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Resistance to UV Irradiation Caused by Inactivation of nurA and herA Genes in Thermus thermophilus.

ABSTRACT: NurA and HerA are thought to be essential proteins for DNA end resection in archaeal homologous recombination systems. Thermus thermophilus, an extremely thermophilic eubacterium, has proteins that exhibit significant sequence similarity to archaeal NurA and HerA. To unveil the cellular function of NurA and HerA in T. thermophilus, we performed phenotypic analysis of disruptant mutants of nurA and herA with or without DNA-damaging agents. The nurA and herA genes were not essential for survival, and their deletion had no effect on cell growth and genome integrity. Unexpectedly, these disruptants of T. thermophilus showed increased resistance to UV irradiation and mitomycin C treatment. Further, these disruptants and the wild type displayed no difference in sensitivity to oxidative stress and a DNA replication inhibitor. T. thermophilus NurA had nuclease activity, and HerA had ATPase. The overexpression of loss-of-function mutants of nurA and herA in the respective disruptants showed no complementation, suggesting their enzymatic activities were involved in the UV sensitivity. In addition, T. thermophilus NurA and HerA interacted with each other in vitro and in vivo, forming a complex with 2:6 stoichiometry. These results suggest that the NurA-HerA complex has an architecture similar to that of archaeal counterparts but that it impairs, rather than promotes, the repair of photoproducts and DNA cross-links in T. thermophilus cells. This cellular function is distinctly different from that of archaeal NurA and HerA.IMPORTANCE Many nucleases and helicases are engaged in homologous recombination-mediated DNA repair. Previous in vitro analyses in archaea indicated that NurA and HerA are the recombination-related nuclease and helicase. However, their cellular function had not been fully understood, especially in bacterial cells. In this study, we performed in vivo analyses to address the cellular function of nurA and herA in an extremely thermophilic bacterium, Thermus thermophilus As a result, T. thermophilus NurA and HerA exhibited an interfering effect on the repair of several instances of DNA damage in the cell, which is in contrast to the results in archaea. This finding will facilitate our understanding of the diverse cellular functions of the recombination-related nucleases and helicases.

SUBMITTER: Fujii Y

PROVIDER: S-EPMC6060359 | biostudies-literature | 2018 Aug

REPOSITORIES: biostudies-literature

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Resistance to UV Irradiation Caused by Inactivation of nurA and herA Genes in Thermus thermophilus.

Fujii Yuki Y Inoue Masao M Fukui Kenji K Kuramitsu Seiki S Masui Ryoji R

Journal of bacteriology 20180725 16

NurA and HerA are thought to be essential proteins for DNA end resection in archaeal homologous recombination systems. Thermus thermophilus, an extremely thermophilic eubacterium, has proteins that exhibit significant sequence similarity to archaeal NurA and HerA. To unveil the cellular function of NurA and HerA in T. thermophilus, we performed phenotypic analysis of disruptant mutants of nurA and herA with or without DNA-damaging agents. The nurA and herA</ ...[more]

PMID: 29844033

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Project description:UnlabelledIn archaea, the NurA nuclease and HerA ATPase/helicase, together with the Mre11-Rad50 complex, function in 3' single-stranded DNA (ssDNA) end processing during homologous recombination (HR). However, bacterial homologs of NurA and HerA have not been characterized. From Deinococcus radiodurans, we identified the manganese-dependent 5'-to-3' ssDNA/double-stranded DNA (dsDNA) exonuclease/endonuclease NurA (DrNurA) and the ATPase HerA (DrHerA). These two proteins stimulated each other's activity through direct protein-protein interactions. The N-terminal HAS domain of DrHerA was the key domain for this interaction. Several critical residues of DrNurA and DrHerA were verified by site-directed mutational analysis. Temperature-dependent activity assays confirmed that the two proteins had mesophilic features, with optimum activity temperatures 10 °C to 15 °C higher than their optimum growth temperatures. Knocking out either nurA or herA affected cell proliferation by shortening the growth phase, especially for growth at a high temperature (37 °C). In addition, both mutant strains displayed almost 10-fold-reduced intermolecular recombination efficiency, indicating that DrNurA and DrHerA might be involved in homologous recombination in vivo. However, single- and double-gene deletions did not show significantly decreased radioresistance. Our results confirmed that the biochemical activities of bacterial NurA and HerA proteins were conserved with archaea. Our phenotypical results suggested that these proteins might have different functions in bacteria.ImportanceDeinococcus radiodurans NurA (DrNurA) was identified as a manganese-dependent 5'-to-3' ssDNA/dsDNA exonuclease/endonuclease, and Deinococcus radiodurans HerA (DrHerA) was identified as an ATPase. Physical interactions between DrNurA and DrHerA explained mutual stimulation of their activities. The N-terminal HAS domain on DrHerA was identified as the interaction domain. Several essential functional sites on DrNurA and DrHerA were characterized. Both DrHerA and DrNurA showed mesophilic biochemical features, with their optimum activity temperatures 10 °C to 15 °C higher than their optimum growth temperatures in vitro. Knockout of nurA or herA led to abnormal cell proliferation and reduced intermolecular recombination efficiency but no obvious effect on radioresistence.

Dataset Information

Resistance to UV Irradiation Caused by Inactivation of nurA and herA Genes in Thermus thermophilus.

Publications

Resistance to UV Irradiation Caused by Inactivation of <i>nurA</i> and <i>herA</i> Genes in Thermus thermophilus.

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