.8 at 37 degrees C. The kinact (h-1) and Kin (mM) were determined for O6-methylguanine (1.66 +/- 0.19, 1.51 +/- 0.32), 6-methoxypurine (1.07 +/- 0.25, 10.6 +/- 4.2), S6-methyl-6-thioguanine (0.63 +/- 0.04, 1.17 +/- 0.18), 6-methylthiopurine (no reaction), Se6-methyl-6-selenoguanine (1.76 +/- 0.28, 10.6 +/- 5.0), 6-methylselenopurine (2.51 +/- 0.62, 15.7 +/- 6.3), O6-methyl-1-deazaguanine (1.71 +/- 0.34, 14.8 +/- 4.4), O6-methyl-3-deazaguanine (1.90 +/- 0.24, 2.54 +/- 0.59), and O6-methyl-7-deazaguanine (1.97 +/- 0.26, 2.56 +/- 0.72). These results indicate that replacement of the nitrogens does not affect the kinact parameter but the Kin is increased upon removal of the exocyclic amino group and the nitrogen at the 1-position. Replacement of the oxygen with sulfur decreases the kinact, and replacement with selenium increases the Kin. The results are consistent with a mechanism in which O6-methylguanine binds to the active site of AGT with hydrogen bonds to the oxygen, the exocyclic amino group, and the nitrogen at the 1-position of the substrate.(ABSTRACT TRUNCATED AT 250 WORDS)     

The partial amino acid sequence of a non-histone chromosomal protein

The amino acid sequence of the first thirty nine residues of the nonhistone chromosomal protein HMG-17 has been determined. Results presented here give a molecular weight of 11,000 for the protein. Some interesting sequence homology with the trout specific histone, histone-T, is noted.     

A system in mouse liver for the repair of O6-methylguanine lesions in methylated DNA.

An activity from mouse liver with catalyzes the disappearance of O6-methylguanine from DNA methylated with methylnitrosourea has been partially purified by ammonium sulfate fractionation and DNA-cellulose chromatography. The activity does not require divalent metal ions and is not affected by EDTA. It is specific for the repair of O6-methylguanine lesions and does not affect the removal of 7-methylguanine, 7-methyladenine or 3-methyladenine. The disappearance of O6-methylguanine is linear with respect to the concentration of protein and is dependent on incubation temperature. The kinetics and substrate dependence experiments suggest that the protein factor is product-inactivated. Amino acid analysis of hydrolysates of protein obtained after incubation of methylated DNA with the protein factor indicates the presence of radiolabeled S-methyl-L-cysteine, suggesting that during the repair of O6-methylguanine from methylated DNA, the methyl group is transferred to a sulfhydryl of a cysteine residue of a protein. This represents the first such demonstration in a mammalian system.     

Purification and partial amino acid sequence of a bovine cartilage-derived collagenase inhibitor

An inhibitor of mammalian collagenase from bovine scapular cartilage has been purified to homogeneity. The inhibitor, extracted from cartilage using 2 M NaCl, was applied to an A-1.5m gel filtration column. Inhibitor eluted at an apparent Mr of 28,000. Further purification was achieved by ion exchange chromatography, gel filtration, and reverse-phase high performance liquid chromatography. A purification of greater than 1,000-fold was achieved. The inhibitor was judged homogeneous by the appearance of a single band on a silver-stained 15% sodium dodecyl sulfate-polyacrylamide gel. Reduced inhibitor had an Mr of 27,400, unreduced inhibitor had an Mr of 23,900. NH2-terminal sequence data were obtained for the first 45 residues. The bovine cartilage-derived inhibitor exhibits greater than 65% homology over the first 23 residues with a collagenase inhibitor purified from human skin fibroblasts maintained in cell culture. This is the first demonstration that collagenase inhibitors extracted directly from tissue may be similar to those obtained from culture medium.     

Purification of the E. coli ogt gene product to homogeneity and its rate of action on O6-methylguanine, O6-ethylguanine and O4-methylthymine in dodecadeoxyribonucleotides.

The E. coli gene ogt encodes the DNA repair protein O6-alkylguanine-DNA-alkyltransferase (O6-AlkG ATase). The protein coding region of the gene was cloned into a multicopy expression vector to obtain high yields of the enzyme (approximately 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 O6-methylguanine (O6-MeG), O6-ethylguanine (O6-EtG) and O4-methylthymine (O4-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 O6-MeG greater than O6-EtG greater than O4-MeT, however, the ogt protein was found to repair O6-MeG, O6-EtG and O4-Met, 1.1, 173 and 84 times, respectively, faster than the ada protein.     

Purification and partial amino acid sequence of proteins from human epidermal keratinocyte conditioned medium

Keratinocytes are the main cell type of the epidermis. They secrete a variety of proteins and peptides that have diverse roles in epidermal physiology. In this report, we present purification and partial amino acid sequence of LEKTI, a serine proteinase inhibitor, and DAN (NO₃) zinc-finger protein, a tumor suppressor protein of neuroblastoma, from human keratinocyte conditioned medium. Epidermal keratinocytes were isolated from human foreskin and serially passaged in a defined medium (MSBM). At confluence of the fourth passage, MSBM medium was replaced with protein-free Dulbecco's modified Eagle medium/F12 (DMEM:F12) 3:1 base medium and collected every 24 h for 4 days. Medium was pooled and concentrated using a stirred cell concentrator. Concentrated medium was diluted 1:1 in 50 mM sodium phosphate, pH 8 buffer, and loaded onto a preparative heparin affinity column. Proteins/peptides were purified from heparin column passthrough by the combination of preparative and analytical FPLC-based gel filtration chromatography and reversed-phase HPLC. Samples electroblotted onto a PVDF support were sequenced by Edman degradation in a gas-phase sequencing system.     

Effect of sequence context on O(6)-methylguanine repair and replication in vivo.

Understanding the origins of mutational hotspots is complicated by the intertwining of several variables. The selective formation, repair, and replication of a DNA lesion, such as O(6)-methylguanine (m(6)G), can, in principle, be influenced by the surrounding nucleotide environment. A nearest-neighbor analysis was used to address the contribution of sequence context on m(6)G repair by the Escherichia coli methyltransferases Ada or Ogt, and on DNA polymerase infidelity in vivo. Sixteen M13 viral genomes with m(6)G flanked by all permutations of G, A, T, and C were constructed and individually transformed into repair-deficient and repair-proficient isogenic cell strains. The 16 genomes were introduced in duplicate into 5 different cellular backgrounds for a total of 160 independent experiments, for which mutations were scored using a recently developed assay. The Ada methyltransferase demonstrated strong 5' and 3' sequence-specific repair of m(6)G in vivo. The Ada 5' preference decreased in the general order: GXN > CXN > TXN > AXN (X = m(6)G, N = any base), while the Ada 3' preference decreased in the order: NX(T/C) > NX(G/A), with mutation frequencies (MFs) ranging from 35% to 90%. The Ogt methyltransferase provided MFs ranging from 10% to 25%. As was demonstrated by Ada, the Ogt methyltransferase repaired m(6)G poorly in an AXN context. When both methyltransferases were removed, the MF was nearly 100% for all sequence contexts, consistent with the view that the replicative DNA polymerase places T opposite m(6)G during replication irrespective of the local sequence environment.     

Expression and cloning of complementary DNA for a human enzyme that repairs O6-methylguanine in DNA

A cell line with an increased resistance to alkylating agents and an extremely high level of O6-methylguanine-DNA methyltransferase activity was isolated after transfection of methyltransferase-deficient Mer- cells with a cDNA library, prepared from methyltransferase-proficient human Mer+ (Raji) cells. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis revealed that a protein, with a molecular weight of approximately 25,000, accepted 3H label from DNA that had been treated with [3H]methylnitrosourea. Since the cDNA for methyltransferase was integrated into the chromosomal DNA, it was recovered by using the polymerase chain reaction. When the cDNA placed in an expression vector p500 was introduced into Mer- cells, the cells acquired an increased resistance to alkylating agents and exhibited a high level of O6-methylguanine-DNA methyltransferase activity. From the transformants the cDNA could be recovered as a part of the autonomously replicating plasmid. The nucleotide sequence of the cDNA was determined, and an open reading frame comprising 207 amino acid residues was found. The molecular weight of methyltransferase, calculated from the predicted amino acid sequence, was 21,700. The predicted amino acid sequence of the human methyltransferase exhibits an intensive homology with those of the bacterial counterparts, Ada and Ogt proteins of Escherichia coli and Dat protein of Bacillus subtilis, especially around possible methyl acceptor sites.