Abstract
DNA polymerase iota (Polι) belongs to the Y-family of DNA polymerases that are involved in DNA damage tolerance through their role in translesion DNA synthesis. Like all other Y-family polymerases, Polι interacts with proliferating cell nuclear antigen (PCNA), Rev1, ubiquitin and ubiquitinated-PCNA and is also ubiquitinated itself. Here, we report that Polι also interacts with the p300 acetyltransferase and is acetylated. The primary acetylation site is K550, located in the Rev1-interacting region. However, K550 amino acid substitutions have no effect on Polι’s ability to interact with Rev1. Interestingly, we find that acetylation of Polι significantly and specifically increases in response to SN2 alkylating agents and to a lower extent to SN1 alkylating and oxidative agents. As we have not observed acetylation of Polι’s closest paralogue, DNA polymerase eta (Polη), with which Polι shares many functional similarities, we believe that this modification might exclusively regulate yet to be determined, and separate function(s) of Polι.
Highlights
DNA polymerase iota (Polι) belongs to the Y-family of DNA polymerases that are involved in DNA damage tolerance through their role in translesion DNA synthesis
We proposed that p300 acetyltransferase inhibition influences polyubiquitination of DNA polymerase iota (Polι), a non-canonical polymerase involved in DNA translesion synthesis (TLS)
We have shown that Polι is acetylated by the p300/CREB-binding protein (CBP) acetyltransferases and that this response is especially strong in cells treated with the SN2 alkylating agents, methyl methanesulphonate (MMS) and dimethyl sulfate (DMS), but not other DNA damaging treatments
Summary
Polι is a TLS polymerase present in mammals, some other vertebrates and some fungi. The cellular function of the enzyme is still far from being understood. In light of the known DNA damage tolerance/repair activities of TLS polymerases, the functional connection of Polι acetylation with DNA rather than protein modifications by SN2 alkylating agents seems more credible. This phenomenon needs to be investigated in detail and will be the subject of future studies. For the immunoprecipitation assay of acetylated Polι and Polη, respective cell extracts including FLAG-tagged proteins were incubated for one hour to overnight at 4 °C with Anti-DYDDDDK (Flag) Affinity Gel (Biotool), washed three times and analyzed directly by SDS-PAGE and western blot. The reaction was stopped by the addition of Laemmli buffer and followed by SDS-PAGE
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