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

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          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.

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          Most cited references29

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          OB(oligonucleotide/oligosaccharide binding)-fold: common structural and functional solution for non-homologous sequences.

          A Murzin (1993)
          A novel folding motif has been observed in four different proteins which bind oligonucleotides or oligosaccharides: staphylococcal nuclease, anticodon binding domain of asp-tRNA synthetase and B-subunits of heat-labile enterotoxin and verotoxin-1. The common fold of the four proteins, which we call the OB-fold, has a five-stranded beta-sheet coiled to form a closed beta-barrel. This barrel is capped by an alpha-helix located between the third and fourth strands. The barrel-helix frameworks can be superimposed with r.m.s. deviations of 1.4-2.2 A, but no similarities can be observed in the corresponding alignment of the four sequences. The nucleotide or sugar binding sites, known for three of the four proteins, are located in nearly the same position in each protein: on the side surface of the beta-barrel, where three loops come together. Here we describe the determinants of the OB-fold, based on an analysis of all four structures. These proposed determinants explain how very different sequences adopt the OB-fold. They also suggest a reinterpretation of the controversial structure of gene 5 ssDNA binding protein, which exhibits some topological and functional similarities with the OB-fold proteins.
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            OB-fold domains: a snapshot of the evolution of sequence, structure and function.

            V Arcus (2002)
            The OB-fold is found in all three kingdoms and is well represented in both sequence and structural databases. The OB-fold is a five-stranded closed beta barrel and the majority of OB-fold proteins use the same face for ligand binding or as an active site. Different OB-fold proteins use this 'fold-related binding face' to, variously, bind oligosaccharides, oligonucleotides, proteins, metal ions and catalytic substrates. Recently, a number of new structures with OB-folds have been reported that augment the variation seen for this set of proteins whilst conserving the characteristic fold and binding face. The conservation of fold and a functional binding face amongst many structures provides a model for investigating the evolutionary trajectory of sequence, structure and function.
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              Applications of network BLAST server.

              The sequence databases continue to grow at an extraordinary rate. Contributions come from both small laboratories and large-scale projects, such as the Merck EST project. This growth has placed new demands on computational sequence comparison tools such as BLAST. Even now it is no longer practical to evaluate some BLAST reports manually; it is necessary to filter the output by, for example, organism, source, or degree of annotation. The new network BLAST service makes such tools possible. It is also possible to present BLAST output in different formats, such as BLANCE. Perhaps most important of all, it becomes simple to call BLAST from another application, making it one step within an integrated system. This makes the automated preparation of sequence evaluations that include BLAST runs possible. In the near future we expect to see a number of applications that use the network BLAST interface to help molecular biologists search against a database that is growing not only in size but in biological richness.
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                Author and article information

                Journal
                Nucleic Acids Res
                nar
                nar
                Nucleic Acids Research
                Oxford University Press
                0305-1048
                1362-4962
                June 2010
                June 2010
                19 February 2010
                19 February 2010
                : 38
                : 11
                : 3709-3720
                Affiliations
                State Key Laboratory of Microbial Resources, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China
                Author notes
                *To whom correspondence should be addressed. Tel: +861064807467; Fax: +861064807461; Email: yanghh@ 123456sun.im.ac.cn
                Correspondence may also be addressed to Huarong Tan. Tel/Fax: +861064807461; Email: tanhr@ 123456sun.im.ac.cn

                The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.

                Article
                gkq085
                10.1093/nar/gkq085
                2887958
                20172959
                06012d7a-e6fa-453d-b554-55bcc946d748
                © The Author(s) 2010. Published by Oxford University Press.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 17 July 2009
                : 15 January 2010
                : 1 February 2010
                Categories
                Nucleic Acid Enzymes

                Genetics
                Genetics

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