Proc. Natl. Acad. Sci. USA Vol. 73, No. 8, pp. 2669-2673, August 1976 Biochemistry
Isolation and characterization of the protein coded by gene A of bacteriophage q5X174 DNA (replicative form DNA replication/superhelical DNA/endonuclease action)
JOH-E IKEDA, ARTURO YUDELEVICH, AND JERARD HURWITZ Department of Developmental Biology and Cancer, Division of Biological Sciences, Albert Einstein College of Medicine, Bronx, New York 10461
Contributed by Jerard Hurwitz, May 24, 1976
ABSTRACT Replication of 4X174 circular replicative form (RFI) DNA by extracts of Escherichia coli infected with bacteriophage kX174 (amber in gene A) requires the kX174 gene A product. This requirement has been used as an assay for the isolation of this protein. The gene A product (purified 4000-fold) caused relaxation of superhelical kX174 RFI and formation of discontinuities in the viral strand of 4X174 RFI uniquely situated in the A region of the genome, and yielded a complex after interacting with kX174 RFI that is active in replication of OX RFI.
Replication of single-stranded DNA (ssDNA) of bacteriophage 4X174 occurs in three stages (1): (a) ssDNA - RF, (b) RF RF, and (c) RF ssDNA, where RF is the replicative form. In stage (a), complementary strand formation depends on host proteins (2), and cell-free systems catalyzing this reaction have been developed (3, 4). In stage (b), RF replication requires, in addition to host proteins, an active form of kX174 gene A product.f In vivo, the A gene protein is a cis-acting endonuclease which results in formation of RFII with a discontinuity in only the viral strand of OX174 RF (6). The endonuclease action has been demonstrated in vitro with partially purified A protein preparations (7). We have described a cell-free system that catalyzes semiconservative replication of RF DNA (8). Extracts lacking A gene product fail to replicate RF, but DNA synthesis is restored when A protein is added. The latter finding has provided a complementation assay for the A protein. We report here the isolation of the A protein and show that it specifically relaxes superhelical OX RFI, forms a single-strand discontinuity in the viral strand of OX RFI uniquely in the A region of the DNA, and interacts with OX RFI to form a complex active in RF replication. MATERIALS AND METHODS Bacterial and Phage Strains. Escherichia coli H514 was obtained from Dr. T. Henry. E. coli HF 4704 was obtained from Dr. R. L. Sinsheimer. kX174 am3 (gene E) was from Dr. R. L. Sinsheimer and OX174 H90 (am in gene A), from Dr. M. Hayashi. Preparation of DNA and Proteins. Ammonium sulfate fractions from uninfected E. coli H514, fraction II from E. coli H514 infected with qX174 H90, and qX174 RFI [3H]DNA, X Abbreviations:
Isolation and characterization of the protein coded by gene A of bacteriophage q5X174 DNA (replicative form DNA replication/superhelical DNA/endonuclease action)
JOH-E IKEDA, ARTURO YUDELEVICH, AND JERARD HURWITZ Department of Developmental Biology and Cancer, Division of Biological Sciences, Albert Einstein College of Medicine, Bronx, New York 10461
Contributed by Jerard Hurwitz, May 24, 1976
ABSTRACT Replication of 4X174 circular replicative form (RFI) DNA by extracts of Escherichia coli infected with bacteriophage kX174 (amber in gene A) requires the kX174 gene A product. This requirement has been used as an assay for the isolation of this protein. The gene A product (purified 4000-fold) caused relaxation of superhelical kX174 RFI and formation of discontinuities in the viral strand of 4X174 RFI uniquely situated in the A region of the genome, and yielded a complex after interacting with kX174 RFI that is active in replication of OX RFI.
Replication of single-stranded DNA (ssDNA) of bacteriophage 4X174 occurs in three stages (1): (a) ssDNA - RF, (b) RF RF, and (c) RF ssDNA, where RF is the replicative form. In stage (a), complementary strand formation depends on host proteins (2), and cell-free systems catalyzing this reaction have been developed (3, 4). In stage (b), RF replication requires, in addition to host proteins, an active form of kX174 gene A product.f In vivo, the A gene protein is a cis-acting endonuclease which results in formation of RFII with a discontinuity in only the viral strand of OX174 RF (6). The endonuclease action has been demonstrated in vitro with partially purified A protein preparations (7). We have described a cell-free system that catalyzes semiconservative replication of RF DNA (8). Extracts lacking A gene product fail to replicate RF, but DNA synthesis is restored when A protein is added. The latter finding has provided a complementation assay for the A protein. We report here the isolation of the A protein and show that it specifically relaxes superhelical OX RFI, forms a single-strand discontinuity in the viral strand of OX RFI uniquely in the A region of the DNA, and interacts with OX RFI to form a complex active in RF replication. MATERIALS AND METHODS Bacterial and Phage Strains. Escherichia coli H514 was obtained from Dr. T. Henry. E. coli HF 4704 was obtained from Dr. R. L. Sinsheimer. kX174 am3 (gene E) was from Dr. R. L. Sinsheimer and OX174 H90 (am in gene A), from Dr. M. Hayashi. Preparation of DNA and Proteins. Ammonium sulfate fractions from uninfected E. coli H514, fraction II from E. coli H514 infected with qX174 H90, and qX174 RFI [3H]DNA, X Abbreviations:
