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Direct Observation of Enzymes Replicating DNA Using a Single-molecule DNA Stretching Assay
Published on: March 23, 2010
Mechanism of DNA elongation during de novo DNA synthesis
Xingguo Liang1, Tomohiro Kato, Hiroyuki Asanuma
1Department of Molecular Design and Engineering, Graduate School of Engineering, Nagoya University, Chikusa, Nagoya 464-8603, Japan. liang@mol.nagoya-u.ac.jp
Abstract:
Under isothermal conditions, short oligodeoxynucleotides (ODNs) were elongated to long DNA by Vent(exo(-)), a thermophilic DNA polymerase, in the presence of dNTPs. Short ODNs (14-28 nt) were designed to form hairpin structures based on the sequence we obtained from de novo DNA synthesis in the presence of restriction enzyme Tsp509I. As short as 14-nt-long DNA could be elongated to longer than 20000 nucleotides by Vent(exo(-)) at 65 degrees C in 1 h. The high efficiency of elongation at very low concentration (<1 nM) supported the THF-SPE (terminal hairpin formation and self-priming extension) mechanism we purposed for DNA elongation during de novo DNA synthesis. The hairpin structure forms at a DNA duplex end as a self-priming complex, followed by strand displacement extension to longer DNA. The highly efficient elongation attributes to the successive repetition of the process of THF-SPE.
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