Host: The Japan Radiation Research Society
DNA replication stalls at lesions such as pyrimidine dimers in a template strand. DNA polymerases from hyperthermophilic archaea recognize the presence of template uracil and stall DNA synthesis. To analyze the recognition mechanism, the binding mode of DNA polymerase B1 of Sulfolobus solfataricus (Pol B1) to uracil-containing DNA was examined by gel-shift assay and atomic force microscopy (AFM). Pol B1 tightly and specifically bound to uracil-DNA and retarded the mobility of DNA on agarose gel. When primer/template DNA was incubated with Pol B1 plus dNTPs, the presence of template uracil significantly inhibited the formation of double-stranded DNA. In the AFM image, single-stranded and double-stranded DNAs were observed as sphere and linear forms, respectively. Omission of dNTPs and primer from the reaction completely blocked the formation of double-stranded DNA and inhibited the formation of intermediates where Pol B1 appeared to bind to uracil-DNA. These results suggest that Pol B1 more efficiently recognizes template uracil by processive sliding on template DNA rather by random diffusion-mediated mechanism. [J Radiat Res 44:410 (2003)]
