Mutations That Extend the Specificity of the Endonuclease Activity of λ Terminase

λ is a double-stranded DNA phage with a 48.5-kb genome. The packaging of λ DNA into a preformed empty shell, the prohead, requires the phage-encoded enzyme terminase. Terminase is a heteromultimer consisting of gpNu1 (181 residues) and gpA (641 residues). Both subunits contain primary sequences which are characteristic of ATPases (Fig. ​(Fig.1)1) (22, 25, 45), and kinetic studies demonstrate that the holoenzyme hydrolyzes ATP and dATP (23, 31, 44). It has been found that each of the terminase subunits possesses an ATPase activity (4, 37). gpNu1 contains a DNA-stimulated, low-affinity site with a Km of 469 μM and a kcat of 84 min−1. gpA contains a high-affinity site with a Km of 4.6 μM and a kcat of 38 min−1 (4, 30, 43). DNA lowers the Km and increases the kcat of the gpNu1 ATPase three- and twofold, respectively (43). FIG. 1 Linear map of domains in gpNu1 and gpA. Relative positions of mutations which affect the endonucleolytic activity of terminase are shown for gpA. ∗, locations of the A-E515G, A-N509K, and A-R504C mutations in the carboxy half of gpA; x, site of ... At its amino terminus, gpNu1 contains a putative helix-turn-helix (HTH) DNA binding motif which is thought to interact with cosB, the terminase binding site (Fig. ​(Fig.1)1) (18, 35). Adjacent to the HTH is the putative ATPase center. The carboxy half of gpNu1 has been shown to contain a domain for interaction with the amino-terminal 48 residues of gpA (21). In the C-terminal third of gpA are the putative ATPase center and a putative basic leucine zipper motif (17). The C-terminal 32 amino acids of gpA contain a specificity domain for interaction with the prohead (Fig. ​(Fig.1)1) (20, 47, 52). The packaging substrate is a concatemer, or end-to-end multimer, of λ chromosomes which is generated at late times after infection. The site containing the DNA packaging signals is called cos. cos consists of three segments, cosQ, cosN, and cosB (Fig. ​(Fig.2).2). The first subsite, cosQ, is required for termination of packaging (13). The second segment, cosN, is the site where terminase introduces staggered nicks to generate the 12-base cohesive ends of virion DNA. cosN contains a 16-bp segment showing partial twofold rotational symmetry; the right half-site is called cosNR, and the left half-site is called cosNL (Fig. ​(Fig.2).2). The twofold rotational symmetry of cosN suggests that symmetrically disposed gpA subunits are responsible for nicking cosNL and cosNR. The third segment, cosB, contains three binding sites for gpNu1, called R3, R2 and R1 (40), and a site, I1, for integration host factor (IHF), a DNA binding-bending protein of the host, Escherichia coli (2, 33, 48, 49). IHF introduces a sharp bend in cosB between R3 and R2 (Fig. ​(Fig.2)2) (33), and packaging of λ DNA is enhanced threefold by IHF (24, 49). It is believed that IHF facilitates cooperative interactions between terminase bound to R3 and terminases bound to R2 and R1. A second DNA binding-bending protein of the host, HU, has also been proposed to play a role in gpNu1-cos interactions (36). Adjacent to cos are the terminase genes Nu1 and A (Fig. ​(Fig.2).2). FIG. 2 The left end of the λ chromosome and expanded views of cosB and cosN. Nu1 and A, the terminase genes, lie downstream from cos. cos consists of three subsites, cosQ, cosN, and cosB. cosB contains the terminase recognition sequences R3, R2, and ... An initial binding step of terminase to λ DNA is followed by nicking at cosNR and cosNL, in that order. Nicking is stimulated by the presence of ATP (12, 29), but ATP hydrolysis is not required (29). Cue and Feiss (12) found that the presence of ATP does not affect the affinity of terminase for DNA and concluded that ATP increases the velocity of the nicking reaction. The first nick is on the bottom strand, at position N1 in cosNR, and the second nick is on the top strand, at N2 in cosNL (Fig. ​(Fig.2)2) (29). The nicked strands are then separated, a step which requires ATP hydrolysis (29, 39). After separation of the cohesive ends, a stable complex forms, called Complex I, which consists of terminase bound to the left end of the chromosome (3, 34, 41), presumably through gpNu1-cosB interactions (12, 40). Terminase of Complex I then binds a prohead to form Complex II, a DNA-terminase-prohead ternary complex, and packaging proceeds. Complex II formation is facilitated by gpFI of λ (5, 16). Mutations in cosN, specifically those in cosNL, affect the nicking activity of terminase; one example is the cosN G2C mutation (Fig. ​(Fig.2).2). cosN G2C reduces the burst size of λ 10-fold due to a defect in the cos cleavage step of packaging (51). The G2C and C11G mutations are rotationally symmetric (Fig. ​(Fig.2).2). The burst size of λ cosN G2C C11G is threefold less than that of λ cosN G2C (51). The cosN C11G mutation alone has no significant effect on the λ burst size or on cos cleavage in vitro. In vitro cos cleavage experiments of these and other rotationally symmetric mutants show that defects in the cosNL sequence are more detrimental to the cleavage activity of terminase than are defects in the cosNR sequence. λ cosN G2C and λ cosN G2C C11G mutants form minute plaques on an IHF+ host strain and do not form plaques on an IHF− strain. We describe here suppressors of the cosN G2C C11G defect that have been found in pseudorevertants of λ cosN G2C C11G.