Kinetic analysis of the endonuclease activity of phage lambda terminase: assembly of a catalytically competent nicking complex is rate-limiting.
Kinetic analysis of the endonuclease activity of phage lambda terminase: assembly of a catalytically competent nicking complex is rate-limiting.
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噬菌体 lambda 终止酶核酸内切酶活性的动力学分析：具有催化能力的切口复合物的组装是限速的。
DOI：
10.1021/bi963044m
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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：
Woods,L;Terpening,C;Catalano,CE
The terminase enzyme from bacteriophage λ is responsible for excision of a single genome from a concatameric DNA precursor and its insertion into an empty viral procapsid. The enzyme possesses a site-specific endonuclease activity which is responsible for excision of the viral genome and the formation of the 12 base-pair single-stranded “sticky” ends of mature λ DNA. We have previously reported a kinetic analysis of the endonuclease activity of λ terminase which showed an enzyme concentration-dependent change in the kinetic time course of the reaction [Tomka, M. A., & Catalano, C. E. (1993b)J. Biol. Chem. 268, 3056−3065]. We presented a model which suggested that the rate-limiting step in the nuclease reaction was the assembly of a catalytically competent prenicking complex. Here, we provide additional evidence for a slow assembly step in the nuclease reaction and demonstrate that the observed rate is affected by protein concentration, but not by the length of the DNA substrate. Consistent with our model, preincubation of terminase with DNA also yields an observable fast phase of the reaction, but only when large (≥3 kb) DNA substrates are used. Finally, we present data which demonstrate that phage λ terminase can efficiently utilize DNA from the closely related phage ø21 as an endonuclease substrate and that the enzyme binds efficiently to thecosBregion of both phage genomes. The implications of these results with respect to the assembly of a catalytically competent nucleoprotein complex required to initiate genome packaging are discussed.
