Author
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Lu, Shunwen |
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Submitted to: Scientific Reports
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 6/11/2014 Publication Date: 6/26/2014 Publication URL: http://handle.nal.usda.gov/10113/59603 Citation: Lu, S. 2014. Zn2+ blocks annealing of complementary single-stranded DNA in a sequence-selective manner. Scientific Reports. 4:5464. Available: http://www.nature.com/srep/2014/140626/srep05464/full/srep05464.html. Interpretive Summary: Zinc is the second most abundant trace element (after iron) essential for all living organisms. Dietary zinc can be taken up by the nucleus which accumulates more than one third (30-40%) of the total amount of intracellular zinc (existing as a divalent cation Zn2+). Within the nucleus, Zn2+ is mainly bound to proteins as structural components or catalytic cofactors, but can be released to free status by reactive oxygen species when the cells undergo oxidative stress. It is unknown yet if the nuclear free Zn2+ could affect annealing of complementary single-stranded (ss) DNA, a crucial step in DNA synthesis, repair and recombination. In this study, the author first developed a simple low-temperature EDTA-free agarose gel electrophoresis procedure and used DNA sequences of a wheat gene to demonstrate that Zn2+ blocks the annealing of complementary ssDNA under near-physiological conditions. It was characterized further that non-coding repetitive DNA common in human and wheat genomes are tolerant to Zn2+ blocking and actually able to provide annealing protection on adjacent Zn2+-sensitive coding-DNA. These findings suggest that Zn2+-ssDNA interaction might be among natural forces driving genome evolution. Further studies may provide new insights into the genetic variations in cereal crops, especially wheat whose genome contains >80% repetitive DNA, some of which is known to be associated with chromosomal rearrangements responsible for economically important traits such as disease resistance/susceptibility. Technical Abstract: A simple low-temperature EDTA-free agarose gel electrophoresis procedure (LTEAGE) coupled with UV-Vis spectrum and fluorescence quenching analyses was developed and the Zn2+-single-stranded (ss) DNA interaction was investigated under near-physiological conditions. It was found that Zn2+ blocked the annealing of complementary ssDNA at micromolar concentrations (50-250 µm) in a sequence-selective manner while other divalent metal ions such as Mg2+ and Mn2+ had no effects under the same conditions. UV-Vis spectrum analysis indicated that Zn2+ interacted with ssDNA with a preference for G+C-rich over A/T-rich simple repetitive sequences common in transposable elements in the genomes of humans/animals and plants such as wheat. Site-specific mutational analysis suggested that Zn2+ might stabilize CG/GC-like helices in the hairpin stems of targeted ssDNA thus preventing Watson-Crick base-pairing between complementary sequences. LTEAGE tests revealed that Zn2+ blocked the end-joining of double-stranded (ds) DNA breaks and prevented the renaturation of long stretches (>1 kb) of denatured dsDNA, in which the Zn2+-tolerant intronic DNA provided annealing protection on otherwise Zn2+-sensitive coding-DNA. These findings imply that the eukaryotic genomes might have recruited repetitive DNA to cope with Zn2+ stress arising coincidentally with the compartmentalization of otherwise cytoplasmic prokaryotic chromosomes into the nucleus, a key milestone in evolution of life. |
