Browsing by Author "SHIMAMOTO, N"

Now showing 1 - 1 of 1
  • No Thumbnail Available
    Item
    ROLE OF POLYMERIC FORMS OF THE BACTERIOPHAGE-PHI-X174 CODED GENE A-PROTEIN IN PHI-XRFI DNA CLEAVAGE
    (1979) IKEDA, JE; YUDELEVICH, A; SHIMAMOTO, N; HURWITZ, J
    Gene A of the .vphi.X174 genome codes for 2 proteins, A and A* of MW 60,000 and 35,000, respectively. The .vphi.X A* protein is formed from a natural internal initiator site within the A gene cistron while the .vphi.X A protein is the product of the entire A gene. These 2 proteins were purified to homogeneity as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Previous studies showed that the .vphi.X A protein is an endonuclease which specifically introduces a discontinuity in the A cistron of the viral strand of supertwisted .vphi.XRF[replicative form]I DNA. In addition to this activity, the .vphi.X A protein also causes relaxation of supertwisted .vphi.XRFI DNA and formation of a .vphi.XRFII DNA .cntdot. .vphi.X A protein complex which has a discontinuity in the A cistron of the viral strand. This isolatable complex supports DNA synthesis when supplemented with extracts of uninfected Escherichia coli which lack .vphi.X A protein and .vphi.XRFI DNA. The .vphi.XRFII DNA .cntdot. .vphi.X A protein complex can be attacked by exonuclease III but is not susceptible to attack by E. coli DNA polymerase I, indicating that the 5''-end of the complex is blocked. Attempts to seal the RFII structure generated from the .vphi.XRFII DNA .cntdot. .vphi.X A protein complex with [phage] T4 DNA ligase in the presence or absence of DNA polymerase were unsuccessful. The .vphi.X A protein does not act catalytically in the cleavage of .vphi.XRFI DNA. Under conditions leading to the quantitative cleavage of .vphi.XRFI DNA, the molar ratio of .vphi.XRFI DNA to added .vphi.X A protein was approximately 1:10. At this molar ratio, cross-linking experiments with dimethyl suberimidate yielded 10 distinct protein bands which were multiples of the monomeric .vphi.X A protein. In the absence of DNA or in the presence of inactive DNA (.vphi.XRFII DNA) no distinct protein bands above a trimer were detected. It is possible in vitro to form a .vphi.XRFII DNA .cntdot. .vphi.X A protein complex with wild-type .vphi.XRFI DNA (.vphi.X A gene+) and with .vphi.XRFI DNA isolated from E. coli (su+) infected with phage .vphi.X H90 (an am mutant in the .vphi.X A gene). Thus, in vitro, in contrast to in vivo studies, .vphi.X A protein is not a cis acting protein. The purified .vphi.X A* protein does not substitute for the .vphi.X A protein in in vitro replication of .vphi.XRFI DNA nor does it interfere with the action of the .vphi.X A protein which binds only to supertwisted .vphi.XRFI DNA. In contrast, the .vphi.X A* protein binds to all duplex DNA preparations tested. This property prevents nucleases of E. coli from hydrolyzing duplex DNA to small MW products.