Abstract
DNA polymerase η (polη) is a Y-family translesion synthesis polymerase that plays a key role in the cellular tolerance toward UV irradiation-induced DNA damage. Here, we identified, for the first time, the phosphorylation of serine 687 (Ser(687)), which is located in the highly conserved nuclear localization signal (NLS) region of human polη and is mediated by cyclin-dependent kinase 2 (CDK2). We also showed that this phosphorylation is stimulated in human cells upon UV light exposure and results in diminished interaction of polη with proliferating cell nuclear antigen (PCNA). Furthermore, we demonstrated that the phosphorylation of Ser(687) in polη confers cellular protection from UV irradiation and increases the efficiency in replication across a site-specifically incorporated cyclobutane pyrimidine dimer in human cells. Based on these results, we proposed a mechanistic model where Ser(687) phosphorylation functions in the reverse polymerase switching step of translesion synthesis: The phosphorylation brings negative charges to the NLS of polη, which facilitates its departure from PCNA, thereby resetting the replication fork for highly accurate and processive DNA replication. Thus, our study, together with previous findings, supported that the posttranslational modifications of NLS of polη played a dual role in polymerase switching, where Lys(682) deubiquitination promotes the recruitment of polη to PCNA immediately prior to lesion bypass and Ser(687) phosphorylation stimulates its departure from the replication fork immediately after lesion bypass.
Highlights
Living cells are constantly exposed to various DNA-damaging agents, such as UV irradiation, chemical carcinogens, and endogenous reactive oxygen species [1]
The Role of serine 687 (Ser687) Phosphorylation of Pol in Its Interaction with proliferating cell nuclear antigen (PCNA)—Considering that Ser687 resides in the PCNAinteracting region of pol that is essential for pol-PCNA interaction during UV damage response [17], we investigated the potential role of Ser687 phosphorylation in the interaction between these two proteins
PCNA is known to be monoubiquitinated in response to UV light exposure, and this ubiquitination stimulates its interaction with pol through the ubiquitin-binding zinc finger (UBZ) domain of the latter protein [16]
Summary
Living cells are constantly exposed to various DNA-damaging agents, such as UV irradiation, chemical carcinogens, and endogenous reactive oxygen species [1]. The resulting DNA damage can block the progression of replication forks and/or induce mutations, which result in cell transformation, From the §Department of Chemistry, University of California, Riverside, California 92521– 0403. A polymerase switching model has been proposed for TLS, where the replicative DNA polymerase ␦ or is switched out for specialized TLS polymerases, a process thought to be mediated by the monoubiquitination of proliferating cell nuclear antigen (PCNA) [4]. During TLS, PCNA becomes monoubiquitinated, which recruits pol to the replication fork through pol’s ubiquitin-binding zinc finger (UBZ) and PCNA-interacting peptide (PIP) domains [13,14,15,16]. After UV irradiation, the monoubiquitination of pol is down-regulated to expose the PCNA-interacting surface, thereby stimulating pol’s interaction with PCNA and facilitating TLS [17]. S687 Phosphorylation of pol in UV Damage Tolerance man pol for further understanding the regulatory mechanisms of this important TLS polymerase
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