Crystal structure of the highly radiation-inducible DinB/YfiT superfamily protein DR0053 from Deinococcus radiodurans R1
Introduction
Deinococcus radiodurans is well known for its extreme resistance to γ-radiation, UV light, oxidative stress, desiccation, and other DNA-damaging agents such as mitomycin C (MMC) [[1], [2], [3], [4], [5]]. Over the decades, extensive studies have revealed that its resistance to multiple stresses results from the concerted actions of various physiological determinants and well-regulated molecular mechanisms [4]. D. radiodurans has diverse DNA repair systems which can efficiently repair DNA double-strand breaks [6,7]. In addition, D. radiodurans has developed efficient enzymatic (catalase, superoxide dismutase, and peroxidase) and non-enzymatic (deinoxanthin, bacillithiol, pyrroloquinoline-quinone, polyphosphate, and Mn2+-metabolite complexes) antioxidant systems to remove reactive oxygen species generated by radiation and desiccation [2,8,9].
Specific expansions of particular protein families are regarded as an important source of bacterial evolution that contributes to specific phenotypes and biological functions of the corresponding organisms [10,11]. For instance, autotrophic archaea possess the expanded ferredoxin family required for iron-dependent respiration, while Mycobacterium tuberculosis has a large number of lipolytic enzymes to degrade host-cell lipids which are used as precursors for its own metabolites and cell-wall components [11]. These findings suggest that protein family expansions are involved in unique metabolic pathways or biochemical mechanisms in relevant organisms.
In D. radiodurans, there are more than 20 specific expansions of protein families [11]. Among them, the largest expansion is the Nudix [nucleoside diphosphates linked to X (any moiety)] superfamily proteins which generally participate in the anti-mutagenic system, such as the removal of damaged macromolecules or preventing oxidative damage to DNA [12,13]. The second largest expansion is the DinB/YfiT-like putative metalloenzyme superfamily (hereafter named as DinB/YfiT protein) whose prototype is Bacillus subtilis DinB (DNA damage inducible gene) [5]. The DinB/YfiT proteins are reportedly induced in response to environmental stresses such as radiation or chemical mutagens. The function of this superfamily is not clear except for speculation based on three conserved His residues which indicates metal dependent enzymatic activities, such as hydrolases or transferases.
Analyses of transcriptome and proteome dynamics in D. radiodurans revealed that many genes and proteins involved in DNA repair, oxidative stress response, and of unknown function are induced after exposure to γ-irradiation [14,15]. The multifunctional DNA repair protein RecA, the single strand binding protein SSB, and several Deinococcus-specific ppr (pleiotropic protein promoting DNA repair) and ddr (DNA damage response) gene products such as PprA and DdrA∼D, are induced by γ-irradiation and are known to be responsible for radiation-resistance [[14], [15], [16], [17], [18]].
In this study, we investigated the expression profiles of 13 dinB genes under various stress conditions and also reported the crystal structure of DR0053, which had the highest gene expression after γ-irradiation and MMC treatment, with a zinc ion bound to its metal binding site.
Section snippets
qRT-PCR
RNA preparation and qRT-PCR were performed as previously described [19]. D. radiodurans R1 was cultivated in TGY medium (0.5% Tryptone, 0.3% Yeast extract, and 0.1% Glucose) at 303 K under standard condition. Cells were grown to log phase (OD600 ∼1.0) under normal or stress conditions (10 kGy γ-irradiation, 60 mM H2O2, and 5 μg/ml MMC for 1 h) according to the previous study [20]. Total RNA was purified using the RNeasy Mini kit (Qiagen) and RNase-free DNase (Qiagen) according to the
Expression profiles of 13 dinB genes under stress conditions
dinB gene expression is known to be induced in response to various environmental stresses. To examine the expression profiles of all 13 dinB genes in D. radiodurans, we performed qRT-PCR using total RNA isolated from wild-type D. radiodurans R1 that had been exposed to γ-radiation (10 kGy), MMC (5 μg/ml), or H2O2 (60 mM) (Fig. 1). The transcript level of dr0053 had increased ∼200-fold higher than the control after exposure to 10 kGy γ-radiation with 1 h of post-irradiation recovery (Fig. 1A).
Conflicts of interest
The authors have no conflicts of interest to report.
Acknowledgements
We thank the beamline staff at the BL-5C beamline, Pohang Light Source (PLS), Republic of Korea for support with data collection. This research was supported by the Nuclear R&D program of Ministry of Science and ICT (MSIT), Republic of Korea.
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