嗜热菌PIF1解旋酶的生化活性研究  

Biochemical Activity of PIF1 Helicase from Thermophilic Bacteria

作　　者：李丹[1] 李海红[1] 戴阳雪 刘娜女[1] 侯锡苗[1] 奚绪光[1] LI Dan;LI Hai-Hong;DAI Yang-Xue;LIU Na-Nv;HOU Xi-Miao;XI Xu-Guang(Department of Biochemistry and Molecular Biology,College of Life Sciences,Northwest A&F University,Yangling 712100,Shaanxi,China)

机构地区：[1]西北农林科技大学生命科学学院生物化学与分子生物学教研室,陕西杨凌712100

出　　处：《中国生物化学与分子生物学报》2021年第9期1220-1232,共13页Chinese Journal of Biochemistry and Molecular Biology

基　　金：国家自然科学基金项目(No.31870788)资助。

摘　　要：小整合频率1(petite integration frequency 1,PIF1)解旋酶广泛存在于生物体内,在核酸的代谢过程中发挥着重要作用。近年来,人们已经报道了多种PIF1解旋酶的生化活性及三维结构,但对极端环境下细菌的PIF1解旋酶的报道仍较少。本文利用多种生物化学与生物物理学技术,对黄石热脱硫弧菌PIF1(Ty.PIF1)解旋酶进行了多方面研究。通过原核表达纯化系统,获得了纯度90%以上,均一性好的Ty.PIF1蛋白。Ty.PIF1在溶液中为单体,分子量约为60 kD。Ty.PIF1具有很高的热稳定性,在65℃以下时,二级结构保持稳定,大于70℃时,二级结构才会发生改变。Ty.PIF1在体外最适解旋温度为45℃,并非黄石热脱硫弧菌生存的最适温度,预示着Ty.PIF1在体内发挥活性时,可能需要其他辅助因子的参与。Ty.PIF1的酶活力温度范围较宽,在20~55℃均具有解旋活性,在55℃能解旋预示了Ty.PIF1具有耐热特性。Ty.PIF1偏向于同含有单链的底物结合,但对单链长度有一定要求,其长度至少大于4 nt;Ty.PIF1也会较好地结合无单链尾链的G4结构,说明Ty.PIF1可能有专门结合G4结构的区域。Ty.PIF1能解开一系列带有5′-单链尾链的类似于复制中间体的底物,且对底物结构有一定的偏好性;Ty.PIF1也可以解开含有G4结构的底物,DNA-RNA杂交链、RNA-loop结构,预示着Ty.PIF1在生物体内有着多种生物学功能。Ty.PIF1解旋带有不同长度5′-单链尾链的双链底物时,尾链长度越长,解旋速率越快,预示着Ty.PIF1可能与Sc.PIF1一样有着较低的解旋持续性。本文将Ty.PIF1的生化活性与其它物种的PIF1解旋酶进行了比较,找出了其中的共性与差异,加深了人们对嗜热菌PIF1解旋酶的认识,为今后研究其它极端环境微生物的PIF1解旋酶提供了一些思路。Petite integration frequency 1(PIF1)helicases are ubiquitous enzymes which play vital roles in nearly all DNA metabolic processes.In recent years,the biochemical activity and three-dimensional structure of several PIF1 helicases have been reported,but there are few reports on the PIF1 helicase of bacteria living in extreme environments.In this paper,a series of biochemical and biophysical techniques were used to study the Thermodesulfovibrio yellowstonii PIF1(Ty.PIF1)helicase in many aspects.Ty.PIF1 was obtained with a purity of over 90%and good uniformity using the prokaryotic expression and purification system.Ty.PIF1 is a monomer with a calculated molecular weight of 60 kD in solution.Ty.PIF1 has high thermal stability.The secondary structure remains stable when the temperature is below 65℃,and the secondary structure changes only when the temperature is above 70℃.The optimal unwinding temperature of Ty.PIF1 in vitro is 45℃,which is not the optimal temperature for the survival of thermodesulfovibrio yellowstonii.It indicates that when Ty.PIF1 exerts its enzymatic activity in vivo,it may require the participation of other cofactors.Ty.PIF1 can exert unwinding activity in a wide temperature range(20-55℃),and the presence of enzyme activity at 55℃indicates that Ty.PIF1 has heat-resistant properties.Ty.PIF1 prefers to bind to substrates containing ssDNA,but there is certain requirement for the length of the ssDNA,which is at least 4 nt in length.Ty.PIF1 can also bind to the G4 structure without ssDNA tail,indicating that Ty.PIF1 may have special G4 structure binding area within its tertiary structure.Ty.PIF1 can unwind a series of substrates similar to replication intermediates with 5′-ssDNA tails,and has a certain preference for the structure of the substrate;Ty.PIF1 can also unwind substrates containing G4 structure,DNA-RNA hybrid chain and RNA-loop structure,suggesting that Ty.PIF1 has a variety of biological functions in organisms.When Ty.PIF1 unwinds double-stranded substrates with 5′-ssDNA tail

关 键 词：小整合频率1解旋酶 热稳定性 结合活性 解旋活性 底物偏好性 

分 类 号：Q71[生物学—分子生物学]