Catalano C E, Tomka M A
School of Pharmacy, University of Colorado Health Sciences Center, Denver 80262, USA.
Biochemistry. 1995 Aug 8;34(31):10036-42. doi: 10.1021/bi00031a027.
One of the final steps in the assembly of bacteriophage lambda is the excision of a single genome from a concatemeric DNA precursor and insertion of this monomer into a preformed capsid. Terminase enzymes are common to all of the double-stranded DNA phages, and in lambda this enzyme is responsible for both excision of a genome monomer from the concatemer and its insertion into the pro-capsid. We have previously demonstrated that the endonuclease activity of lambda terminase (cos-cleavage) was stoichiometric with enzyme and postulated that this was due to formation of a stable, postcleavage enzyme.DNA intermediate (complex I) (Tomka & Catalano, 1993b). Bacteriophage lambda gpFI protein is required for efficient assembly of the virus, and current models suggest that this protein increases the rate of pro-capsid binding to complex I. We show here that gpFI markedly stimulates cos-cleavage by lambda terminase, even in the absence of viral pro-capsids. Importantly, the observed increase in nicking activity did not result from an increase in the rate of cos-cleavage, but rather by an increase in turnover by the enzyme. These data suggest that gpFI destabilizes complex I, thus allowing terminase release from cos and catalytic turnover by the enzyme. The implications of these results with respect to terminase assembly onto viral DNA, nicking of the duplex, and subsequent translocation during packaging are discussed.
噬菌体λ组装的最后步骤之一是从串联DNA前体中切除单个基因组，并将该单体插入预先形成的衣壳中。末端酶是所有双链DNA噬菌体共有的，在λ噬菌体中，这种酶负责从串联体中切除基因组单体并将其插入原衣壳。我们之前已经证明，λ噬菌体末端酶的内切核酸酶活性（cos切割）与酶呈化学计量关系，并推测这是由于形成了一种稳定的切割后酶-DNA中间体（复合体I）（托姆卡和卡塔拉诺，1993b）。噬菌体λ gpFI蛋白是病毒有效组装所必需的，目前的模型表明，这种蛋白提高了原衣壳与复合体I结合的速率。我们在此表明，即使在没有病毒原衣壳的情况下，gpFI也能显著刺激λ噬菌体末端酶的cos切割。重要的是，观察到的切口活性增加并非源于cos切割速率的提高，而是源于酶的周转增加。这些数据表明，gpFI使复合体I不稳定，从而使末端酶从cos释放并实现酶的催化周转。本文讨论了这些结果对于末端酶组装到病毒DNA上、双链切口以及包装过程中后续转位的意义。
