The role of the carboxyl-terminal tail in human O6-methylguanine DNA methyltransferase substrate specificity and temperature sensitivity

S. E. Morgan, M. R. Kelley, R. O. Pieper

Research output: Contribution to journalArticle

20 Scopus citations

Abstract

The human O6-methylguanine DNA methyltransferase (MGMT) repairs O6-methylguanine (O6-MG) in DNA at a much lower rate than the Escherichia coli Ada protein, and only MGMT repairs the altered base, O6-benzylguanine (O6-BG). The diversity in DNA repair properties between MGMT and Ada may be a result of divergent amino acid sequences outside their common proline-cysteine-histidine-arginine-valine (PCHRV) acceptor site. One notable sequence difference is an MGMT 28-amino acid carboxyl-terminal tail which is highly conserved among all mammalian alkyltransferases. The role of this tail sequence in substrate specificity was assessed by expressing full-length MGMT and Ada proteins, and mutant MGMT proteins lacking either 10 or 28 amino acids from the carboxyl terminus, as GST fusion proteins in alkyltransferase-deficient E. coli cells, and comparing rates of repair of O6-MG containing DNA and O6-BG by these fusion proteins at 4°C and 37°C. The MGMT carboxyl-terminal tail was not required for repair of O6-MG in DNA at 37°C although the deletion of this tail sequence reversibly inhibited the ability of MGMT to repair O6-MG in DNA at 4°C. Therefore, the absence of this region affects the ability of the protein to repair O6-MG in DNA at lower temperatures. Furthermore, removal of the tail sequence from MGMT decreased the rate of O6-BG repair 5-fold. We conclude that the 28-amino acid carboxyl-terminal MGMT tail, while not required for activity, modulates the rate of MGMT repair at reduced temperatures and plays a role in substrate specificity.

Original languageEnglish (US)
Pages (from-to)19802-19809
Number of pages8
JournalJournal of Biological Chemistry
Volume268
Issue number26
StatePublished - Jan 1 1993

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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