RESUMO
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.
Assuntos
Proteínas de Bactérias/fisiologia , DNA Helicases/fisiologia , Replicação do DNA , DNA Bacteriano/biossíntese , Proteínas de Ligação a DNA/fisiologia , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Cromatografia de Afinidade , DNA Primase/metabolismo , DnaB Helicases , Escherichia coli/genética , Substâncias Macromoleculares , Mutagênese , Mutagênese Sítio-Dirigida , Mutação de Sentido Incorreto , Conformação de Ácido Nucleico , Conformação Proteica , Estrutura Terciária de Proteína , Técnicas do Sistema de Duplo-HíbridoRESUMO
The current models that have been proposed to explain the mechanism of replication termination are (i) passive arrest of a replication fork by the terminus (Ter) DNA-terminator protein complex that impedes the replication fork and the replicative helicase in a polar fashion and (ii) an active barrier model in which the Ter-terminator protein complex arrests a fork not only by DNA-protein interaction but also by mechanistically significant terminator protein-helicase interaction. Despite the existence of some evidence supporting in vitro interaction between the replication terminator protein (RTP) and DnaB helicase, there has been continuing debate in the literature questioning the validity of the protein-protein interaction model. The objective of the present work was two-fold: (i) to reexamine the question of RTP-DnaB interaction by additional techniques and different mutant forms of RTP, and (ii) to investigate if a common domain of RTP is involved in the arrest of both helicase and RNA polymerase. The results validate and confirm the RTP-DnaB interaction in vitro and suggest a critical role for this interaction in replication fork arrest. The results also show that the Tyr(33) residue of RTP plays a critical role both in the arrest of helicase and RNA polymerase.
