PROBING OF CONFORMATIONAL-CHANGES IN HUMAN O-6-ALKYLGUANINE-DNA ALKYLTRANSFERASE PROTEIN IN ITS ALKYLATED AND DNA-BOUND STATES BY LIMITED PROTEOLYSIS

Human O-6-alkylguanine-DNA alkyl transferase (hAGT) is a DNA repair pr otein that protects cells from alkylation damage by transferring an al kyl group from the O-6-position of guanine to a cysteine residue in th e active site (-PCHR-) of the protein. The structure of the hAGT prote in (23 kDa) has been probed by limited proteolysis with trypsin and Gl u-C endoproteases and analysis of the polypeptide fragments by SDS/PAG E. The native hAGT protein had limited accessibility to digestion with trypsin and Glu-C in spite of a number of potential cleavage sites. I nitial cleavage by trypsin occurred at residue Lys-193 to give a 21 kD a polypeptide fragment, and this polypeptide underwent further cleavag e at residues Arg-128 and Lys-165. These trypsin-cleavage sites became more accessible to digestion in the presence of double-stranded DNA ( dsDNA), indicating that hAGT undergoes a change in its conformation on binding to DNA. However, the trypsin cutting site at the Arg-l28 posi tion was less available for digestion in the presence of single-strand ed DNA (ssDNA), suggesting that the hAGT protein has a different confo rmation when bound to ssDNA compared with dsDNA. When protease digesti on was carried out on wild-type protein, preincubated with the low-mol ecular-mass pseudosubstrate O-6-benzylguanine, increased susceptibilit y to proteases was observed. A mutant C145A hAGT protein, which cannot repair O-6-alkylguanine because the Cys-145 acceptor site in the acti ve site of the protein is changed to Ala, showed identical trypsin cle avage to the wild type, but its digestion was not affected by O-6-benz ylguanine. These results suggest that alkylation of hAGT leads to an a ltered conformation. The acquisition of increased susceptibility to pr oteases upon DNA binding and alkylation demonstrates that hAGT undergo es considerable conformational changes in its structure upon binding t o DNA and after repair of alkylation damage.