Yang Q, Hanagan A, Catalano C E
Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Health Sciences Center, Denver 80262, USA.
Biochemistry. 1997 Mar 11;36(10):2744-52. doi: 10.1021/bi9622682.
A critical step in the assembly of bacteriophage lambda is the excision of a single genome from a concatemeric DNA precursor and insertion of genomic DNA into an empty viral capsid. DNA packaging is mediated by the lambda proteins gpNu1 and gpA, which form an enzyme complex known as terminase. Initiation of the packaging process requires assembly of the terminase subunits onto cos, the lambda DNA packaging sequence, and nicking of the duplex, thus forming the 12-base-pair "sticky" ends of the mature genome. We have utilized gel-retardation techniques to examine the interaction of gpNu1, gpA, and terminase holoenzyme with DNA. Our data demonstrate that gpNu1 interacts specifically with cos-containing DNA, forming three gel-retarded complexes. Similarly, the larger gpA subunit binds to DNA, forming two complexes; however, this subunit forms similar complexes with DNA substrates of random sequence. All of the nucleoprotein complexes examined are disrupted by elevated concentrations of NaCl and we suggest that altered DNA binding is responsible for the extreme salt sensitivity of the endonuclease activity of the enzyme [Tomka, M. A., & Catalano, C. E. (1993) J. Biol. Chem. 268, 3056-3065]. DNA binding by each subunit is strongly affected by the presence of the other, with 10- and 3-fold increases in the affinity of gpNu1 and gpA, respectively, for DNA. Moreover, our data suggest that the terminase subunits interact in solution prior to DNA binding. Finally, we provide evidence that complex I, the first stable intermediate in the packaging pathway, is composed of the mature left genome end bound to the terminase subunits and demonstrate that dissociation of the complex is quite slow (t1/2 > 8 h). The significance of these data with respect to terminase-mediated genome packaging is discussed.
噬菌体λ组装过程中的关键一步是从串联DNA前体中切除单个基因组，并将基因组DNA插入空的病毒衣壳中。DNA包装由λ蛋白gpNu1和gpA介导，它们形成一种称为末端酶的酶复合物。包装过程的启动需要将末端酶亚基组装到cos（λDNA包装序列）上，并切割双链，从而形成成熟基因组的12个碱基对的“粘性”末端。我们利用凝胶阻滞技术研究了gpNu1、gpA和末端酶全酶与DNA的相互作用。我们的数据表明，gpNu1与含cos的DNA特异性相互作用，形成三种凝胶阻滞复合物。同样，较大的gpA亚基与DNA结合，形成两种复合物；然而，该亚基与随机序列的DNA底物形成类似的复合物。所有检测的核蛋白复合物都被高浓度的NaCl破坏，我们认为DNA结合的改变是该酶内切酶活性对盐极度敏感的原因[Tomka，M.A.，& Catalano，C.E.（1993）J.Biol.Chem.268，3056 - 3065]。每个亚基与DNA的结合都受到另一个亚基的强烈影响，gpNu1和gpA对DNA的亲和力分别增加了10倍和3倍。此外，我们的数据表明，末端酶亚基在与DNA结合之前就在溶液中相互作用。最后，我们提供证据表明，复合物I是包装途径中的第一个稳定中间体，由与末端酶亚基结合的成熟基因组左端组成，并证明该复合物的解离非常缓慢（t1/2 > 8小时）。讨论了这些数据对于末端酶介导的基因组包装的意义。
