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Regulation of Human MutYH DNA Glycosylase by the E3 Ubiquitin Ligase Mule

J. Dorn,E. Ferrari,Ralph Imhof,Nathalie Ziegler,U. Hübscher

Published 2014 in Journal of Biological Chemistry

ABSTRACT

Background: Regulation of MutYH DNA glycosylase is essential for genomic integrity. Results: MutYH is ubiquitinated by the E3 ligase Mule changing its protein levels and recruitment to chromatin. MutYH levels in cancer cells influence the mutagenic potential of reactive oxygen species. Conclusion: Ubiquitination of MutYH is an important regulatory step. Significance: We identified the residues that are targets for ubiquitination by Mule. Oxidation of DNA is a frequent and constantly occurring event. One of the best characterized oxidative DNA lesions is 7,8-dihydro-8-oxoguanine (8-oxo-G). It instructs most DNA polymerases to preferentially insert an adenine (A) opposite 8-oxo-G instead of the appropriate cytosine (C) thus showing miscoding potential. The MutY DNA glycosylase homologue (MutYH) recognizes A:8-oxo-G mispairs and removes the mispaired A giving way to the canonical base excision repair that ultimately restores undamaged guanine (G). Here we characterize for the first time in detail a posttranslational modification of the human MutYH DNA glycosylase. We show that MutYH is ubiquitinated in vitro and in vivo by the E3 ligase Mule between amino acids 475 and 535. Mutation of five lysine residues in this region significantly stabilizes MutYH, suggesting that these are the target sites for ubiquitination. The endogenous MutYH protein levels depend on the amount of expressed Mule. Furthermore, MutYH and Mule physically interact. We found that a ubiquitination-deficient MutYH mutant shows enhanced binding to chromatin. The mutation frequency of the ovarian cancer cell line A2780, analyzed at the HPRT locus can be increased upon oxidative stress and depends on the MutYH levels that are regulated by Mule. This reflects the importance of tightly regulated MutYH levels in the cell. In summary our data show that ubiquitination is an important regulatory mechanism for the essential MutYH DNA glycosylase in human cells.

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