The terminase of bacteriophage SPP1, constituted by a large (G2P) and a small (G1P) subunit, is essential for the initiation of DNA packaging. A hexa-histidine G2P (H6-G2P), which is functional in vivo, possesses endonuclease, ATPase, and double-stranded DNA binding activities. H6-G2P introduces a cut with preference at the 5-RCGG2CW-3 sequence. Distamycin A, which is a minor groove binder that mimics the architectural structure generated by G1P at pac, enhances the specific cut at both bona fide 5-CTATTGCGG2C-3 sequences within pacC of SPP1 and SF6 phages. H6-G2P hydrolyzes rATP or dATP to the corresponding rADP or dADP and P i . H6-G2P interacts with two discrete G1P domains (I and II). Full-length G1P and G1P⌬N62 (lacking domain I) stimulate 3.5-and 1.9-fold, respectively, the ATPase activity of H6-G2P. The results presented suggest that a DNA structure, artificially promoted by distamycin A or facilitated by the assembly of G1P at pacL and/or pacR, stimulates H6-G2P cleavage at both target sites within pacC. In the presence of two G1P decamers per H6-G2P monomer, the H6-G2P endonuclease is repressed, and the ATPase activity stimulated. Based on these results, we propose a model that can account for the role of terminase in headful packaging.Initiation of packaging of double-stranded viral DNA (dsDNA) 1 involves the specific interaction of the prohead with viral DNA in a process mediated by a phage-encoded terminase protein (1-4). The terminase enzymes are usually hetero-oligomers composed of a small and a large subunit. The role of the terminase small subunit is to specifically recognize the packaging initiation site (cos or pac) and to form a nucleoprotein structure that helps to position the terminase large subunit to cleave at the pac or cos site (1-6). Two general principles for packaging of concatemeric DNA into a virus head have been proposed. The first implies a site-specific packaging that shows some constraint in DNA size, in which the recognition sequence (termed cos in phage ) plays an important role in initiation and termination of packaging. This packaging process is wellcharacterized in coliphages , T3, and T7 (1-6). The second principle implies headful packaging, in which the packaging initiates at a specific site (termed pac in phage SPP1) but with the capacity of the prohead playing a predominant role in the termination step. Within the poorly characterized headful packaging mechanism, phages P1, P22, T4, and SPP1 are included (1, 3, 7).The terminase enzyme of Bacillus subtilis phages SPP1 and SF6 is composed of a small (G1P) and a large (G2P) subunit (8). The terminase initiates unidirectional DNA packaging from a concatemeric DNA substrate by binding to pac DNA, introducing a 1-bp staggered cut, within the 83-bp pacC subsite, and encapsidating the DNA from the cleaved end into an empty prohead until no more DNA can be inserted (headful) (8 -11). DNA packaging terminates when the DNA inside the prohead is separated from the concatemer by a cutting process (headful cut). The headful cleavage...
Initiation of headful packaging of SPP1 DNA concatemers involves the interaction of the terminase, G1P and G2P, and the portal protein, G6P. G1P, which specifically recognizes the non-adjacent pacL and pacR subsites and directs loading of G2P to pacC, interacts with G6P. G2P, which has endonuclease, DNA binding, and ATPase activities, interacts with G1P and does it transiently with G6P. The stoichiometry of G1P on the G1P⅐G2P complex promotes the transition from a G2P endonuclease to an ATPase. G6P does not alter the endonuclease activity of G2P. Both G1P and G6P, which do not have endogenous ATPase activity, synergistically enhance and modulate the ATPase activity of G2P. Based on these results, we propose a model in which the modulation of the ATPase and endonuclease activities of G2P accounts for the role of the terminase in headful packaging.Many cellular processes require the action of a biological motor protein that converts chemical energy into mechanical force or directional movement. Packaging of viral head-to-tail concatemeric dsDNA 1 into viruses involves the specific interaction of virus DNA with the pre-assembled procapsid and subsequent translocation of the former into the latter, by the action of a DNA translocase, to render a highly condensed structure (1-4). DNA translocases are molecular motor proteins that use the energy of nucleoside triphosphate hydrolysis to package concatemeric dsDNA onto an empty procapsid. This is a common feature shared by the DNA packaging machinery of many bacterial, pox, and herpes viruses. Two general modes for packaging of concatemeric dsDNA into the capsid of a bacterial virus (also termed bacteriophage or phage) have been proposed. The first implies a site-specific packaging in which the recognition sequence (termed cos in phage ) plays an important role in initiation and termination of DNA encapsidation. This packaging process, which generates unit-length encapsidated molecules, is well characterized in phages , T3, and T7 (1-4). The second mode implies headful packaging, in which the encapsidation initiates at a specific site in the genome (termed pac in phage SPP1), but with the capacity of the procapsid playing a predominant role in the termination step. The sequential headful packaging mechanism generates a heterogeneous population of terminally redundant and partially circularly permuted DNA molecules as in the cases of SPP1, P1, P22, or in the case of T4 whose DNA is totally permuted and terminally redundant (Refs. 1 and 3 and Fig. 1).Bacillus subtilis phage SPP1 replication results in the formation of large head-to-tail concatemeric dsDNA. To initiate DNA packaging, the terminase, composed of small G1P and large G2P subunits, recognizes and cleaves the concatemeric DNA within the pac site (see Refs. 5 and 6 and Fig. 1). The SPP1 packaging site is divided into three discrete subsites: pacL, defining the non-encapsidated or left DNA end, pacC, the cleavage site, and pacR, the encapsidated or right DNA end (Refs. 7 and 8 and Fig. 1A). The terminase, bound to...
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