Kitabayashi Masao, Nishiya Yoshiaki, Esaka Muneharu, Itakura Mitsuo, Imanaka Tadayuki
Tsuruga Institute of Biotechnology, Toyobo Co., Ltd., 10-24 Toyo-cho, Tsuruga, Fukui 914-0047, Japan.
Biosci Biotechnol Biochem. 2002 Oct;66(10):2194-200. doi: 10.1271/bbb.66.2194.
The gene encoding the proliferating cell nuclear antigen (PCNA), a sliding clamp of DNA polymerases, was cloned from an euryarchaeote, Thermococcus kodakaraensis KOD1. The PCNA homologue, designated Tk-PCNA, contained 249 amino acid residues with a calculated molecular mass of 28,200 Da and was 84.3% identical to that from Pyrococcus furiosus. Tk-PCNA was overexpressed in Escherichia coli and purified. This protein stimulated the primer extension abilities of the DNA polymerase from T. kodakaraensis KOD1 'KOD DNA polymerase'. The stimulatory effect of Tk-PCNA was observed when a circular DNA template was used and was equally effective on both circular and linear DNA. The Tk-PCNA improved the sensitivity of PCR without adverse effects on fidelity with the KOD DNA polymerase. This is the first report in which a replication-related factor worked on PCR.
编码增殖细胞核抗原（PCNA）的基因从广古菌科的嗜热栖热菌KOD1中克隆得到，PCNA是DNA聚合酶的滑动夹。PCNA同源物命名为Tk-PCNA，含有249个氨基酸残基，计算分子量为28200道尔顿，与激烈热球菌的PCNA同源性为84.3%。Tk-PCNA在大肠杆菌中过量表达并纯化。该蛋白刺激了嗜热栖热菌KOD1的DNA聚合酶（“KOD DNA聚合酶”）的引物延伸能力。当使用环状DNA模板时可观察到Tk-PCNA的刺激作用，对环状和线性DNA均有同等效果。Tk-PCNA提高了PCR的灵敏度，且对KOD DNA聚合酶的保真度无不利影响。这是关于复制相关因子作用于PCR的首次报道。
