Interactions of human O6-alkylguanine-DNA alkyltransferase (AGT) with short single-stranded DNAs.
Ontology highlight
ABSTRACT:
SUBMITTER: Rasimas JJ
PROVIDER: S-EPMC1941669 | biostudies-literature | 2007 Feb
REPOSITORIES: biostudies-literature
Publications
Interactions of human O6-alkylguanine-DNA alkyltransferase (AGT) with short single-stranded DNAs.
Rasimas Joseph J JJ Kar Sambit R SR Pegg Anthony E AE Fried Michael G MG
The Journal of biological chemistry 20061130 5
The O6-alkylguanine-DNA alkyltransferase (AGT) repairs O6-alkylguanine and O4-alkylthymine adducts in single-stranded and duplex DNAs. Here we characterize the binding of AGT to single-stranded DNAs ranging in length from 5 to 78 nucleotides (nt). Binding is moderately cooperative (37.9 +/- 3.0 <or= omega <or= 89.8 +/- 8.9), resulting in an all-or-nothing association pattern on short templates. This cooperativity contrasts with the isolated binding seen in recent crystal structures of AGT-DNA co ...[more]
Similar Datasets
Project description:O(6)-alkylguanine-DNA alkyltransferase (AGT) is a ubiquitous enzyme with an amino acid sequence that is conserved in Eubacteria, Archaea, and Eukarya. It repairs O(6)-alkylguanine and O(4)-alkylthymine adducts in single-stranded and duplex DNAs. In performing these functions, AGT must partition between adduct-containing sites and the large excess of adduct-free DNA distributed throughout the genome. Here, we characterize the binding of human AGT to linear double-stranded, adduct-free DNAs ranging in length from 11 bp to 2686 bp. Moderately cooperative binding (22.6 +/- 3.7 < or = omega < or = 145.0 +/- 37.0) results in an all-or-nothing association pattern on short templates. The apparent binding site size S(app) (mean = 4.39 +/- 0.02 bp) oscillates with increasing template length. Oscillations in cooperativity factor omega have the same frequency but are of opposite phase to S(app), with the result that the most stable protein-protein and protein-DNA interactions occur at the highest packing densities. The oscillation period (4.05 +/- 0.02 bp/protein) is nearly identical to the occluded binding site size obtained at the highest measured binding density (4 bp/protein) and is significantly smaller than the contour length ( approximately 8 bp) occupied in crystalline complexes. A model in which protein molecules overlap along the DNA contour is proposed to account for these features. High AGT densities resulting from cooperative binding may allow efficient search for lesions in the context of chromatin remodeling and DNA replication.
| S-EPMC2801566 | biostudies-literature
Project description:This article summarizes the current understanding of known variant forms of the MGMT gene that encode an altered protein. Epidemiological studies have been carried out to test whether these alterations are associated with altered cancer risk. Laboratory studies using recombinant proteins and cells expressing the known variants have investigated the possible effects of these sequence alterations on the ability of the encoded O(6)-alkylguanine-DNA alkyltransferase protein to protect cells from alkylation damage and to respond to therapeutic inactivators currently undergoing trials for cancer chemotherapy.
| S-EPMC2030992 | biostudies-literature
Project description:The mutagenic and cytotoxic effects of many alkylating agents are reduced by O(6)-alkylguanine-DNA alkyltransferase (AGT). In humans, this protein not only protects the integrity of the genome, but also contributes to the resistance of tumors to DNA-alkylating chemotherapeutic agents. Here we describe and test models for cooperative multiprotein complexes of AGT with single-stranded and duplex DNAs that are based on in vitro binding data and the crystal structure of a 1:1 AGT-DNA complex. These models predict that cooperative assemblies contain a three-start helical array of proteins with dominant protein-protein interactions between the amino-terminal face of protein n and the carboxy-terminal face of protein n+3, and they predict that binding duplex DNA does not require large changes in B-form DNA geometry. Experimental tests using protein cross-linking analyzed by mass spectrometry, electrophoretic and analytical ultracentrifugation binding assays, and topological analyses with closed circular DNA show that the properties of multiprotein AGT-DNA complexes are consistent with these predictions.
| S-EPMC2736636 | biostudies-literature
Project description:O (6)-Alkylguanine-DNA alkyltransferase (AGT) plays an important role by protecting cells from alkylating agents. This reduces the frequency of carcinogenesis and mutagenesis initiated by such agents, but AGT also provides a major resistance mechanism to some chemotherapeutic drugs. To improve our understanding of the AGT-mediated repair reaction and our understanding of the spectrum of repairable damage, we have studied the ability of AGT to repair interstrand cross-link DNA damage where the two DNA strands are joined via the guanine- O (6) in each strand. An oligodeoxyribonucleotide containing a heptane cross-link was repaired with initial formation of an AGT-oligo complex and further reaction of a second AGT molecule yielding a hAGT dimer and free oligo. However, an oligodeoxyribonucleotide with a butane cross-link was a very poor substrate for AGT-mediated repair, and only the first reaction that forms an AGT-oligo complex could be detected. Models of the reaction of these substrates in the AGT active site show that the DNA duplex is forced apart locally to repair the first guanine. This reaction is greatly hindered with the butane cross-link, which is mostly buried in the active site pocket and limited in conformational flexibility. This limitation also prevents the adoption of a conformation for the second reaction to repair the AGT-oligo complex. These results are consistent with the postulated mechanism of AGT repair that involves DNA binding and flipping of the substrate nucleotide and indicate that hAGT can repair some types of interstrand cross-link damage.
| S-EPMC2632579 | biostudies-literature
Project description:Isoelectric focusing, CD, steady-state and time-resolved fluorescence spectroscopy were used to compare the native recombinant human DNA-repair protein O6-alkylguanine-DNA alkyltransferase (AGT) with AGT derivatives methylated or benzylated on Cys145 or modified by site-directed mutagenesis at the active centre (Met145 mutant). The AGT protein is approximately spherical with highly constrained Trp residues, but is not stabilized by disulphide bridges. In contrast with native AGT, alkylated AGT precipitated at 25 degrees C but remained monomeric at 4 degrees C. As revealed by isoelectric focusing, pI changed from 8.2 (AGT) to 8. 4 (Cys145-methylated AGT) and 8.6 (Cys145-benzylated AGT). The alpha-helical content of the Met145 mutant was decreased by approx. 5% and Trp residues were partially liberated. Although non-covalent binding of O6-benzylguanine did not alter the secondary structure of AGT, its alpha-helical content was increased by approx. 2% on methylation and by approx. 4% on benzylation, altogether indicating a small conformational change in AGT on undergoing alkylation. No signal sequences have been found in AGT that mark it for polyubiquitination. Therefore the signal for AGT degradation remains to be discovered.
| S-EPMC1218433 | biostudies-other
Project description:O(6)-Benzylguanine is an irreversible inactivator of O(6)-alkylguanine-DNA alkyltransferase currently in clinical trials to overcome alkyltransferase-mediated resistance to certain cancer chemotherapeutic alkylating agents. In order to produce more soluble alkyltransferase inhibitors, we have synthesized three aminomethyl-substituted O(6)-benzylguanines and the three methyl analogs and found that the substitution of aminomethyl at the meta-position greatly enhances inactivation of alkyltransferase, whereas para-substitution has little effect and ortho-substitution virtually eliminates activity. Molecular modeling of their interactions with alkyltransferase provided a molecular explanation for these results. The square of the correlation coefficient (R(2)) obtained between E-model scores (obtained from GLIDE XP/QPLD docking calculations) vs log(ED(50)) values via a linear regression analysis was 0.96. The models indicate that the ortho-substitution causes a steric clash interfering with binding, whereas the meta-aminomethyl substitution allows an interaction of the amino group to generate an additional hydrogen bond with the protein.
| S-EPMC2645950 | biostudies-literature
Project description:Alkyltransferase-like proteins (ATLs) are a novel class of DNA repair proteins related to O(6)-alkylguanine-DNA alkyltransferases (AGTs) that tightly bind alkylated DNA and shunt the damaged DNA into the nucleotide excision repair pathway. Here, we present the first structure of a bacterial ATL, from Vibrio parahaemolyticus (vpAtl). We demonstrate that vpAtl adopts an AGT-like fold and that the protein is capable of tightly binding to O(6)-methylguanine-containing DNA and disrupting its repair by human AGT, a hallmark of ATLs. Mutation of highly conserved residues Tyr(23) and Arg(37) demonstrate their critical roles in a conserved mechanism of ATL binding to alkylated DNA. NMR relaxation data reveal a role for conformational plasticity in the guanine-lesion recognition cavity. Our results provide further evidence for the conserved role of ATLs in this primordial mechanism of DNA repair.
| S-EPMC2859536 | biostudies-literature
Project description:A cDNA encoding the human O6-alkylguanine-DNA alkyltransferase (ATase; EC 2.1.1.63; methylated-DNA: protein-cysteine methyltransferase) has been manipulated to generate a C-terminally deleted protein which retains full methyl-transfer activity. The elimination of 22 amino-acid residues from the C-terminus was achieved by endonuclease-SacI digestion of the 623 bp cDNA coding sequence and ligation of a SacI/HindIII linker containing an in-frame stop codon. The truncated protein was characterized by its reduced molecular mass in immunoblots probed with an antiserum against the full-length protein and by fluorography after incubation with [3H]methylated calf thymus DNA. The rate of methyl transfer was virtually identical for the full-length and truncated ATases. The construction of such a truncated, yet still functional, ATase, with a molecular mass of 19.7 kDa should facilitate a detailed n.m.r. structural study and help to determine the functional significance of the C-terminal domain of mammalian ATases.
| S-EPMC1132851 | biostudies-other
Project description:O(6)-POB-dG (O(6)-[4-oxo-4-(3-pyridyl)but-1-yl]deoxyguanosine) are promutagenic nucleobase adducts that arise from DNA alkylation by metabolically activated tobacco-specific nitrosamines such as 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosonicotine (NNN). If not repaired, O(6)-POB-dG adducts cause mispairing during DNA replication, leading to G → A and G → T mutations. A specialized DNA repair protein, O(6)-alkylguanine-DNA-alkyltransferase (AGT), transfers the POB group from O(6)-POB-dG in DNA to a cysteine residue within the protein (Cys145), thus restoring normal guanine and preventing mutagenesis. The rates of AGT-mediated repair of O(6)-POB-dG may be affected by local DNA sequence context, potentially leading to adduct accumulation and increased mutagenesis at specific sites within the genome. In the present work, isotope dilution high performance liquid chromatography-electrospray ionization-tandem mass spectrometry (HPLC-ESI(+)-MS/MS)-based methodology was developed to investigate the influence of DNA sequence on the kinetics of AGT-mediated repair of O(6)-POB-dG adducts. In our approach, synthetic DNA duplexes containing O(6)-POB-dG at a specified site are incubated with recombinant human AGT protein for defined periods of time. Following spiking with D(4)-O(6)-POB-dG internal standard and mild acid hydrolysis to release O(6)-POB-guanine (O(6)-POB-G) and D(4)-O(6)-POB-guanine (D(4)-O(6)-POB-G), samples are purified by solid phase extraction (SPE), and O(6)-POB-G adducts remaining in DNA are quantified by capillary HPLC-ESI(+)-MS/MS. The new method was validated by analyzing mixtures containing known amounts of O(6)-POB-G-containig DNA and the corresponding unmodified DNA duplexes and by examining the kinetics of alkyl transfer in the presence of increasing amounts of AGT protein. The disappearance of O(6)-POB-dG from DNA was accompanied by pyridyloxobutylation of AGT Cys-145 as determined by HPLC-ESI(+)-MS/MS of tryptic peptides. The applicability of the new approach was shown by determining the second order kinetics of AGT-mediated repair of O(6)-POB-dG adducts placed within a DNA duplex representing modified rat H-ras sequence (5'-AATAGTATCT[O(6)-POB-G]GAGCC-3') opposite either C or T. Faster rates of alkyl transfer were observed when O(6)-POB-dG was paired with T rather than with C (k = 1.74 × 10(6) M(-1) s(-1) vs 1.17 × 10(6) M(-1) s(-1)).
| S-EPMC3221886 | biostudies-literature
Project description:1,2,3,4-Diepoxybutane (DEB) is a key carcinogenic metabolite of the important industrial chemical 1,3-butadiene. DEB is a bifunctional alkylating agent capable of reacting with DNA and proteins. Initial DNA alkylation by DEB produces N7-(2'-hydroxy-3',4'-epoxybut-1'-yl)-guanine monoadducts, which can react with another nucleophilic site to form cross-linked adducts. A recent report revealed a strong correlation between cellular expression of the DNA repair protein O6-alkylguanine DNA alkyltransferase (AGT) and the cytotoxic and mutagenic activity of DEB, suggesting that DEB induces AGT-DNA cross-links (Valadez, J. G., et al. (2004) Activation of bis-electrophiles to mutagenic conjugates by human O6-alkylguanine-DNA alkyltransferase. Chem. Res. Toxicol. 17, 972-982). The purpose of our study was to analyze the formation and structures of DEB-induced AGT-DNA conjugates and to identify specific amino acid residues within the protein involved in cross-linking. DNA-protein cross-link formation was detected by SDS-PAGE when 32P-labeled double-stranded oligodeoxynucleotides were exposed to DEB in the presence of either wild-type hAGT or a C145A hAGT mutant. Capillary HPLC-electrospray ionization mass spectrometry (ESI-MS) analysis of hAGT that had been treated with N7-(2'-hydroxy-3',4'-epoxybut-1'-yl)-deoxyguanosine (dG monoepoxide) revealed the ability of the protein to form either one or two butanediol-dG cross-links, corresponding to mass shifts of +353 and +706 Da, respectively. HPLC-ESI+ -MS/MS sequencing of the tryptic peptides obtained from dG monoepoxide-treated protein indicated that the two cross-linking sites were the alkyl acceptor site, Cys145, and a neighboring active site residue, Cys150. The same two amino acid residues of hAGT became covalently cross-linked to DNA following DEB treatment. Modification of Cys145 was further confirmed by HPLC-ESI+ -MS/MS analysis of dG monoepoxide-treated synthetic peptide GNPVPILIPCHR which represents the active site tryptic fragment of hAGT (C = Cys145). The replacement of the catalytic cysteine residue with alanine in the C145A hAGT mutant abolished DEB-induced cross-linking at this site, while the formation of conjugates via neighboring Cys150 was retained. The exact chemical structure of the cross-linked lesion was established as 1-(S-cysteinyl)-4-(guan-7-yl)-2,3-butanediol by HPLC-ESI+ -MS/MS analysis of the amino acids resulting from the total digestion of modified proteins analyzed in parallel with an authentic standard. AGT-DNA cross-linking is a likely mechanism of DEB-mediated cytotoxicity in cells expressing this important repair protein.
| S-EPMC3213031 | biostudies-literature