ASSEMBLY OF A NUCLEOPROTEIN COMPLEX REQUIRED FOR DNA PACKAGING BY BACTERIOPHAGE-LAMBDA

A critical step in the assembly of bacteriophage lambda is the excisio n of a single genome from a concatemeric DNA precursor and insertion o f genomic DNA into an empty viral capsid. DNA packaging is mediated by the lambda proteins gpNu1 and epA, which form an enzyme complex known as terminase. Initiation of the packaging process requires assembly o f the terminase subunits onto cos, the a DNA packaging sequence, and n icking of the duplex, thus forming the 12-base-pair ''sticky'' ends of the mature genome. We have utilized gel-retardation techniques to exa mine the interaction of gpNu1, gpA, and terminase holoenzyme with DNA. Our data demonstrate that gpNu1 interacts specifically with cos-conta ining DNA, forming three gel-retarded complexes. Similarly, the larger gpA subunit binds to DNA, forming two complexes; however, this subuni t forms similar complexes with DNA substrates of random sequence. All of the nucleoprotein complexes examined are disrupted by elevated conc entrations of NaCl and we suggest that altered DNA binding is responsi ble for the extreme salt sensitivity of the endonuclease activity of t he enzyme [Tomka, M. A., & Catalano, C. E. (1993) J. Biol. Chem. 268, 3056-3065]. DNA binding by each subunit is strongly affected by the pr esence 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. Fina lly, we provide evidence that complex I, the first stable intermediate in the packaging pathway, is composed of the mature left genome end b ound to the terminase subunits and demonstrate that dissociation of th e complex is quite slow (t(1/2) > 8 h). The significance of these data with respect to terminase-mediated genome packaging is discussed.