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Published February 17, 2009 | Published + Supplemental Material
Journal Article Open

Two proteolytic pathways regulate DNA repair by cotargeting the Mgt1 alkylguanine transferase


O^6-methylguanine (O^6meG) and related modifications of guanine in double-stranded DNA are functionally severe lesions that can be produced by many alkylating agents, including N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), a potent carcinogen. O^6meG is repaired through its demethylation by the O^6-alkylguanine-DNA alkyltransferase (AGT). This protein is called Mgmt (or MGMT) in mammals and Mgt1 in the yeast Saccharomyces cerevisiae. AGT proteins remove methyl and other alkyl groups from an alkylated O^6 in guanine by transferring the adduct to an active-site cysteine residue. The resulting S-alkyl-Cys of AGT is not restored back to Cys, so repair proteins of this kind can act only once. We report here that S. cerevisiae Mgt1 is cotargeted for degradation, through a degron near its N terminus, by 2 ubiquitin-mediated proteolytic systems, the Ubr1/Rad6-dependent N-end rule pathway and the Ufd4/Ubc4-dependent ubiquitin fusion degradation (UFD) pathway. The cotargeting of Mgt1 by these pathways is synergistic, in that it increases not only the yield of polyubiquitylated Mgt1, but also the processivity of polyubiquitylation. The N-end rule and UFD pathways comediate both the constitutive and MNNG-accelerated degradation of Mgt1. Yeast cells lacking the Ubr1 and Ufd4 ubiquitin ligases were hyperresistant to MNNG but hypersensitive to the toxicity of overexpressed Mgt1. We consider ramifications of this discovery for the control of DNA repair and mechanisms of substrate targeting by the ubiquitin system.

Additional Information

©2009 by the National Academy of Sciences. Contributed by Alexander Varshavsky, December 9, 2008 (received for review November 22, 2008). Published online before print January 21, 2009, doi:10.1073/pnas.0812316106 We thank M. Longtine (Oklahoma State University, Stillwater, OK) for pFA6a-KanMX6 and pFA6a-13MYC-TRP1, Y. Xie (Wayne State University, Detroit, MI) for a collection of Ub ligase mutants, J. Li and J. Shan (Weill Cornell Medical College, New York, NY) for their assistance with Mgt1 modeling, and the present and former members of the Varshavsky laboratory, particularly J. Sheng, for their advice and assistance. This work was supported by from the National Institutes of Health Grants GM31530 and DK39520 (to A.V.), the Sandler Program for Asthma Research, and the Ellison Medical Foundation. Author contributions: C.-S.H., A.S., and A.V. designed research; C.-S.H. and A.S. performed research; C.-S.H., A.S., and A.V. analyzed data; and C.-S.H., A.S., and A.V. wrote the paper. The authors declare no conflict of interest. This article contains supporting information online at www.pnas.org/cgi/content/full/0812316106/DCSupplemental.

Attached Files

Published - Hwang2009p10010.1073pnas.0812316106.pdf

Supplemental Material - Hwang0812316106SI.pdf


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