5 research outputs found

    Protease Activity of PprI Facilitates DNA Damage Response: Mn(2+)-Dependence and Substrate Sequence-Specificity of the Proteolytic Reaction

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    <div><p>The extremophilic bacterium <i>Deinococcus radiodurans</i> exhibits an extraordinary resistance to ionizing radiation. Previous studies established that a protein named PprI, which exists only in the <i>Deinococcus-Thermus</i> family, acts as a general switch to orchestrate the expression of a number of DNA damage response (DDR) proteins involved in cellular radio-resistance. Here we show that the regulatory mechanism of PprI depends on its Mn(2+)-dependent protease activity toward DdrO, a transcription factor that suppresses DDR genes’ expression. Recognition sequence-specificity around the PprI cleavage site is essential for DNA damage repair <i>in vivo</i>. PprI and DdrO mediate a novel DNA damage response pathway differing from the classic LexA-mediated SOS response system found in radiation-sensitive bacterium <i>Escherichia coli</i>. This PprI-mediated pathway in <i>D</i>. <i>radiodurans</i> is indispensable for its extreme radio-resistance and therefore its elucidation significantly advances our understanding of the DNA damage repair mechanism in this amazing organism.</p></div

    The uncleavable mutant strain MR109E.

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    <p>(A) The proteolytic product <i>in vitro</i> was assayed by western blotting. The total or N-terminal fragment of the cleaved substrate could be recognized by purified rabbit anti-DdrO polyclonal antibody. (B) A time-course-level of DdrO in the wild type R1 and YR1 mutant after gamma radiation assayed by western blotting. The first lane from left was the sample prior to ionizing radiation. The DdrO band of wild type R1 disappeared immediately after radiation and reappear one hour later, while the band of YR1 was constantly present. (C) A time-course-level of DdrO in the <i>pprI</i> complemented strain YR1-PprI and the <i>pprI</i> site-mutant YR1-PprI(H118L). (D) A time-course-level of DdrO in the wild type R1 and MR109E mutant after gamma radiation. The bands for MR109E remain unchanged after irradiation, similar to the result of YR1. (E) Survival rate of <i>D</i>. <i>radiodurans</i> under gamma radiation, (F) UV radiation and (G) H<sub>2</sub>O<sub>2</sub> stress. R1 (open rectangle), wild type strain; MR109E (open circular), <i>ddrO</i> uncleavable mutant strain; YR1 (open triangle), <i>pprI</i>-knockout strain; CMR109E (inverted triangle), strain MR109E with the pRADK containing of the wild type <i>ddrO</i> gene. Data are the mean of triplicate experiments (error bars indicate standard deviation [SD]).</p

    RDRM sites in the promoter regions were imperative for the binding of DdrO.

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    <p>The promoter regions (P<i>recA-</i>, P<i>ddrO-</i>, P<i>ddrB-</i>, P<i>pprA-</i>, P<i>ddrA-</i>, P<i>drssB-</i>, P<i>recQ-</i>) absent the RDRM sequence fail to bind DdrO.</p

    DdrO binds to the promoter regions of DDR genes <i>in vitro</i>.

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    <p>(A) Gel mobility shift assay using purified, tag removed DdrO and DDR gene promoter regions. The concentration of DNA substrates is 1.6 μM and the DdrO concentrations are 0, 6, 8, 12, and 16 μM, respectively. (B) Analysis of the interaction between DdrO and promoter regions. DNA band density on the GMSA gel was analyzed by Quantity One software. And the data represent the average of three independent experiments. (C) Detecting the DdrO binding capability of RDRM containing promoter regions. CK was a blank control without adding DdrO.</p