Study of the structure of human O6-alkylguanine-DNA alkyl transferase (hAGT) protein in alkylated and DNA bound states by limited proteolysis

S. Kanugula, K. V. Goodtzova, Anthony Pegg

Research output: Contribution to journalArticle

Abstract

hAGT plays a critical role in protecting cells from alkylating agents by transferring alkylgroups from the O6-position of guanine in DNA to a cysteinc residue (Cys145). The structure of hAGT was probed by subjecting it to limited proteolysis and analysing the polypeptide tragments by SDS-PAGE Despite the presence of a number of potential cleavage sites. hAGT in its native state had limited accessibility to digestion. Initial cleavage by trypsin occurred at the C-terminal part of the molecute (1yrs193) and this polypeptide fragment underwent further cleavages at Arg128 and Lys1165 These trypsin cleavage sites become more accessible to digestion in the presence of double stranded DNA. Interestingly, the cleavage site at Arg128 became less available to digestion in the presence of single stranded DNA suggesting hAGT protein has a different conformation when bound to ssDNA compared to dsDNA. The inactive mutant C145A hAGT showed identical behavior to wild type AGT indicating that it can be used for studies of substrate binding. Incubation with a low M. W. pseudosubstrate, O6 benzylyuanine (BG), inactivates hAGT and forms S-benzylcysteine at Cys 145. Incubation of wild type hAGT with BG led to increased sensitivity to PnrOteases but no change in sensitivity was seen with the mutant C145A indicating that the alteration was due to alkylation at the active site. The acquisition of increased susceptibility to proteases upon DNA binding and alkylation indicate bAGT undergoes considerable conformational changes in its structure on DNA binding and after the repair reaction. These changes are likely to be important in both DNA repair and the removal of the alkylated form of the protein. Supported by grants CA- 18137 and CA-717876.

Original languageEnglish (US)
Pages (from-to)A1190
JournalFASEB Journal
Volume11
Issue number9
StatePublished - Dec 1 1997

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Biochemistry
  • Molecular Biology
  • Genetics

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