Dataset Information

A Unique B-Family DNA Polymerase Facilitating Error-Prone DNA Damage Tolerance in Crenarchaeota.

ABSTRACT: Sulfolobus islandicus codes for four DNA polymerases: three are of the B-family (Dpo1, Dpo2, and Dpo3), and one is of the Y-family (Dpo4). Western analysis revealed that among the four polymerases, only Dpo2 exhibited DNA damage-inducible expression. To investigate how these DNA polymerases could contribute to DNA damage tolerance in S. islandicus, we conducted genetic analysis of their encoding genes in this archaeon. Plasmid-borne gene expression revealed that Dpo2 increases cell survival upon DNA damage at the expense of mutagenesis. Gene deletion studies showed although dpo1 is essential, the remaining three genes are dispensable. Furthermore, although Dpo4 functions in housekeeping translesion DNA synthesis (TLS), Dpo2, a B-family DNA polymerase once predicted to be inactive, functions as a damage-inducible TLS enzyme solely responsible for targeted mutagenesis, facilitating GC to AT/TA conversions in the process. Together, our data indicate that Dpo2 is the main DNA polymerase responsible for DNA damage tolerance and is the primary source of targeted mutagenesis. Given that crenarchaea encoding a Dpo2 also have a low-GC composition genome, the Dpo2-dependent DNA repair pathway may be conserved in this archaeal lineage.

SUBMITTER: Feng X

PROVIDER: S-EPMC7390963 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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A Unique B-Family DNA Polymerase Facilitating Error-Prone DNA Damage Tolerance in Crenarchaeota.

Feng Xu X Liu Xiaotong X Xu Ruyi R Zhao Ruiliang R Feng Wenqian W Liao Jianglan J Han Wenyuan W She Qunxin Q

Frontiers in microbiology 20200723

<i>Sulfolobus islandicus</i> codes for four DNA polymerases: three are of the B-family (Dpo1, Dpo2, and Dpo3), and one is of the Y-family (Dpo4). Western analysis revealed that among the four polymerases, only Dpo2 exhibited DNA damage-inducible expression. To investigate how these DNA polymerases could contribute to DNA damage tolerance in <i>S. islandicus</i>, we conducted genetic analysis of their encoding genes in this archaeon. Plasmid-borne gene expression revealed that Dpo2 increases cell ...[more]

PMID: 32793138

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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

A Unique B-Family DNA Polymerase Facilitating Error-Prone DNA Damage Tolerance in Crenarchaeota.

Publications

A Unique B-Family DNA Polymerase Facilitating Error-Prone DNA Damage Tolerance in Crenarchaeota.

Similar Datasets

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