Posttranslational Regulation of Human DNA Polymerase i

Background: Many proteins are subject to posttranslational regulation, such as ubiquitination. Results: Human DNA polymerase iota (pol iota) can be monoubiquitinated at >27 unique sites, and exposure to naphthoquinones results in polyubiquitination of pol iota. Conclusion: Ubiquitination sites are located across the entire pol iota polypeptide as well as various structural motifs. Significance: Ubiquitination at these sites is likely to alter cellular functions of pol iota in vivo. Human DNA polymerases (pols) eta and i are Y-family DNA polymerase paralogs that facilitate translesion synthesis past damaged DNA. Both pol eta and poli can be monoubiquitinated in vivo. Pol eta 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 poli 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. Poli 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, poli becomes transiently polyubiquitinated via Lys(11)- and Lys(48)-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 poli occurs via the interplay between lysine acetylation and ubiquitination of ubiquitin itself at Lys(11) and Lys(48) rather than oxidative damage per se.