Weiditch Sasha A, Seraphim Thiago V, Houry Walid A, Kanelis Voula
Department of Cell and Systems Biology, University of Toronto, Toronto, ON, Canada, M5S 3G5; Department of Chemical and Physical Sciences, University of Toronto Mississauga, Mississauga, ON, Canada, L5L 1C6.
Department of Biochemistry, University of Toronto, Toronto, Ontario, M5G 1M1, Canada.
Protein Expr Purif. 2019 Aug;160:45-55. doi: 10.1016/j.pep.2019.03.017. Epub 2019 Apr 4.
Packaging the viral genome in the head of double-stranded DNA viruses, such as bacteriophages, requires the activity of a terminase. The bacteriophage terminase consists of a small terminase subunit (TerS), which binds the viral DNA, and a large terminase subunit (TerL) that possesses the ATPase and nuclease activities for packaging the DNA in the phage head. Some phages require additional components for DNA packaging, such as the HNH endonuclease gp74 in the bacteriophage HK97. Gp74 enhances the activity of terminase-mediated digestion of the cohesive (cos) site that connects individual genomes in phage concatemeric DNA, a pre-requisite to DNA packaging, and this enhancement requires an intact HNH motif in gp74. Testing of whether gp74 alters the terminase DNA binding or enzymatic activities requires obtaining isolated samples of pure TerS and TerL, which has been challenging owing to the poor solubility of these proteins. To this end, we developed methods to obtain purified TerS and TerL proteins that are active. TerS is expressed solubly in E. coli as a fusion with SUMO, which can be removed during purification to yield a TerS nonamer (TerS). Homogenous samples of a TerL monomer are also obtained, but the homogeneity of the sample depends on the solution conditions, as seen for other terminases. DNA binding, ATPase, and nuclease assays demonstrate that our preparations of TerS and TerL are functional, and that they also function with gp74. Purified TerS and TerL enable studies into the molecular basis by which gp74 regulates terminase activity in phage maturation.
将病毒基因组包装到双链DNA病毒（如噬菌体）的头部需要末端酶的活性。噬菌体末端酶由一个结合病毒DNA的小末端酶亚基（TerS）和一个具有ATP酶和核酸酶活性以将DNA包装到噬菌体头部的大末端酶亚基（TerL）组成。一些噬菌体需要其他成分来进行DNA包装，例如噬菌体HK97中的HNH核酸内切酶gp74。Gp74增强了末端酶介导的对粘性（cos）位点的消化活性，该位点连接噬菌体串联体DNA中的各个基因组，这是DNA包装的先决条件，而这种增强需要gp74中完整的HNH基序。测试gp74是否改变末端酶的DNA结合或酶活性需要获得纯TerS和TerL的分离样品，由于这些蛋白质的溶解性较差，这一直具有挑战性。为此，我们开发了获得有活性的纯化TerS和TerL蛋白的方法。TerS在大肠杆菌中作为与SUMO的融合蛋白可溶表达，在纯化过程中可以去除SUMO以产生TerS九聚体（TerS）。也获得了TerL单体的均匀样品，但样品的均匀性取决于溶液条件，这与其他末端酶的情况相同。DNA结合、ATP酶和核酸酶测定表明，我们制备的TerS和TerL具有功能，并且它们也能与gp74一起发挥作用。纯化的TerS和TerL能够研究gp74在噬菌体成熟过程中调节末端酶活性的分子基础。
