极端嗜热古菌———芝田硫化叶菌DNA 连接酶的生化性质

极端嗜热古菌———芝田硫化叶菌 DNA 连接酶 (Ssh 连接酶 ) 的最适辅因子为 ATP ，在 dATP 存在时，该酶也能表现出较弱的连接活性 . ATP 或 dATP 都能够使该酶发生腺苷化，腺苷化的 Ssh 连接酶能够将腺苷基团转移至含切刻的 DNA 上 . 电泳迁移率改变实验表明， Ssh 连接酶能够结合双链 DNA ，且与含切刻及不含切刻的 DNA 结合的亲和力相同，但不结合单链 DNA. 酵母双杂交实验显示，硫磺矿硫化叶菌 ( 与芝田硫化叶菌亲缘关系很近 ) 的 DNA 连接酶，与该菌所含的 3 个增殖细胞核抗原 (PCNA) 同源蛋白中的一个 (PCNA-1) 有相互作用，而与另外 2 个同源蛋白 (PCNA-like 和 PCNA-2) 则无相互作用 . 在古菌中高度保守的 Sac10b 蛋白家族成员 Ssh10b 能够激活 Ssh 连接酶的活性，而硫化叶菌中的主要染色体蛋白——— 7 ku DNA 结合蛋白 (Ssh7) 则对该酶活性没有影响 .

Biochemical Properties of DNA Ligase From The Hyperthermophilic Archaeon Sulfolobus shibatae

DNA ligase from the hyperthermophilic crenarcheon Sulfolobus shibatae (Ssh ligase) was optimally active in the presence of ATP and partially active in the presence of dATP. The enzyme was adenylated by both ATP and dATP, and the adenylate moiety covalently linked to the active site of the ligase was transferable to nicked DNA. Electrophoretic gel mobility shift assays revealed that the enzyme bound a duplex DNA fragment with a nick and that without a nick with similar affinity, but displayed little affinity for single-stranded DNA. Sso ligase, a closely related homologue of Ssh ligase from Sulfolobus solfataricus, interacted with PCNA-1, one of the three PCNA homologues found in the organism, as detected by yeast two-hybrid assays. No interaction of the enzyme with the other two PCNA homologues (PCNA-like and PCNA-2) was detected. Ssh10b, a member of the highly conserved Sac10b protein family of Archaea, stimulated DNA ligation by Ssh ligase, whereas Ssh7, a major Sulfolobus chromatin protein showed no effect on the activity of the ligase.