|Budget Amount *help
¥6,700,000 (Direct Cost : ¥6,700,000)
Fiscal Year 1990 : ¥1,000,000 (Direct Cost : ¥1,000,000)
Fiscal Year 1989 : ¥5,700,000 (Direct Cost : ¥5,700,000)
T3 phage DNA is synthesized as a concatemer in which untit length molecules are jointed together in a head-to-tail Fashion Through terminally redundant (TR) sequences. During head assembly, mature monomers are cut from the concatemer, in a manner tightly coupled to DNA packaging (DNA maturation), and translocated into the prohead with the aid of packaging proteins, gp18 and gp19. T3 phage can package and transduce plasmid DNA carrying DNA sequences necessary for DNA packaging (pac signal). The pac signal is composed of the target sequence for the cutting, TR and its flanking sequences from the concatemer, and the secondary origin of DNA replication, located at the right end of T3 genome. When linearized DNA carrying pac signal was incubated in a defined in vitro system for packaging T3 DNA, DNA was cleaved at the left end of the TR sequences when DNA is packaged left-ward, demonstrating that the cleavage corresponds to the termination cleavage, separating monomeric DNAs from a concatemer. From the results, it is concluded that the DNA maturation is composed of the initiation cleavage at the right end of the TR sequence, creating the DNA end required for initiation of DNA packaging, and the termination cleavage. The initiation cleavage is highly specific and therefore determines the packaging specificity. gp19 is an ATP binding protein. Mutations were introduced into the consensus ATP binding domains of gp19 and mutant proteins were overproduced, purified and characterized. A mutant protein was active in DNA packaging but defective in DNA cleavage although the target sequence for cleavage was recognized. The results indicate that gp19 plays a central role in DNA maturation.