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Identification, cloning and characterization of a new DNA- binding protein from the hyperthermophilic methanogen Methanopyrus kandleri

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Pavlov,  N. A.
Department of Molecular Biology, MPI for biophysical chemistry, Max Planck Society;

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Cherny,  D. I.
Department of Molecular Biology, MPI for biophysical chemistry, Max Planck Society;

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Subramaniam,  V.
Department of Molecular Biology, MPI for biophysical chemistry, Max Planck Society;

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Citation

Pavlov, N. A., Cherny, D. I., Nazimov, I. V., Slesarev, A. I., & Subramaniam, V. (2002). Identification, cloning and characterization of a new DNA- binding protein from the hyperthermophilic methanogen Methanopyrus kandleri. Nucleic Acids Research, 30(3), 685-694. Retrieved from http://nar.oxfordjournals.org/content/30/3/685.full.pdf+html.


Cite as: http://hdl.handle.net/11858/00-001M-0000-0012-F449-4
Abstract
Three novel DNA-binding proteins with apparent molecular masses of 7, 10 and 30 kDa have been isolated from the hyperthermophilic methanogen Methanopyrus kandleri. The proteins were identified using a blot overlay assay that was modified to emulate the high ionic strength intracellular environment of M.kandleri proteins. A 7 kDa protein, named 7kMk, was cloned and expressed in Escherichia coli. As indicated by CD spectroscopy and computer-assisted structure prediction methods, 7kMk is a substantially alpha-helical protein possibly containing a short N-terminal beta-strand. According to analytical gel filtration chromatography and chemical crosslinking, 7kMk exists as a stable dimer, susceptible to further oligomerization. Electron microscopy showed that 7kMk bends DNA and also leads to the formation of loop-like structures of similar to43.5+/- 3.5 nm (136+/- 11 bp for B-form DNA) circumference. A topoisomerase relaxation assay demonstrated that looped DNA is negatively supercoiled under physiologically relevant conditions (high salt and temperature). A BLAST search did not yield 7kMk homologs at the amino acid sequence level, but based on a multiple alignment with ribbon-helix-helix (RHH) transcriptional regulators, fold features and self-association properties of 7kMk we hypothesize that it could be related to RHH proteins.