Mechanism of termination of DNA replication of Escherichia coli involves helicase-contrahelicase interaction.
Using yeast forward and reverse two-hybrid analyses, we have discovered that the replication terminator protein Tus of Escherichia coli physically interacts with DnaB helicase in vivo. We have confirmed this protein-protein interaction in vitro. We show further that replication termination involves protein-protein interaction between Tus and DnaB at a critical region of Tus protein, called the L1 loop. Several mutations located in the L1 loop region not only reduced the protein-protein interaction but also eliminated or reduced the ability of the mutant forms of Tus to arrest DnaB at a Ter site. At least one mutation, E49K, significantly reduced Tus-DnaB interaction and almost completely eliminated the contrahelicase activity of Tus protein in vitro without significantly reducing the affinity of the mutant form of Tus for Ter DNA, in comparison with the wild-type protein. The results, considered along with the crystal structure of Tus-Ter complex, not only elucidate further the mechanism of helicase arrest but also explain the molecular basis of polarity of replication fork arrest at Ter sites.
Mulugu
S
S
Department of Microbiology, Duke University Medical Center, Durham, NC 27710, USA.
Potnis
A
A
Shamsuzzaman
Taylor
J
J
Alexander
K
K
Bastia
D
D
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D002846:Chromatography, Affinity; D004265:DNA Helicases / Q000502:physiology*; D019915:DNA Primase / Q000378:metabolism; D004261:DNA Replication; D004269:DNA, Bacterial / Q000096:biosynthesis*; D004268:DNA-Binding Proteins / Q000502:physiology*; D053488:DnaB Helicases; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D020798:Two-Hybrid System Techniques
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D046911:Macromolecular Substances; C061910:tus protein, Bacteria; C489236:tus protein, E coli; D019915:DNA Primase; D004265:DNA Helicases; D053488:DnaB Helicases