Minimum size of active human O6-methylguanine-dna methyltransference (MGMT); substrate binding parallels mgmt activity

Tapas Hazra, R. Roy, D. T. Grabowski, S. Mitra

Research output: Contribution to journalArticle

Abstract

O6-methylguanine-DNA methyltransferase (MGMT), a ubiquitous DNA repair protein, repairs the mutagenic adduct O6-alkylguanine by accepting the alkyl group on a unique cysteine residue in DNA. In an effort to identify the minimum size required for MGMT activity, its cDNA was sequentially deleted at one or both termini and the methyltransferase activities of the encoded polypeptides were determined. Up to 8 and 31 residues could be removed from the NH2- and COO--termini of the protein, respectively, without loss of activity. Although, removal of residue 9 (Arg) from the NH2-terminus or residue 32 (Leu) from the COO--terminus abolished activity, these residues are not essential for activity in the full length protein, but may by involved in maintaining structure. The second order rate constant of the MGMT reaction (5×107 M- min-1) was not significantly altered in the active truncated mutants. Specific complexes between MGMT's and O6-methylguanine-containing oligonucleotide were only observed in electrophoretic mobility shift assays with active, truncated proteins. Furthermore, conservative substitution of the alkylacceptor cysteine with serine induced a significant conformational change associated with loss of both methyltransferase activity and substrate binding.

Original languageEnglish (US)
JournalFASEB Journal
Volume10
Issue number6
StatePublished - 1996

Fingerprint

methyltransferases
Methyltransferases
Substrates
Cysteine
cysteine
DNA
Proteins
Repair
proteins
Electrophoretic mobility
Electrophoretic Mobility Shift Assay
DNA repair
oligonucleotides
Oligonucleotides
DNA Repair
serine
Serine
Rate constants
Assays
polypeptides

ASJC Scopus subject areas

  • Agricultural and Biological Sciences (miscellaneous)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Biochemistry
  • Cell Biology

Cite this

Minimum size of active human O6-methylguanine-dna methyltransference (MGMT); substrate binding parallels mgmt activity. / Hazra, Tapas; Roy, R.; Grabowski, D. T.; Mitra, S.

In: FASEB Journal, Vol. 10, No. 6, 1996.

Research output: Contribution to journalArticle

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AU - Grabowski, D. T.

AU - Mitra, S.

PY - 1996

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