Carcinogenesis, Vol. 23, No. 5, 823-830,
May 2002
© 2002 Oxford University Press
CARCINOGENESIS |
Degradation of the alkylated form of the DNA repair protein, O6-alkylguanine-DNA alkyltransferase
Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, The Milton S. Hershey Medical Center, PO Box 850, 500 University Drive, Hershey, PA 17033-0850, USA
O6-Alkylguanine-DNA alkyltransferase (AGT) is a DNA repair protein that removes alkyl groups from DNA by transferring them to an internal Cys-145 residue. As the S-alkylcysteine is not converted back to cysteine, the protein can only act once and the resulting alkylated AGT molecule is rapidly degraded. The mechanism underlying the disappearance of the alkylated AGT has been studied in vivo in CHO cells and in vitro in reticulocyte lysates by using the pseudosubstrate O6-benzylguanine (BG) and mutant forms of AGT. The wild-type AGT was stable but was ubiquitinated and degraded rapidly by the proteasome after treatment with BG or with an oligodeoxyribonucleotide, which contained O6-methylguanine. Mutants C145F (and other mutants with bulky substituents at position 145), which have alterations that cause a steric alteration at the active site and also prevent hydrogen bonding involving Cys-145 resembled the alkylated AGT and were ubiquitinated and degraded rapidly irrespective of treatment with BG. Mutant M134F, which causes a steric alteration without interfering directly with the hydrogen-bonding network involving Cys-145, partially destabilized AGT and its degradation was increased further by reaction with BG. Mutant C145S, which maintains the hydrogen-binding network and causes no distortion, was not rapidly degraded. The results indicate that the conformational change resulting in the opening of the asparagine hinge region in the structure, which is brought about by formation of an S-alkyl adduct, leads to an increased recognition by a ubiquitin ligase targeting the protein for degradation. This is a novel type of post-translational modification causing ubiquitination.
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