Alteration of arginine-128 to alanine abolishes the ability of human O6-alkylguanine-DNA alkyltransferase to repair methylated DNA but has no effect on its reaction with O6-benzylguanine

Kanugula, Sreenivas; Goodtzova, Karina; Edara, Suvarchala; Pegg, Anthony E.

doi:10.1021/bi00021a024

Cited by 54 publications

(68 citation statements)

References 26 publications

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“…This value agrees well with previous measurements (13) and is consistent with the value (M r ϭ 21,614) predicted from the sequence of this variant of the protein. The preparations used were Ͼ95% active in transfer of methyl groups from O 6 -[ 3 H]methylguanine-labeled calf thymus DNA to AGT and Ͼ95% active in debenzoylating O 6 -benzylguanine as described previously (25,26). AGT concentrations were measured spectrophotometrically using a molar extinction coefficient, ⑀ 280 ϭ 3.93 ϫ 10 4 M Ϫ1 cm Ϫ1 , calculated from the data of Roy et al (27).…”

Section: Methodsmentioning

confidence: 99%

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

Rasimas

Kar

Pegg

et al. 2007

Journal of Biological Chemistry

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Section: Methodsmentioning

confidence: 99%

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

Rasimas

Kar

Pegg

et al. 2007

Journal of Biological Chemistry

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“…A substrate oligonucleotide with the same sequence and a methyl substitution at the O 6 -position of the 3Ј-most guanine (shown above in bold type) was purchased from Synthegen LLC (Houston, TX). When duplex DNA was required, the oligonucleotide samples were combined and annealed as described (17). The DNA samples were labeled at the 5Ј termini with 32 P as described by Maxam and Gilbert (18) and transferred into 10 mM Tris (pH 8.0 at 21°C) using Sephadex G-25 centrifuge columns (Amersham Biosciences).…”

Section: Methodsmentioning

confidence: 99%

DNA-binding Mechanism ofO 6-Alkylguanine-DNA Alkyltransferase

Rasimas

Pegg

Fried³

2003

Journal of Biological Chemistry

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The mutagenic and cytotoxic effects of many endogenous and exogenous alkylating agents are mitigated by the actions of O 6 -alkylguanine-DNA alkyltransferase (AGT). In humans this protein protects the integrity of the genome, but it also contributes to the resistance of tumors to DNA-alkylating chemotherapeutic agents. Here we report properties of the interaction between AGT and short DNA oligonucleotides. We show that although AGT sediments as a monomer in the absence of DNA, it binds cooperatively to both single-stranded and double-stranded deoxyribonucleotides. This strong cooperative interaction is only slightly perturbed by active site mutation of AGT or by alkylation of either AGT or DNA. The stoichiometry of complex formation with 16-mer oligonucleotides, assessed by analytical ultracentrifugation and electrophoretic mobility shift assays, is 4:1 on single-stranded and duplex DNA and is unchanged by several active site mutations or by protein or DNA alkylation. These results have significant implications for the mechanisms by which AGT locates and interacts with repairable alkyl lesions to effect DNA repair. O6 -Alkylguanine-DNA alkyltransferase is a ubiquitous repair protein that plays a vital role in minimizing the mutagenic effects of alkylating agents (1-4). It catalyzes the stoichiometric transfer of a variety of alkyl substituents from the O 6 -position of guanine to an active site cysteine, preventing incorrect base pairing caused by these adducts. More than 100 alkyltransferases are now known, and the crystal structures are available for three family members: the Ada-C protein from Escherichia coli (5), the human alkyltransferase (hAGT) 1 (6), and the protein from the thermophilic archaeon, Pyrococcus kodakaraensis (7). All of the known alkyltransferases lack the ability to dealkylate themselves, and no dealkylation activity has been found in cell extracts to date. On this basis, it is widely thought that alkyltransferase participates in a single reaction and is then irreversibly inactivated. Given the apparently nonenzymatic nature of the protein, the protection afforded by alkyltransferase is likely to depend on the regulation of its synthesis and degradation and on its ability to efficiently locate repairable lesions throughout the genome.The mechanisms by which AGT interacts with adduct-containing and adduct-free DNAs are poorly understood. Two contrasting mechanisms have been proposed to date. In the first, single AGT proteins bind normal and lesion-containing DNA, and the distribution of AGT between normal and lesion sites depends on a difference in binding affinity. This model is consistent with the observation that a single AGT monomer is necessary and sufficient to dealkylate a single O 6 -alkyl guanine adduct within a DNA duplex (3). It is supported by the observation that single AGT-DNA complexes are detected by gel shift assay when AGT binds short DNA molecules. These complexes have been interpreted as having a 1:1 AGT:DNA stoichiometry, and the binding affinities have been calculated ...

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“…Purification of AGT-AGT was expressed and purified to homogeneity by ammonium sulfate precipitation, chromatography on Mono-S, and gel-filtration as described previously (10,20). 6 -Benzylguanine-The purified alkyltransferase proteins were incubated in 50 mM Tris-HCl, pH 7.5, 0.…”

Section: Methodsmentioning

confidence: 99%

Repair of O6-Benzylguanine by the Escherichia coli Ada and Ogt and the Human O6-Alkylguanine-DNA Alkyltransferases

Goodtzova

Kanugula

Edara

et al. 1997

Journal of Biological Chemistry

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O 6 -Methylguanine is removed from DNA via the transfer of the methyl group to a cysteine acceptor site present in the DNA repair protein O 6 -alkylguanine-DNA alkyltransferase. The human alkyltransferase is inactivated by the free base O 6 -benzylguanine, raising the possibility that substantially larger alkyl groups could also be accepted as substrates. However, the Escherichia coli alkyltransferase, Ada-C, is not inactivated by O 6 -benzylguanine. The Ada-C protein was rendered capable of reaction by the incorporation of two sitedirected mutations converting Ala 316 to a proline (A316P) and Trp 336 to alanine (W336A) or glycine (W336G). These changes increase the space at the active site of the protein where Cys 321 is buried and thus permit access of the O 6 -benzylguanine inhibitor. Reaction of the mutant A316P/W336A-Ada-C with O 6 -benzylguanine was greatly stimulated by the presence of DNA, providing strong support for the concept that binding of DNA to the Ada-C protein activates the protein. The Ada-C protein was able to repair O 6 -benzylguanine in a 16-mer oligodeoxyribonucleotide. However, the rate of repair was very slow, whereas the E. coli Ogt, the human alkyltransferase, and the mutant A316P/W336A-Ada-C alkyltransferases reacted very rapidly with this 16-mer substrate and preferentially repaired it when incubated with a mixture of the methylated and benzylated 16-mers. These results show that benzyl groups are better substrates than methyl groups for alkyltransferases provided that steric factors do not prevent binding of the substrate in the correct orientation for alkyl group transfer.

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Alteration of arginine-128 to alanine abolishes the ability of human O6-alkylguanine-DNA alkyltransferase to repair methylated DNA but has no effect on its reaction with O6-benzylguanine

Cited by 54 publications

References 26 publications

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

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

DNA-binding Mechanism ofO 6-Alkylguanine-DNA Alkyltransferase

Repair of O6-Benzylguanine by the Escherichia coli Ada and Ogt and the Human O6-Alkylguanine-DNA Alkyltransferases

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