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      In vitro synthesis of uniform poly(dG)–poly(dC) by Klenow exo fragment of polymerase I

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          Abstract

          In this paper, we describe a production procedure of the one-to-one double helical complex of poly(dG)–poly(dC), characterized by a well-defined length (up to 10 kb) and narrow size distribution of molecules. Direct evidence of strands slippage during poly(dG)–poly(dC) synthesis by Klenow exo fragment of polymerase I is obtained by fluorescence resonance energy transfer (FRET). We show that the polymer extension results in an increase in the separation distance between fluorescent dyes attached to 5′ ends of the strands in time and, as a result, losing communication between the dyes via FRET. Analysis of the products of the early steps of the synthesis by high-performance liquid chromatography and mass spectroscopy suggest that only one nucleotide is added to each of the strand composing poly(dG)–poly(dC) in the elementary step of the polymer extension. We show that proper pairing of a base at the 3′ end of the primer strand with a base in sequence of the template strand is required for initiation of the synthesis. If the 3′ end nucleotide in either poly(dG) or poly(dC) strand is substituted for A, the polymer does not grow. Introduction of the T-nucleotide into the complementary strand to permit pairing with A-nucleotide results in the restoration of the synthesis. The data reported here correspond with a slippage model of replication, which includes the formation of loops on the 3′ ends of both strands composing poly(dG)–poly(dC) and their migration over long-molecular distances (μm) to 5′ ends of the strands.

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          Most cited references 40

          • Record: found
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          Fluorescence energy transfer as a spectroscopic ruler.

           L Stryer (1977)
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            Slippage synthesis of simple sequence DNA.

            The analysis of slippage synthesis of simple sequence DNA in vitro sheds some light on the question of how simple sequences arise in vivo. We show that it is possible to synthesize all types of repetitious di- and trinucleotide motifs starting from short primers and a polymerase in vitro. The rate of this synthesis depends on a sequence specific slippage rate, but is independent of the length of the fragments being synthesized. This indicates that only the ends of the DNA fragments are involved in determining this rate and that slippage is accordingly a short range effect. Slippage synthesis occurs also on a fixed template where only one strand is free to move, a situation which resembles chromosome replication in vivo. It seems therefore likely that slippage during replication is the cause of the observed length polymorphism of simple sequence stretches between individuals of a population.
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              G-quadruplex DNA structures--variations on a theme.

               T Simonsson (2001)
              To be functional, nucleic acids need to adopt particular three-dimensional structures. For a long time DNA was regarded as a rigid and passive molecule with the sole purpose to store genetic information, but experimental data has now accumulated that indicates the full dynamic repertoire of this macromolecule. During the last decade, four-stranded DNA structures known as G-quadruplexes, or DNA tetraplexes, have emerged as a three-dimensional structure of special interest. Motifs for the formation of G-quadruplex DNA structures are widely dispersed in eukaryotic genomes, and are abundant in regions of biological significance, for example, at telomeres, in the promoters of many important genes, and at recombination hotspots, to name but a few in man. Here I explore the plethora of G-quadruplex DNA structures, and discuss their possible biological functions as well as the proteins that interact with them.
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                Author and article information

                Journal
                Nucleic Acids Res
                Nucleic Acids Research
                Nucleic Acids Research
                Oxford University Press
                0305-1048
                1362-4962
                2005
                2005
                26 January 2005
                : 33
                : 2
                : 525-535
                Affiliations
                1Department of Biochemistry, George S. Wise Faculty of Life Sciences Ramat Aviv, 69978 Israel
                2Nanotechnology Center Ramat Aviv, 69978 Israel
                3School of Chemistry, Tel Aviv University Ramat Aviv, 69978 Israel
                Author notes
                *To whom correspondence should be addressed. Tel: +972 3 640 7138; Fax: +972 3 640 6834; Email: s2shak@ 123456post.tau.ac.il
                Article
                10.1093/nar/gki178
                548336
                15673713
                © The Author 2005. Published by Oxford University Press. All rights reserved

                The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the open access version of this article for non-commercial purposes provided that: the original authorship is properly and fully attributed; the Journal and Oxford University Press are attributed as the original place of publication with the correct citation details given; if an article is subsequently reproduced or disseminated not in its entirety but only in part or as a derivative work this must be clearly indicated. For commercial re-use, please contact journals.permissions@ 123456oupjournals.org .

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                Genetics

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