极端嗜热古菌——芝田硫化叶菌DNA连接酶的生化性质  

Biochemical Properties of DNA Ligase From The Hyperthermophilic Archaeon Sulfolobus shibatae

作　　者：赖小勤 黄力[1] 

机构地区：[1]中国科学院微生物研究所,微生物资源前期开发国家重点实验室,北京100080

出　　处：《生物化学与生物物理进展》2005年第2期122-128,共7页Progress In Biochemistry and Biophysics

基　　金：国家自然科学基金项目资助(39925001; 30030010);中国科学院知识创新工程资助项目(KSCX2-SW-112).~~

摘　　要：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.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.

关 键 词：古菌 硫化叶菌 DNA连接酶 DNA结合 Saclob家族蛋白 PCNA 

分 类 号：Q78[生物学—分子生物学]