Kinetic characterization of the strand separation ("helicase") activity of the DNA packaging enzyme from bacteriophage lambda.
Kinetic characterization of the strand separation ("helicase") activity of the DNA packaging enzyme from bacteriophage lambda.
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来自 lambda 噬菌体的 DNA 包装酶的链分离（“解旋酶”）活性的动力学表征。
DOI：
10.1021/bi970689t
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发表时间：
1997
期刊：
影响因子：
--
通讯作者：
Catalano,CE
中科院分区：
文献类型：
--
作者：
Yang,Q;Catalano,CE
Bacteriophage λ is assembled from preformed viral capsids (proheads), tails, and genomes that are excised from a concatemeric DNA precursor. The enzyme responsible for insertion of the genome into the precapsid is known as terminase. This enzyme possesses site-specific endonuclease, ATPase, and DNA strand separation (“helicase”) catalytic activities, which work in concert to excise and package a single viral genome during phage assembly. We have previously characterized the endonuclease [Tomka, M. A., & Catalano, C. E. (1993)J. Biol. Chem. 268, 3056−3065] and ATPase [Tomka, M. A., & Catalano, C. E. (1993)Biochemistry 32, 11992−11997] catalytic activities of λ terminase and present here similar studies on the strand separation activity of the enzyme. Strand separation requires terminase, divalent metal, and adenosine nucleotides with a hydrolyzable β,γ-phosphate bond. Two apparent binding sites for ATP-mediated strand separation were identified, one of which appears to be distinct from the high- and low-affinity sites previously observed for ATP hydrolysis [Hwang, Y., Catalano, C. E., & Feiss, M. (1995)Biochemistry 35, 2796−2803]. Salt stimulates the reaction at low concentrations but is strongly inhibitory at elevated concentrations, presumably due to impaired DNA binding. The above results are identical with either a complex DNA mixture (a nicked, annealed DNA duplex in the presence of excess nonspecific DNA) or a purified DNA substrate; however, a kinetic analysis of the reaction revealed that the observed rate was ≈5-fold greater with the purified DNA substrate. Moreover, whileEscherichia coliintegration host factor (IHF) stimulates terminase-mediated strand separation with both substrates, the observed stimulation is more pronounced with the complex DNA mixture (10-fold rate increase) than the purified DNA substrate (5-fold rate increase). Our data are consistent with a model where IHF binding to the terminase assembly site forms a binary protein·DNA complex readily distinguishable from bulk DNA. The implications of these results to the process of DNA packaging in bacteriophage λ are discussed.
