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Characterization of EndoTT, a novel single-stranded DNA-specific endonuclease from Thermoanaerobacter tengcongensis.

ABSTRACT: EndoTT encoded by tte0829 of Thermoanaerobacter tengcongensis binds and cleaves single-stranded (ss) and damaged double-stranded (ds) DNA in vitro as well as binding dsDNA. In the presence of a low concentration of NaCl, EndoTT cleaved ss regions of damaged dsDNA efficiently but did not cleave DNA that was entirely ss or ds. At high concentrations of NaCl or MgCl(2) or ATP, there was also specific cleavage of ssDNA. This suggested a preference for ss/ds junctions to stimulate cleavage of the DNA substrates. EndoTT has six specific sites (a-f) in the oriC region (1-70 nt) of T. tengcongensis. Substitutions of nucleotides around site c prevented cleavage by EndoTT of both sites c and d, implying that the cleavage specificity may depend on both the nucleotide sequence and the secondary structure of the ssDNA. A C-terminal sub-fragment of EndoTT (residues 107-216) had both endonucleolytic and DNA-binding activity, whereas an N-terminal sub-fragment (residues 1-110) displayed only ssDNA-binding activity. Site-directed mutations showed that G(170), R(172) and G(177) are required for the endonuclease activity of EndoTT, but not for DNA-binding, whereas D(171), R(178) and G(189) are partially required for the DNA-binding activity.

SUBMITTER: Zhang S

PROVIDER: S-EPMC2887958 | biostudies-literature | 2010 Jun

REPOSITORIES: biostudies-literature

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Characterization of EndoTT, a novel single-stranded DNA-specific endonuclease from Thermoanaerobacter tengcongensis.

Zhang Shuli S Liu Bo B Yang Haihua H Tian Yuqing Y Liu Gang G Li Lei L Tan Huarong H

Nucleic acids research 20100219 11

EndoTT encoded by tte0829 of Thermoanaerobacter tengcongensis binds and cleaves single-stranded (ss) and damaged double-stranded (ds) DNA in vitro as well as binding dsDNA. In the presence of a low concentration of NaCl, EndoTT cleaved ss regions of damaged dsDNA efficiently but did not cleave DNA that was entirely ss or ds. At high concentrations of NaCl or MgCl(2) or ATP, there was also specific cleavage of ssDNA. This suggested a preference for ss/ds junctions to stimulate cleavage of the DNA ...[more]

PMID: 20172959

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Project description:BackgroundAlthough cold shock responses and the roles of cold shock proteins in microorganisms containing multiple cold shock protein genes have been well characterized, related studies on bacteria possessing a single cold shock protein gene have not been reported. Thermoanaerobacter tengcongensis MB4, a thermophile harboring only one known cold shock protein gene (TtescpC), can survive from 50° to 80 °C, but has poor natural competence under cold shock at 50 °C. We therefore examined cold shock responses and their effect on natural competence in this bacterium.ResultsThe transcriptomes of T. tengcongensis before and after cold shock were analyzed by RNA-seq and over 1200 differentially expressed genes were successfully identified. These genes were involved in a wide range of biological processes, including modulation of DNA replication, recombination, and repair; energy metabolism; production of cold shock protein; synthesis of branched amino acids and branched-chain fatty acids; and sporulation. RNA-seq analysis also suggested that T. tengcongensis initiates cell wall and membrane remodeling processes, flagellar assembly, and sporulation in response to low temperature. Expression profiles of TtecspC and failed attempts to produce a TtecspC knockout strain confirmed the essential role of TteCspC in the cold shock response, and also suggested a role of this protein in survival at optimum growth temperature. Repression of genes encoding ComEA and ComEC and low energy metabolism levels in cold-shocked cells are the likely basis of poor natural competence at low temperature.ConclusionOur study demonstrated changes in global gene expression under cold shock and identified several candidate genes related to cold shock in T. tengcongensis. At the same time, the relationship between cold shock response and poor natural competence at low temperature was preliminarily elucidated. These findings provide a foundation for future studies on genetic and molecular mechanisms associated with cold shock and acclimation at low temperature.

Dataset Information

Characterization of EndoTT, a novel single-stranded DNA-specific endonuclease from Thermoanaerobacter tengcongensis.

Publications

Characterization of EndoTT, a novel single-stranded DNA-specific endonuclease from Thermoanaerobacter tengcongensis.

Similar Datasets

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