Posttranslational Regulation of Human DNA Polymerase ι

• Published in: The Journal of biological chemistry Vol. 290; no. 45; pp. 27332 - 27344
• Main Authors: McIntyre, Justyna, McLenigan, Mary P., Frank, Ekaterina G., Dai, Xiaoxia, Yang, Wei, Wang, Yinsheng, Woodgate, Roger
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 06.11.2015; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Amino Acid Substitution; Binding Sites - genetics; DNA and Chromosomes; DNA Damage; DNA polymerase ι; DNA Repair; DNA synthesis; DNA-Directed DNA Polymerase - chemistry; DNA-Directed DNA Polymerase - genetics; DNA-Directed DNA Polymerase - metabolism; HEK293 Cells; Humans; Lysine - chemistry; Models, Molecular; Molecular Sequence Data; mutagenesis; Mutagenesis, Site-Directed; posttranslational modification (PTM); Protein Conformation; Protein Processing, Post-Translational; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - metabolism; Tandem Mass Spectrometry; ubiquitin; Ubiquitination; Y-family DNA polymerase; posttranslational modification (PTM); DNA synthesis; DNA polymerase ι; DNA repair; mutagenesis; ubiquitin; Y-family DNA polymerase
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M115.675769

Human DNA polymerases (pols) η and ι are Y-family DNA polymerase paralogs that facilitate translesion synthesis past damaged DNA. Both polη and polι can be monoubiquitinated in vivo. Polη has been shown to be ubiquitinated at one primary site. When this site is unavailable, three nearby lysines may become ubiquitinated. In contrast, mass spectrometry analysis of monoubiquitinated polι revealed that it is ubiquitinated at over 27 unique sites. Many of these sites are localized in different functional domains of the protein, including the catalytic polymerase domain, the proliferating cell nuclear antigen-interacting region, the Rev1-interacting region, and its ubiquitin binding motifs UBM1 and UBM2. Polι monoubiquitination remains unchanged after cells are exposed to DNA-damaging agents such as UV light (generating UV photoproducts), ethyl methanesulfonate (generating alkylation damage), mitomycin C (generating interstrand cross-links), or potassium bromate (generating direct oxidative DNA damage). However, when exposed to naphthoquinones, such as menadione and plumbagin, which cause indirect oxidative damage through mitochondrial dysfunction, polι becomes transiently polyubiquitinated via Lys11- and Lys48-linked chains of ubiquitin and subsequently targeted for degradation. Polyubiquitination does not occur as a direct result of the perturbation of the redox cycle as no polyubiquitination was observed after treatment with rotenone or antimycin A, which both inhibit mitochondrial electron transport. Interestingly, polyubiquitination was observed after the inhibition of the lysine acetyltransferase KATB3/p300. We hypothesize that the formation of polyubiquitination chains attached to polι occurs via the interplay between lysine acetylation and ubiquitination of ubiquitin itself at Lys11 and Lys48 rather than oxidative damage per se. Background: Many proteins are subject to posttranslational regulation, such as ubiquitination. Results: Human DNA polymerase ι (polι) can be monoubiquitinated at >27 unique sites, and exposure to naphthoquinones results in polyubiquitination of polι. Conclusion: Ubiquitination sites are located across the entire polι polypeptide as well as various structural motifs. Significance: Ubiquitination at these sites is likely to alter cellular functions of polι in vivo.