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Regulation of the error-prone DNA polymerase Pol? by oncogenic signaling and its contribution to drug resistance.

ABSTRACT: The DNA polymerase Pol? plays a key role in translesion synthesis, an error-prone replication mechanism. Pol? is overexpressed in various tumor types. Here, we found that melanoma and lung and breast cancer cells experiencing stress from oncogene inhibition up-regulated the expression of Pol? and shifted its localization from the cytoplasm to the nucleus. This effect was phenocopied by inhibition of the kinase mTOR, by induction of ER stress, or by glucose deprivation. In unstressed cells, Pol? is continually transported out of the nucleus by exportin-1. Inhibiting exportin-1 or overexpressing Pol? increased the abundance of nuclear-localized Pol?, particularly in response to the BRAFV600E-targeted inhibitor vemurafenib, which decreased the cytotoxicity of the drug in BRAFV600E melanoma cells. These observations were analogous to how Escherichia coli encountering cell stress and nutrient deprivation can up-regulate and activate DinB/pol IV, the bacterial ortholog of Pol?, to induce mutagenesis that enables stress tolerance or escape. However, we found that the increased expression of Pol? was not excessively mutagenic, indicating that noncatalytic or other functions of Pol? could mediate its role in stress responses in mammalian cells. Repressing the expression or nuclear localization of Pol? might prevent drug resistance in some cancer cells.

SUBMITTER: Temprine K

PROVIDER: S-EPMC7428051 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Regulation of the error-prone DNA polymerase Polκ by oncogenic signaling and its contribution to drug resistance.

Temprine Kelsey K Campbell Nathaniel R NR Huang Richard R Langdon Erin M EM Simon-Vermot Theresa T Mehta Krisha K Clapp Averill A Chipman Mollie M White Richard M RM

Science signaling 20200428 629

The DNA polymerase Polκ plays a key role in translesion synthesis, an error-prone replication mechanism. Polκ is overexpressed in various tumor types. Here, we found that melanoma and lung and breast cancer cells experiencing stress from oncogene inhibition up-regulated the expression of Polκ and shifted its localization from the cytoplasm to the nucleus. This effect was phenocopied by inhibition of the kinase mTOR, by induction of ER stress, or by glucose deprivation. In unstressed cells, Polκ ...[more]

PMID: 32345725

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Project description:UnlabelledEscherichia coli's DNA polymerase IV (Pol IV/DinB), a member of the Y family of error-prone polymerases, is induced during the SOS response to DNA damage and is responsible for translesion bypass and adaptive (stress-induced) mutation. In this study, the localization of Pol IV after DNA damage was followed using fluorescent fusions. After exposure of E. coli to DNA-damaging agents, fluorescently tagged Pol IV localized to the nucleoid as foci. Stepwise photobleaching indicated ∼60% of the foci consisted of three Pol IV molecules, while ∼40% consisted of six Pol IV molecules. Fluorescently tagged Rep, a replication accessory DNA helicase, was recruited to the Pol IV foci after DNA damage, suggesting that the in vitro interaction between Rep and Pol IV reported previously also occurs in vivo. Fluorescently tagged RecA also formed foci after DNA damage, and Pol IV localized to them. To investigate if Pol IV localizes to double-strand breaks (DSBs), an I-SceI endonuclease-mediated DSB was introduced close to a fluorescently labeled LacO array on the chromosome. After DSB induction, Pol IV localized to the DSB site in ∼70% of SOS-induced cells. RecA also formed foci at the DSB sites, and Pol IV localized to the RecA foci. These results suggest that Pol IV interacts with RecA in vivo and is recruited to sites of DSBs to aid in the restoration of DNA replication.ImportanceDNA polymerase IV (Pol IV/DinB) is an error-prone DNA polymerase capable of bypassing DNA lesions and aiding in the restart of stalled replication forks. In this work, we demonstrate in vivo localization of fluorescently tagged Pol IV to the nucleoid after DNA damage and to DNA double-strand breaks. We show colocalization of Pol IV with two proteins: Rep DNA helicase, which participates in replication, and RecA, which catalyzes recombinational repair of stalled replication forks. Time course experiments suggest that Pol IV recruits Rep and that RecA recruits Pol IV. These findings provide in vivo evidence that Pol IV aids in maintaining genomic stability not only by bypassing DNA lesions but also by participating in the restoration of stalled replication forks.

Dataset Information

Regulation of the error-prone DNA polymerase Pol? by oncogenic signaling and its contribution to drug resistance.

Publications

Regulation of the error-prone DNA polymerase Polκ by oncogenic signaling and its contribution to drug resistance.

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