Human AGT Research Articles

Phosphorylation of Thr9 Affects the Folding Landscape of the N-Terminal Segment of Human AGT Enhancing Protein Aggregation of Disease-Causing Mutants.

The mutations G170R and I244T are the most common disease cause in primary hyperoxaluria type I (PH1). These mutations cause the misfolding of the AGT protein in the minor allele AGT-LM that contains the P11L polymorphism, which may affect the folding of the N-terminal segment (NTT-AGT). The NTT-AGT is phosphorylated at T9, although the role of this event in PH1 is unknown. In this work, phosphorylation of T9 was mimicked by introducing the T9E mutation in the NTT-AGT peptide and the full-length protein. The NTT-AGT conformational landscape was studied by circular dichroism, NMR, and statistical mechanical methods. Functional and stability effects on the full-length AGT protein were characterized by spectroscopic methods. The T9E and P11L mutations together reshaped the conformational landscape of the isolated NTT-AGT peptide by stabilizing ordered conformations. In the context of the full-length AGT protein, the T9E mutation had no effect on the overall AGT function or conformation, but enhanced aggregation of the minor allele (LM) protein and synergized with the mutations G170R and I244T. Our findings indicate that phosphorylation of T9 may affect the conformation of the NTT-AGT and synergize with PH1-causing mutations to promote aggregation in a genotype-specific manner. Phosphorylation should be considered a novel regulatory mechanism in PH1 pathogenesis.

Influence of C5‐Substituents on Repair of O4‐Methyl Adducts of Pyrimidines by O6‐Alkylguanine DNA Alkyltransferases

AbstractThe DNA repair protein O6‐alkylguanine‐DNA alkyltransferase (AGT), found in numerous organisms, can remove methyl groups from the O6‐ and O4‐atoms of 2′‐deoxyguanosine and thymidine in DNA. AGT variants demonstrate different repair efficiencies towards these lesions. To understand the influence of C5 nucleobase substituents on O4‐methyl removal by AGTs, DNA duplexes containing 5‐chloro‐, 5‐bromo‐ 5‐iodo‐ and 5‐trifluoromethyl‐O4‐methyl‐2′‐deoxyuridine were studied. UV thermal denaturation revealed a stability reduction of 11 °C for the O4‐methyl halogen series and 5‐trifluoromethyl analog relative to their controls. For the 5‐chloro analog efficient repair was observed by human and E.coli AGTs. For the larger halogens (5‐bromo and 5‐iodo) and 5‐trifluoromethyl analog, human AGT showed moderate repair of the O4‐methyl adduct. E.coli OGT and Ada‐C readily repaired most adducts with reduced efficiency for the larger groups, except C5‐iodo. These results suggest electronic contributions and favourable interactions of the C5‐halogens within the AGT active site contribute to efficient dealkylation.

Abstract 13451: Upregulation of CaMKII and Contrast Changes of Cardiac β 1 - and β 3 -Adrenergic Signaling Pathways in a Humanized Angiotensinogen Model of Hypertension

Background: The transgenic rats expressing the human AGT gene [TGR (hAGT) L1623] (hAogen) is an established humanized model of hypertension. Recently we further found that this model is associated with declines in cardiac contractile function and β-adrenergic receptor (AR) reserve. However, the molecular basis is unclear. It has been suggested that pivotal restructuring of β-AR system is a critical determinant of the dysfunctional β-AR regulation in several cardiovascular diseases, including hypertension. But the alterations of subtypes of β-ARs have not been systemically evaluated in this model. Furthermore, there is significant crosstalk between β-AR signaling and CaMKII activation presenting CaMKII as a possible downstream mediator of detrimental β-AR signaling in the abnormal contractile phenotype of cardiac hypertrophy. However, the changes in cardiac CaMKII in this model are unknown. We tested the hypothesis that in hAogen, CaMKII and β 3 -AR are upregulated in the myocardium, but β 1 -AR is down-regulated, which are important causes of cardiac dysfunction and β-AR desensitization. Methods: We compared LV myocyte CaMKIIδ expression and activity and β 1 - and β 3 -AR expression and determined myocyte functional performance and [Ca 2+ ] i transient ([Ca 2+ ] iT) response to β -AR stimulation in myocytes obtained from 6 Sprague Dawley (SD) control and 6 haogen male adult rats. Results: Compared with SD, in hAogen LV myocytes, β 3 -AR protein level significantly increased (26%, 0.29 vs 0.23), but β 1 -AR protein level (26%, 0.4 vs 0.54) significantly decreased. The CaMKIIδ protein levels (16%, 0.50 vs 0.43) and CaMKIIδ phosphorylation (at Thr287) (35%, 0.27 vs 0.20) were significantly increased. These changes were followed by significantly reduced cell contraction (dL/dt max , 102.1vs 138.8μm/s), relaxation (dR/dt max , 94.0 vs 104.7 μm/s) and [Ca 2+ ] iT (0.15 vs 0.21). ISO-stimulated increases in dL/dt max (42% vs 60%), dR/dt max (36% vs 51%) and [Ca 2+ ] iT (20% vs 31%) were also significantly reduced. Conclusions: The upregulation of LV myocyte CaMKIIδ and contrast changes in β 3 -AR and β 1 -AR may be the important cause of the abnormal contractile phenotype and β-AR desensitization in this humanized angiotensinogen model of hypertension of TGR (hAGT) L1623.

RNA interference therapeutics targeting angiotensinogen ameliorate preeclamptic phenotype in rodent models.

Preeclampsia, with the hallmark features of new-onset hypertension and proteinuria after 20 weeks of gestation, is a major cause of fetal and maternal morbidity and mortality. Studies have demonstrated a role for the renin-angiotensin system (RAS) in its pathogenesis; however, small-molecule RAS blockers are contraindicated because of fetal toxicity. We evaluated whether siRNA targeting maternal hepatic angiotensinogen (Agt) could ameliorate symptoms of preeclampsia without adverse placental or fetal effects in 2 rodent models. The first model used a cross of females expressing human Agt with males expressing human renin, resulting in upregulation of the circulating and uteroplacental RAS. The second model induced ischemia/reperfusion injury and subsequent local and systemic inflammation by surgically reducing placental blood flow midgestation (reduced uterine perfusion pressure [RUPP]). These models featured hypertension, proteinuria, and fetal growth restriction, with altered biomarkers. siRNA treatment ameliorated the preeclamptic phenotype in both models, reduced blood pressure, and improved intrauterine growth restriction, with no observed deleterious effects on the fetus. Treatment also improved the angiogenic balance and proteinuria in the transgenic model, and it reduced angiotensin receptor activating antibodies in both. Thus, an RNAi therapeutic targeting Agt ameliorated the clinical sequelae and improved fetal outcomes in 2 rodent models of preeclampsia.

Human AGT -p.Met268Thr and coronary heart disease risk: a case-control study and meta-analysis

Summary Background. Polymorphisms in genes, which is involved in the renin–angiotensin system, play an important role in the pathogenesis of coronary heart disease (CHD). Polymorphism of c.803T>C in the human angiotensinogen gene results in methionine (M) to threonine (T) substitution at codon 268 (p.Met268Thr), which traditionally has been known as M235T. This polymorphism may contribute to cardiovascular diseases. Objectives. The aim of this study was to investigate the association between p.Met268Thr polymorphism in the angiotensinogen gene and coronary heart disease (CHD) through a case-control study, which is followed by a meta-analysis. Material and methods. In the case-control study, c.803T>C genotyping of 217 subjects (102 CHD cases vs 115 controls) was investigated by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. In the meta-analysis, 31 studies were included, reflecting 12,028 people with CHD and 16,362 healthy controls. Results. The data from the case-control study revealed that MT (OR, 1.875; 95%CI, 1.060–3.316; p = 0.031) and TT (OR, 3.389; 95%CI, 1.251–9.179; p = 0.016) genotypes are significantly associated with CHD. The meta-analysis revealed a significant association in the recessive model (OR, 1.156; 95%CI, 1.011–1.321; p = 0.034). Conclusions. Although the pooled OR of the meta-analysis showed that there is an increased risk of CHD conferred by p.Met268Thr of the AGT gene, this association was weak, which could be attributed to a bias in publications. © Copyright by Wydawnictwo Continuo.

Altering Residue 134 Confers an Increased Substrate Range of Alkylated Nucleosides to the E. coli OGT Protein.

O6-Alkylguanine-DNA alkyltransferases (AGTs) are proteins responsible for the removal of mutagenic alkyl adducts at the O6-atom of guanine and O4-atom of thymine. In the current study we set out to understand the role of the Ser134 residue in the Escherichia coli AGT variant OGT on substrate discrimination. The S134P mutation in OGT increased the ability of the protein to repair both O6-adducts of guanine and O4-adducts of thymine. However, the S134P variant was unable, like wild-type OGT, to repair an interstrand cross-link (ICL) bridging two O6-atoms of guanine in a DNA duplex. When compared to the human AGT protein (hAGT), the S134P OGT variant displayed reduced activity towards O6-alkylation but a much broader substrate range for O4-alkylation damage reversal. The role of residue 134 in OGT is similar to its function in the human homolog, where Pro140 is crucial in conferring on hAGT the capability to repair large adducts at the O6-position of guanine. Finally, a method to generate a covalent conjugate between hAGT and a model nucleoside using a single-stranded oligonucleotide substrate is demonstrated.

Fluorine at the C5 Position of 2′‐Deoxyuridine Enhances Repair of a O4‐Methyl Adduct by O6‐Alkylguanine DNA Alkyltransferases

Alkylation damage at the O6‐ and O4‐atoms of 2′‐deoxyguanosine (dG) and thymidine (T), respectively, can be removed by O6‐alkylguanine‐DNA alkyltransferases (AGTs). Previous studies have shown that human AGT (hAGT) repairs small adducts poorly at the O4‐atom of T, in comparison to the E. coli variants (OGT and Ada‐C). The C5 methyl group of the thymine nucleobase is suspected to contribute to hAGT repair proficiency possibly due to steric effects in the protein active site. In the present study, repair of oligonucleotides containing a 5‐fluoro‐O4‐methyl‐2′‐deoxyuridine (dFU‐Me) insert by hAGT, E. coli AGT variants (OGT and Ada‐C) and a chimeric hAGT/OGT protein was evaluated. All AGT variants, particularly hAGT and the hAGT/OGT chimera, demonstrated improved proficiency at removing the O4‐methyl group from substrates containing dFU‐Me, relative to the thymidine and 2′‐deoxyuridine counterparts.

Site-specific covalent capture of human O6-alkylguanine-DNA-alkyltransferase using single-stranded intrastrand cross-linked DNA.

A methodology is reported to conjugate human O6-alkylguanine-DNA-alkyltransferase (hAGT) to the 3'-end of DNA in excellent yields with short reaction times by using intrastrand cross-linked (IaCL) DNA probes. This strategy exploited the substrate specificity of hAGT to generate the desired DNA-protein covalent complex. IaCL DNA linking two thymidine residues, or linking a thymidine residue to a 2'-deoxyguanosine residue (either in a 5'→3' or 3'→5' fashion), lacking a phosphodiester linkage at the cross-linked site, were prepared using a phosphoramidite strategy followed by solid-phase synthesis. All duplexes containing the model IaCL displayed a reduction in thermal stability relative to unmodified control duplexes. The O4-thymidine-alkylene-O4-thymidine and the (5'→3') O6-2'-deoxyguanosine-alkylene-O4-thymidine IaCL DNA adducts were not repaired by any of the AGTs evaluated (human AGT and Escherichia coli homologues, OGT and Ada-C). The (5'→3') O4-thymidine-alkylene-O6-2'-deoxyguanosine IaCL DNA containing a butylene or heptylene tethers were efficiently repaired by the human variant, whereas Ada-C was capable of modestly repairing the heptylene IaCL adduct. The IaCL strategy has expanded the toolbox for hAGT conjugation to DNA strands, without requiring the presence of a complementary DNA sequence. Finally, hAGT was functionalized with a fluorescently-labelled DNA sequence to demonstrate the applicability of this conjugation method.

Production of angiotensin within the SFO is sufficient to increase ERK1/2 and CREB activity in the SFO and PVN

Release of angiotensin II (ANG) within the brain increases fluid intake and sodium appetite. We sought to determine the sufficiency of ANG production in the subfornical organ (SFO) to increase fluid intake and sodium appetite by breeding mice expressing human renin under the neuron specific promoter, synapsin (sR), with mice expressing a conditionally inducible human AGT (hAGT) construct (ARed; double transgenic sRARed). Injection of adenovirus encoding Cre‐recombinase (AdCRE) into the lateral ventricle (ICV) of sRARed mice significantly increases hAGT transcript production within the SFO compared to other cardiovascular brain regions (p<0.05, n=12). One month after ICV AdCRE in sRARed mice total fluid intake and sodium appetite (0.436±0.04 mL/g/day; 1.721±0.087 mEq/day) increased compared to littermate controls (0.176±0.04 mL/g/day, P<0.001; 1.257 mEq/day, p<0.05; n=6) in a two‐bottle choice paradigm (water vs. 0.3M NaCl). After ICV AdCRE we observed a relative increase in immunostaining for ERK1/2 and CREB, but not p‐38 MAPK activity in the SFO and paraventricular nucleus (PVN) of sRARedcompared to littermate controls. In conclusion, we demonstrate that activation of ANG production specifically in the SFO is sufficient to increase ERK1/2 and CREB activity in the SFO and PVN, and to increase total fluid intake and sodium appetite.

DNA–protein crosslinks processed by nucleotide excision repair and homologous recombination with base and strand preference in E. coli model system

Bis-electrophiles including dibromoethane and epibromohydrin can react with O6-alkylguanine-DNA alkyltransferase (AGT) and form AGT-DNA crosslinks in vitro and in vivo. The presence of human AGT (hAGT) paradoxically increases the mutagenicity and cytotoxicity of bis-electrophiles in cells. Here we establish a bacterial system to study the repair mechanism and cellular responses to DNA–protein crosslinks (DPCs) in vivo. Results show that both nucleotide excision repair (NER) and homologous recombination (HR) pathways can process hAGT-DNA crosslinks with HR playing a dominant role. Mutation spectra show that HR has no strand preference but NER favors processing of the DPCs in the transcribed strand; UvrA, UvrB and Mfd can interfere with small size DPCs but only UvrA can interfere with large size DPCs in the transcribed strand processed by HR. Further, we found that DPCs at TA deoxynucleotide sites are very inefficiently processed by NER and the presence of NER can interfere with these DNA lesions processed by HR. These data indicate that NER and HR can process DPCs cooperatively and competitively and NER processes DPCs with base and strand preference. Therefore, the formation of hAGT-DNA crosslinks can be a plausible and specific system to study the repair mechanism and effects of DPCs precisely in vivo.

Preclinical evaluation of Laromustine for use in combination with radiation therapy in the treatment of solid tumors

Purpose: These studies explored questions related to the potential use of Laromustine in the treatment of solid tumors and in combination with radiotherapy.Materials and methods: The studies used mouse EMT6 cells (both parental and transfected with genes for O6-alkylguanine-DNA transferase [AGT]), repair-deficient human Fanconi Anemia C and Chinese hamster VC8 (BRCA2-/-) cells and corresponding control cells, and EMT6 tumors in mice assayed using cell survival and tumor growth assays.Results: Hypoxia during Laromustine treatment did not protect EMT6 cells or human fibroblasts from this agent. Rapidly proliferating EMT6 cells were more sensitive than quiescent cultures. EMT6 cells expressing mouse or human AGT, which removes O6-alkyl groups from DNA guanine, thereby protecting against G-C crosslink formation, increased resistance to Laromustine. Crosslink-repair-deficient Fanconi Anemia C and VC8 cells were hypersensitive to Laromustine, confirming the importance of crosslinks as lethal lesions. In vitro, Laromustine and radiation produced additive toxicities to EMT6 cells. Studies using tumor cell survival and tumor growth assays showed effects of regimens combining Laromustine and radiation that were compatible with additive or subadditive interactions. Conclusions: The effects of Laromustine on solid tumors and with radiation are complex and are influenced by microenvironmental and proliferative heterogeneity within these malignancies.

The study of angiotensinogen gene polymorphism by microchip electrophoresis

ObjectiveTo investigate the AGT gene polymorphism distribution and the relationship between the AGT gene polymorphism and essential hypertension (EH) in Shenyang Hans population using the new microchip-based method, and to...

Three new polymorphisms in human angiotensinogen gene promoter and their role in blood pressure regulation

Human AGT gene has an A/G polymorphism at -6 and -6A allele is associated with increased blood pressure. However, transgenic mice containing 1.2 Kb of the promoter with -6A of the hAGT gene show neither increased plasma AGT level nor increased blood pressure compared to -6G. We have found that hAGT gene has three additional SNPs (A/G at –1670, C/G at -1562 and T/G at -1561). Variants -1670A, -1562C, and -1561T almost always occur with -6A (-6A haplotype); and variants -1670G, -1562G, and -1561G almost always occur with -6G (-6G haplotype). We show that these polymorphisms affect the binding of HNF-1á and GR to the promoter and reporter construct containing 1.8 Kb hAGT gene promoter with -6A haplotype has four fold increased glucocorticoid induced promoter activity as compared to -6G haplotype. In order to understand the physiological significance of these haplotypes in an in vivo situation, we have generated double transgenic mice containing either -6A or -6G haplotype of the hAGT gene and human renin gene. Our ChIP assay shows that HNF1á and GR have stronger affinity to the chromatin obtained from the liver of transgenic mice containing -6A haplotype. Our studies also show that transgenic mice containing -6A haplotype have increased plasma AGT level and increased blood pressure as compared to -6G haplotype. Our studies explain the molecular mechanism involved in association of -6A allele of the hAGT gene with hypertension.

Quantitative relationship between guanine O6-alkyl lesions produced by Onrigin™ and tumor resistance by O6-alkylguanine-DNA alkyltransferase

O 6-Alkylguanine-DNA alkyltransferase (AGT) mediates tumor resistance to alkylating agents that generate guanine O 6-chloroethyl (Onrigin™ and carmustine) and O 6-methyl (temozolomide) lesions; however, the relative efficiency of AGT protection against these lesions and the degree of resistance to these agents that a given number of AGT molecules produces are unclear. Measured from differential cytotoxicity in AGT-ablated and AGT-intact HL-60 cells containing 17,000 AGT molecules/cell, AGT produced 12- and 24-fold resistance to chloroethylating (90CE) and methylating (KS90) analogs of Onrigin™, respectively. For 50% growth inhibition, KS90 and 90CE generated 5,600 O 6-methylguanines/cell and ∼300 O 6-chloroethylguanines/cell, respectively. AGT repaired O 6-methylguanines until the AGT pool was exhausted, while its repair of O 6-chloroethylguanines was incomplete due to progression of the lesions to AGT-irreparable interstrand DNA cross-links. Thus, the smaller number of O 6-chloroethylguanine lesions needed for cytotoxicity accounted for the marked degree of resistance (12-fold) to 90CE produced by AGT. Transfection of human or murine AGT into AGT deficient transplantable tumor cells (i.e., EMT6, M109 and U251) generated transfectants expressing AGT ranging from 4,000 to 700,000 molecules/cell. In vitro growth inhibition assays using these transfectants treated with 90CE revealed that AGT caused a concentration dependent resistance up to a level of ∼10,000 AGT molecules/cell. This finding was corroborated by in vivo studies where expression of 4,000 and 10,000 murine AGT molecules/cell rendered EMT6 tumors partially and completely resistant to Onrigin™, respectively. These studies imply that the antitumor activity of Onrigin™ stems from guanine O 6-chloroethylation and define the threshold concentration of AGT that negates its antineoplastic activity.

Alkyltransferase-mediated toxicity of bis-electrophiles in mammalian cells

The primary function of O 6-alkylguanine-DNA alkyltransferase (AGT) is to maintain genomic integrity in the face of damage by both endogenous and exogenous alkylating agents. However, paradoxically, bacterial and mammalian AGTs have been shown to increase cytotoxicity and mutagenicity of dihaloalkanes and other bis-electrophiles when expressed in bacterial cells. We have extended these studies to mammalian cells using CHO cells that lack AGT expression and CHO cells stably transfected with a plasmid that expresses human AGT. The cytotoxicity of 1,2-dibromoethane, dibromomethane and epibromohydrin was significantly increased by the presence of AGT but cytotoxicity of butadiene diepoxide was not affected. Mutations caused by these agents were assessed using hypoxanthine-guanine phosphoribosyltransferase (HPRT) as a reporter gene. There was a small (c. 2–3-fold) but statistically significant AGT-mediated increase in mutations caused by 1,2-dibromoethane, dibromomethane and epibromohydrin. Analysis of the mutation spectrum induced by 1,2-dibromoethane showed that the presence of AGT also altered the types of mutations with an increase in total base substitution mutants due to a rise in transversions at both G:C and A:T sites. AGT expression also led to mutations arising from the transcribed strand, which were not seen in cells lacking AGT. Although the frequency of deletion mutations was decreased by AGT expression, the formation of large deletions (≥3 exons) was increased. This work demonstrates that interaction of AGT with some bis-electrophiles can cause mutagenicity and diminished cell survival in mammalian cells. It is consistent with the hypothesis that DNA–AGT cross-links, which have been characterized in experiments with purified AGT protein and such bis-electrophiles, can be formed in mammalian cells.

Development of an O6-alkylguanine–DNA alkyltransferase assay based on covalent transfer of the benzyl moiety from [benzene- 3H] O6-benzylguanine to the protein

Although it is known that (i) O 6-alkylguanine–DNA alkyltransferase (AGT) confers tumor cell resistance to guanine O 6-targeting drugs such as cloretazine, carmustine, and temozolomide and that (ii) AGT levels in tumors are highly variable, measurement of AGT activity in tumors before treatment is not a routine clinical practice. This derives in part from the lack of a reliable clinical AGT assay; therefore, a simple AGT assay was devised based on transfer of radioactive benzyl residues from [benzene- 3H] O 6-benzylguanine ([ 3H]BG) to AGT. The assay involves incubation of intact cells or cell homogenates with [ 3H]BG and measurement of radioactivity in a 70% methanol precipitable fraction. Approximately 85% of AGT in intact cells was recovered in cell homogenates. Accuracy of the AGT assay was confirmed by examination of AGT levels by Western blot analysis with the exception of false-positive results in melanin-containing cells due to [ 3H]BG binding to melanin. Second-order kinetic constants for human and murine AGT were 1100 and 380 M −1 s −1, respectively. AGT levels in various human cell lines ranged from less than 500 molecules/cell (detection limit) to 45,000 molecules/cell. Rodent cell lines frequently lacked AGT expression, and AGT levels in rodent cells were much lower than in human cells.

DNA binding, nucleotide flipping, and the helix-turn-helix motif in base repair by O6-alkylguanine-DNA alkyltransferase and its implications for cancer chemotherapy

O 6-Alkylguanine-DNA alkyltransferase (AGT) is a crucial target both for the prevention of cancer and for chemotherapy, since it repairs mutagenic lesions in DNA, and it limits the effectiveness of alkylating chemotherapies. AGT catalyzes the unique, single-step, direct damage reversal repair of O 6-alkylguanines by selectively transferring the O 6-alkyl adduct to an internal cysteine residue. Recent crystal structures of human AGT alone and in complex with substrate DNA reveal a two-domain α/β fold and a bound zinc ion. AGT uses its helix-turn-helix motif to bind substrate DNA via the minor groove. The alkylated guanine is then flipped out from the base stack into the AGT active site for repair by covalent transfer of the alkyl adduct to Cys145. An asparagine hinge (Asn137) couples the helix-turn-helix DNA binding and active site motifs. An arginine finger (Arg128) stabilizes the extrahelical DNA conformation. With this newly improved structural understanding of AGT and its interactions with biologically relevant substrates, we can now begin to unravel the role it plays in preserving genetic integrity and discover how it promotes resistance to anticancer therapies.

Interactions of Human O6-Alkylguanine-DNA Alkyltransferase (AGT) with Short Single-stranded DNAs

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 complexes. The statistical binding site size S (mean = 5.2 +/- 0.1) oscillates with increasing template length. The oscillation period (4.10 +/- 0.02 nt/protein) is nearly identical to the binding site size obtained at the highest known binding density (S = 4 nt/protein) and is significantly smaller than the contour length (approximately 8 bp) occupied in crystalline complexes. A model in which AGT proteins overlap along the DNA contour is proposed to account for these features. Oscillations in intrinsic binding constant Ki and cooperativity factor omega have the same frequency but are of opposite phase to S with the result that the most stable protein-protein and protein-DNA interactions occur at the highest packing densities. We hypothesize that modest binding cooperativity and high binding densities are adaptations that allow AGT to efficiently search for lesions in the context of chromatin remodeling and DNA replication.

Kinetics of O6-Methyl-2‘-deoxyguanosine Repair by O6-Alkylguanine DNA Alkyltransferase within K-ras Gene-Derived DNA Sequences

O6-Methyl-2‘-deoxyguanosine (O6-Me-dG) is a potent mutagenic DNA adduct that can be induced by a variety of methylating agents, including tobacco-specific nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). O6-Me-dG is directly repaired by the specialized DNA repair protein, O6-alkylguanine DNA alkyltransferase (AGT), which transfers the O6-alkyl group from the modified guanine to a cysteine thiol within the active site of the protein. Previous investigations suggested that AGT repair of O6-alkylguanines may be sequence-dependent as a result of flanking nucleobase effects on DNA conformation and energetics. In the present work, a novel high-performance/pressure liquid chromatography−electrospray ionization tandem mass spectrometry (HPLC−ESI+−MS/MS)-based approach was developed to analyze the kinetics of AGT-mediated repair of O6-Me-dG adducts placed at different sites within the double-stranded DNA sequence representing codons 8−17 of the K-ras protooncogene, 5‘-G1TA G2TT G3G4A G5CT G6G7T G8G9C G10TA G11G12C AAG13 AG14T-3‘, where G5, G6, G7, G8, G9, G10, or G11 was replaced with O6-Me-dG. The second guanine of K-ras codon 12 (G7 in our numbering system) is a major mutational hotspot for G → A transitions observed in lung tumors of smokers and in neoplasms induced in laboratory animals by exposure to methylating agents. O6-Me-dG-containing duplexes were incubated with human recombinant AGT protein, and the reactions were quenched at specific times. Following acid hydrolysis to release purines, isotope dilution HPLC−ESI−MS/MS was used to determine the amounts of O6-Me-G remaining in DNA. The relative extent of demethylation for O6-Me-dG adducts located at G5, G6, G7, G8, G9, G10, or G11 following a 10 s incubation with AGT showed little variation as a function of sequence position. Furthermore, the second-order rate constants for the repair of O6-Me-dG adducts located at the first and second positions of the K-ras codon 12 (5‘-G6G7T-3‘) were similar (1.4 × 107 M-1 s-1 vs 7.4 × 106 M-1 s-1, respectively), suggesting that O6-Me-dG repair by AGT is not the determining factor for K-ras codon 12 mutagenesis following exposure to methylating agents. The new HPLC−ESI−MS/MS assay developed in this work is a valuable tool which will be used to further explore the role of local sequence environment and endogenous DNA modifications in shaping mutational spectra of NNK and other methylating agents.

Protein Labeling And Localization In Cells Using A Self Labeling Protein Tag

Autofluorescent proteins like GFP have become a basic tool for todays cell biologist. We will discuss the utility of an alternative approach, based on covalently self-labeling protein tags. One of these tags is the SNAP-tag which is based on the human protein AGT. This tag reacts covalently with a wide range of substrates that are based on benzylguanine (BG). Three cell permeable fluorescent substrates are available so far which allow covalent labeling of SNAP-tag fusion proteins inside living cells with blue, green, and red fluorophores. In addition non-cell permeable fluorescent labels exist. The applicability of the concept for cellular studies will be discussed using the following examples: 1) Labeling of SNAP-PKC-gamma fusion protein in CHO-cells and proof of intracellular redistribution upon stimulation with a phorbol ester. 2) Exclusive extracellular labeling of SNAP-NK1 expressed in CHO-cells and its internalization upon stimulation with substance P. 3) The sequential (pulse chase) labeling of a SNAP-actin with two different colors inside living cells with the clear identification of resting and of extending actin filaments. A further result of interest is the fixation stability of labels introduced using self labeling protein tags, and the possibility of labeling a tag in fixed cells, which is feasible with the SNAP-tag. These applications will be used to demonstrate the potential of self-labeling protein tags in cell biology. This work was in part funded by a CTI-grant from the Swiss government (CTI-grant 6687).