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ClpR protein-like regulator specifically recognizes RecA protein-independent promoter motif and broadly regulates expression of DNA damage-inducible genes in mycobacteria.

ABSTRACT: The RecA-dependent DNA damage response pathway (SOS response) appears to be the major DNA repair mechanism in most bacteria, but it has been suggested that a RecA-independent mechanism is responsible for controlling expression of most damage-inducible DNA repair genes in Mycobacterium tuberculosis. The specific reparative responses and molecular mediators involved in the DNA repair mechanism remain largely unclear in this pathogen and its related species. In this study, a mycobacterial ClpR-like regulator, corresponding to Rv2745c in M. tuberculosis and to Ms2694 in M. smegmatis mc(2)155, was found to interact with the promoter regions of multiple damage-inducible DNA repair genes. Specific binding of the ClpR-like factor to the conserved RecA-independent promoter RecA-NDp motif was then confirmed using in vitro electrophoretic mobility shift assays as well as in vivo chromatin immunoprecipitation experiments. The ClpR knock-out experiments, in combination with quantitative real time PCR assays, demonstrated that the expression of these RecA-independent genes were significantly down-regulated in the mutant strain of M. smegmatis in response to a DNA-damaging agent compared with the wild type strain. Furthermore, the ClpR-like factor was shown to contribute to mycobacterial genomic stability. These results enhance our understanding of the function of the ClpR regulator and the regulatory mechanism of RecA-independent DNA repair in mycobacteria.

SUBMITTER: Wang Y

PROVIDER: S-EPMC3173126 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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ClpR protein-like regulator specifically recognizes RecA protein-independent promoter motif and broadly regulates expression of DNA damage-inducible genes in mycobacteria.

Wang Yi Y Huang Yuanxia Y Xue Chaolun C He Yang Y He Zheng-Guo ZG

The Journal of biological chemistry 20110719 36

The RecA-dependent DNA damage response pathway (SOS response) appears to be the major DNA repair mechanism in most bacteria, but it has been suggested that a RecA-independent mechanism is responsible for controlling expression of most damage-inducible DNA repair genes in Mycobacterium tuberculosis. The specific reparative responses and molecular mediators involved in the DNA repair mechanism remain largely unclear in this pathogen and its related species. In this study, a mycobacterial ClpR-like ...[more]

PMID: 21771781

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Project description:Anthrax toxin activator (AtxA) is the master virulence gene regulator of Bacillus anthracis It regulates genes on the chromosome as well as the pXO1 and pXO2 plasmids. It is not clear how AtxA regulates these genes, and direct binding of AtxA to its targets has not been shown. It has been previously suggested that AtxA and other proteins in the Mga/AtxA global transcriptional regulators family bind to the curvature of their DNA targets, although this has never been experimentally proven. Using electrophoretic mobility shift assays, we demonstrate that AtxA binds directly to the promoter region of pagA upstream of the RNA polymerase binding site. We also demonstrate that in vitro, CO2 appears to have no role in AtxA binding. However, phosphomimetic and phosphoablative substitutions in the phosphotransferase system (PTS) regulation domains (PRDs) do appear to influence AtxA binding and pagA regulation. In silico, in vitro, and in vivo analyses demonstrate that one of two hypothesized stem-loops located upstream of the RNA polymerase binding site in the pagA promoter region is important for AtxA binding in vitro and pagA regulation in vivo Our study clarifies the mechanism by which AtxA interacts with one of its targets.IMPORTANCE Anthrax toxin activator (AtxA) regulates the major virulence genes in Bacillus anthracis The bacterium produces the anthrax toxins, and understanding the mechanism of toxin production may facilitate the development of therapeutics for B. anthracis infection. Since the discovery of AtxA 25?years ago, the mechanism by which it regulates its targets has largely remained a mystery. Here, we provide evidence that AtxA binds to the promoter region of the pagA gene encoding the main central protective antigen (PA) component of the anthrax toxin. These data suggest that AtxA binding plays a direct role in gene regulation. Our work also assists in clarifying the role of CO2 in AtxA's gene regulation and provides more evidence for the role of AtxA phosphorylation in virulence gene regulation.

Dataset Information

ClpR protein-like regulator specifically recognizes RecA protein-independent promoter motif and broadly regulates expression of DNA damage-inducible genes in mycobacteria.

Publications

ClpR protein-like regulator specifically recognizes RecA protein-independent promoter motif and broadly regulates expression of DNA damage-inducible genes in mycobacteria.

Similar Datasets

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