Characterisation and nucleotide sequence ofogt, the O6-alkyiguanine-DNA-alkyltransferase gene ofE.coli

Potter, Philip M.; Wilkinson, Mark C.; Fitton, John E.; Carr, Francis J.; Brennand, John; Cooper, Donald P.; Margison, Geoffrey P.

doi:10.1093/nar/15.22.9177

Cited by 161 publications

(89 citation statements)

References 29 publications

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“…The additional bands may have been generated by either functionally active ATase fragments expressed from additional ATG codons in frame with the alkyl-acceptor cysteins residue or by proteolytic cleavage of the 25 kDa ada 06-AlkG ATase. Multiple protein bands following fluorographic analysis have been previously identified when the truncated ada gene was overexpressed in E. coli (Potter et al, 1987). The endogenous Chinese hamster ATase protein is also about 25 kDa (Morten et al, 1992) but the expression in clone SB was unlikely to have been upregulation of the hamster ATase gene since no G418 resistant clones produced from the transfection of the pZipneoSV(X)l parent plasmid had elevated levels of ATase activity.…”

Section: Resultsmentioning

confidence: 99%

“…Prokaryotes have a specific repair mechanism for 06-AlkG and 04-AlkT which involves the transfer of the alkyl group to a cysteine residue within an ATase protein in an autoinactivating process (Olsson & Lindahl, 1980;Margison et al, 1985;Potter et al, 1987). E.coli has two genes which encode ATase enzymes; ada (Sedgwick, 1983;Margison et al, 1985) and ogt (Potter et al, 1987).…”

mentioning

confidence: 99%

“…E.coli has two genes which encode ATase enzymes; ada (Sedgwick, 1983;Margison et al, 1985) and ogt (Potter et al, 1987). The ada gene encodes a 39 kDa protein which is composed of two subfragments sized 18 and 20 kDa capable of repairing 06-AlkG, 04-AlkT and the S stereoisomer of alkylphosphotriesters (AlkPT), respectively.…”

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confidence: 99%

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Expression in mammalian cells of the Escherichia coli O6 alkylguanine-DNA-alkyltransferase gene ogt reduces the toxicity of alkylnitrosoureas

Harris

Margison²

1993

Br J Cancer

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V79 Chinese hamster cells expressing either the O6-alkylguanine-DNA-alkyltransferase (ATase) encoded by the E. coli ogt gene or a truncated version of the E. coli ada gene have been exposed to various alkylnitrosoureas to investigate the contribution of ATase repairable lesions to the toxicity of these compounds. Both ATases are able to repair O6-alkylguanine (O6-AlkG) and O4-alkylthymine (O4-AlkT) but the ogt ATase is more efficient in the repair of O4-methylthymine (O4-MeT) and higher alkyl derivatives of O6-AlkG than is the ada ATase. Expression of the ogt ATase provided greater protection against the toxic effects of the alkylating agents then the ada ATase particularly with N-ethyl-N-nitrosourea (ENU) and N-butyl-N-nitrosourea (BNU) to which the ada ATase expressing cells were as sensitive as parent vector transfected cells. Although ogt was expressed at slightly higher levels than the truncated ada in the transfected cells, this could not account for the differential protection observed. For-N-methyl-N-nitrosourea (MNU) the increased protection in ogt-transfected cells is consistent with O4-MeT acting as a toxic lesion. For the longer chain alkylating agents and chloroethylating agents, the protection afforded by the ogt protein may be a consequence of the more efficient repair of O6-AlkG, O4-AlkT or both of these lesions in comparison with the ada-encoded ATase. Images Figure 2 Figure 3

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Section: Resultsmentioning

confidence: 99%

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confidence: 99%

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Expression in mammalian cells of the Escherichia coli O6 alkylguanine-DNA-alkyltransferase gene ogt reduces the toxicity of alkylnitrosoureas

Harris

Margison²

1993

Br J Cancer

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“…Thus, benzyl groups are actually preferred substrates for both of these alkyltransferases. E. coli contains a second alkyltransferase gene (1,26,27). Its product, the Ogt protein, is a constitutive alkyltransferase.…”

Section: Repair Of O 6 -Benzylguaninementioning

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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“…1) is the responsibility of the methyltransferase (MTase) proteins Ada and Ogt [6]. Both of these proteins can repair O 6 mG directly by transferring the methyl group to the sulfhydryl group of an active site cysteine residue [13][14][15][16]. In the case where MTase repair of O 6 mG occurs after one round of replication, a mismatched G:T base pair results.…”

Section: Introductionmentioning

confidence: 99%

Mismatch repair proteins collaborate with methyltransferases in the repair of O6-methylguanine

et al. 2008

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DNA repair is essential for combatting the adverse effects of damage to the genome. One example of base damage is O 6 -methylguanine (O 6 mG), which stably pairs with thymine during replication and thereby creates a promutagenic O 6 mG:T mismatch. This mismatch has also been linked with cellular toxicity. Therefore, in the absence of repair, O 6 mG:T mismatches can lead to cell death or result in G:C→A:T transition mutations upon the next round of replication. Cysteine thiolate residues on the Ada and Ogt methyltransferase (MTase) proteins directly reverse the O 6 mG base damage to yield guanine. When a cytosine is opposite the lesion, MTase repair restores a normal G:C pairing. However, if replication past the lesion has produced an O 6 mG:T mismatch, MTase conversion to a G:T mispair must still undergo correction to avoid mutation. Two mismatch repair pathways in E. coli that convert G:T mispairs to native G:C pairings are methyl-directed mismatch repair (MMR) and very short patch repair (VSPR). This work examined the possible roles that proteins in these pathways play in coordination with the canonical MTase repair of O 6 mG:T mismatches. The possibility of this repair network was analyzed by probing the efficiency of MTase repair of a single O 6 mG residue in cells deficient in individual mismatch repair proteins (Dam, MutH, MutS, MutL, or Vsr). We found that MTase repair in cells deficient in Dam or MutH showed wild-type levels of MTase repair. In contrast, cells lacking any of the VSPR proteins MutS, MutL, or Vsr showed a decrease in repair of O 6 mG by the Ada and Ogt MTases. Evidence is presented that the VSPR pathway positively influences MTase repair of O 6 mG:T mismatches, and assists the efficiency of restoring these mismatches to native G:C base pairs.

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Characterisation and nucleotide sequence ofogt, the O⁶-alkyiguanine-DNA-alkyltransferase gene ofE.coli

Cited by 161 publications

References 29 publications

Expression in mammalian cells of the Escherichia coli O6 alkylguanine-DNA-alkyltransferase gene ogt reduces the toxicity of alkylnitrosoureas

Expression in mammalian cells of the Escherichia coli O6 alkylguanine-DNA-alkyltransferase gene ogt reduces the toxicity of alkylnitrosoureas

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

Mismatch repair proteins collaborate with methyltransferases in the repair of O6-methylguanine

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