Bacteriophage P1 Ban protein is a hexameric DNA helicase that interacts with and substitutes for Escherichia coli DnaB.
Since the ban gene of bacteriophage P1 suppresses a number of conditionally lethal dnaB mutations in Escherichia coli, it was assumed that Ban protein is a DNA helicase (DnaB analogue) that can substitute for DnaB in the host replication machinery. We isolated and sequenced the ban gene, purified the product, and analysed the function of Ban protein in vitro and in vivo. Ban hydrolyses ATP, unwinds DNA and forms hexamers in the presence of ATP and magnesium ions. Since all existing conditionally lethal dnaB strains bear DnaB proteins that may interfere with the protein under study, we constructed a dnaB null strain by using a genetic set-up designed to provoke the conditional loss of the entire dnaB gene from E.coli cells. This novel tool was used to show that Ban restores the viability of cells that completely lack DnaB at 30 degrees C, but not at 42 degrees C. Surprisingly, growth was restored by the dnaB252 mutation at a temperature that is restrictive for ban and dnaB252 taken separately. This indicates that Ban and DnaB are able to interact in vivo. Complementary to these results, we demonstrate the formation of DnaB-Ban hetero-oligomers in vitro by ion exchange chromatography. We discuss the interaction of bacterial proteins and their phage-encoded analogues to fulfil functions that are essential to phage and host growth.
Lemonnier
Marc
M
Departamento de Microbiología Molecular, Centro de Investigaciones Biológicas (CSIC), Velázquez 144, 28006 Madrid, Spain. mlm@cib.csic.es
Ziegelin
Günter
G
Reick
Tobias
T
Muñoz Gómez
Ana
A
Díaz-Orejas
Ramón
R
Lanka
Erich
E
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D017101:Bacteriophage P1 / Q000201:enzymology / Q000235:genetics*; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004279:DNA, Viral / Q000737:chemistry / Q000235:genetics; D019281:Dimerization; D053488:DnaB Helicases; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D001426:Bacterial Proteins; D004279:DNA, Viral; D014764:Viral Proteins; D004265:DNA Helicases; D053488:DnaB Helicases; C017048:ban protein, Enterobacteria phage P1