Kiyonari Shinichi, Kamigochi Toru, Ishino Yoshizumi
Department of Genetic Resources Technology, Faculty of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka-shi, Fukuoka, 812-8581, Japan.
Extremophiles. 2007 Sep;11(5):675-84. doi: 10.1007/s00792-007-0083-0. Epub 2007 May 9.
Proliferating cell nuclear antigen (PCNA) is known as a DNA sliding clamp that acts as a platform for the assembly of enzymes involved in DNA replication and repair. Previously, it was reported that a crenarchaeal PCNA formed a heterotrimeric structure, and that each PCNA subunit has distinct binding specificity to PCNA-binding proteins. Here we describe the PCNA-binding properties of a DNA ligase from the hyperthermophilic crenarchaeon Aeropyrum pernix K1. Based on our findings on the Pyrococcus furiosus DNA ligase-PCNA interaction, we predicted that the aromatic residue, Phe132, in the DNA-binding domain of A. pernix DNA ligase (ApeLig) would play a critical role in binding to A. pernix PCNA (ApePCNA). Surface plasmon resonance analyses revealed that the ApeLig F132A mutant does not interact with an immobilized subunit of ApePCNA. Furthermore, we could not detect any stimulation of the ligation activity of the ApeLig F132A protein by ApePCNA in vitro. These results indicated that the phenylalanine, which is located in our predicted PCNA-binding region in ApeLig, has a critical role for the physical and functional interaction with ApePCNA.
增殖细胞核抗原（PCNA）是一种DNA滑动夹，作为参与DNA复制和修复的酶组装的平台。此前有报道称，泉古菌PCNA形成异源三聚体结构，且每个PCNA亚基对PCNA结合蛋白具有不同的结合特异性。在此，我们描述了嗜热泉古菌火球菌K1中一种DNA连接酶的PCNA结合特性。基于我们对嗜热栖热菌DNA连接酶与PCNA相互作用的研究结果，我们预测火球菌DNA连接酶（ApeLig）DNA结合结构域中的芳香族残基苯丙氨酸132（Phe132）在与火球菌PCNA（ApePCNA）结合中起关键作用。表面等离子体共振分析表明，ApeLig F132A突变体不与固定化的ApePCNA亚基相互作用。此外，我们在体外未检测到ApePCNA对ApeLig F132A蛋白连接活性的任何刺激。这些结果表明，位于我们预测的ApeLig中PCNA结合区域的苯丙氨酸在与ApePCNA的物理和功能相互作用中起关键作用。
