Alkylation damage repair protein O6-alkylguanine-DNA alkyltransferase from the hyperthermophiles Aquifex aeolicus and Archaeoglobus fulgidus

Sreenivas Kanugula, Anthony Pegg

Research output: Contribution to journalArticle

9 Citations (Scopus)

Abstract

AGT (O6-alkylguanine DNA alkyltransferase) is an important DNA-repair protein that protects cells from killing and mutagenesis by alkylating agents. The AGT genes from two extremely thermophilic organisms, the bacterium Aquifex aeolicus and the archaeon Archaeoglobus fulgidus were PCR-derived and cloned into an expression vector. The nucleotide sequence of the Aq. aeolicus AGT encodes a 201-amino-acid protein with a molecular mass of 23 000 Da and Ar. fulgidus AGT codes for a 147-amino-acid protein with a molecular mass of 16718 Da. The Aq. aeolicus and Ar. fulgidus AGTs were expressed at high levels in Escherichia coli fused to an N-terminal polyhistidine tag that allowed single-step isolation and purification by metal-affinity chromatography. Both AGTs formed inclusion bodies and were not soluble under native purification conditions. Therefore AGT isolation was performed under protein-denaturation conditions in the presence of 8.0 M urea. Soluble AGT was obtained by refolding the AGT in the presence of calf thymus DNA. Both AGTs were active in repairing O6-methylguanine and, at a lower rate, O4-methylthymine in DNA. They exhibited thermostability and optimum activity at high temperature. The thermostable AGTs, particularly that from Aq. aeolicus, were readily inactivated by the low-molecular-mass inhibitor O6-benzylguanine, which is currently in clinical trials to enhance cancer chemotherapy.

Original languageEnglish (US)
Pages (from-to)449-455
Number of pages7
JournalBiochemical Journal
Volume375
Issue number2
DOIs
StatePublished - Oct 15 2003

Fingerprint

Archaeoglobus fulgidus
Alkylation
Molecular mass
Repair
Purification
Protein Denaturation
Amino Acids
Proteins
Alkylating Agents
Inclusion Bodies
Archaea
Affinity Chromatography
Affinity chromatography
Mutagenesis
DNA Repair
Denaturation
Chemotherapy
Urea
DNA
Metals

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

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title = "Alkylation damage repair protein O6-alkylguanine-DNA alkyltransferase from the hyperthermophiles Aquifex aeolicus and Archaeoglobus fulgidus",
abstract = "AGT (O6-alkylguanine DNA alkyltransferase) is an important DNA-repair protein that protects cells from killing and mutagenesis by alkylating agents. The AGT genes from two extremely thermophilic organisms, the bacterium Aquifex aeolicus and the archaeon Archaeoglobus fulgidus were PCR-derived and cloned into an expression vector. The nucleotide sequence of the Aq. aeolicus AGT encodes a 201-amino-acid protein with a molecular mass of 23 000 Da and Ar. fulgidus AGT codes for a 147-amino-acid protein with a molecular mass of 16718 Da. The Aq. aeolicus and Ar. fulgidus AGTs were expressed at high levels in Escherichia coli fused to an N-terminal polyhistidine tag that allowed single-step isolation and purification by metal-affinity chromatography. Both AGTs formed inclusion bodies and were not soluble under native purification conditions. Therefore AGT isolation was performed under protein-denaturation conditions in the presence of 8.0 M urea. Soluble AGT was obtained by refolding the AGT in the presence of calf thymus DNA. Both AGTs were active in repairing O6-methylguanine and, at a lower rate, O4-methylthymine in DNA. They exhibited thermostability and optimum activity at high temperature. The thermostable AGTs, particularly that from Aq. aeolicus, were readily inactivated by the low-molecular-mass inhibitor O6-benzylguanine, which is currently in clinical trials to enhance cancer chemotherapy.",
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Alkylation damage repair protein O6-alkylguanine-DNA alkyltransferase from the hyperthermophiles Aquifex aeolicus and Archaeoglobus fulgidus. / Kanugula, Sreenivas; Pegg, Anthony.

In: Biochemical Journal, Vol. 375, No. 2, 15.10.2003, p. 449-455.

Research output: Contribution to journalArticle

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