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¹ Division of Genetics and Mutagenesis, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo, 158-8501 Japan
² Radiation Hazards Research Group, Research Center of Radiation Safety, National Institute of Radiological Sciences 4-9-1, Anagawa, Inage-ku, Chiba-shi, Chiba, 263-8555 Japan
³ Department of Food and Nutrition, Aobagakuen Junior College, 3-12-9 Setagaya, Setagaya-ku, Tokyo, 154-0017 Japan
⁴ Istituto di Biochimica delle Proteine, Consiglio Nazionale delle Ricerche, Via P. Castellino, 111. 80131-Napoli, Italy

Deamination of cytosine to uracil is a hydrolytic reaction that is greatly accelerated at high temperatures. The resulting uracil pairs with adenine during DNA replication, thereby inducing G:C to A:T transitions in the progeny. Interestingly, B-family DNA polymerases from hyperthermophilic Archaea recognize the presence of uracil in DNA and stall DNA synthesis. To better understand the recognition mechanism, the binding modes of DNA polymerase B1 of Sulfolobus solfataricus (Pol B1) to uracil-containing DNA were examined by gel mobility shift assays and atomic force microscopy. Although PolB1 per se specifically binds to uracil-containing single-stranded DNA, the binding efficiency was substantially enhanced by the initiation of DNA synthesis. Analysis by the atomic force microscopy showed a number of double-stranded DNA (dsDNA) in the products of DNA synthesis. The generation of ds DNA was significantly inhibited, however, by the presence of template uracil, and intermediates where monomeric forms of Pol B1 appeared to bind to uracil-containing DNA were observed. These results suggest that Pol B1 more efficiently recognizes uracil in DNA during DNA synthesis rather than during random diffusion in solution, and that single molecules of Pol B1 bind to template uracil and stall DNA synthesis.

^aPresent address: Department of Molecular Biotechnology, Hiroshima University, 1-3-1 Kagamiyama, Hiroshima 739-8530, Japan

^* Correspondence: E-mail: nohmi{at}nihs.go.jp