Unraveling the Mechanism of the Primosome Assembly Process

Abstract

E. Coli primosome is a multiprotein-DNA complex present at the crossroads of DNA replication and repair where it is used to restart stalled replication fork at the damage sites. The assembly and function of the primosome is controlled by synchronized action of seven proteins, PriA, PriB, DnaT, PriC, DnaB, DnaC, DnaG, and nucleic acid. The presence of two helicases: PriA and DnaB, ensures bidirectional mechanical translocation and/or unwinding while DnaG primase synthesizes oligoribonucleotide primers used by DNA polymerase III holoenzyme.In spite of intensive studies on primosome structure and function over the last two decades, many basic aspects of the primosome assembly process remain ambiguous. Fundamental characteristics, such as the stoichiometry and interactions within the primosome are not well understood. As the result, the molecular mechanism of the assembly process of the primosome is still lacking.Here, using fluorescence intensity, fluorescence anisotropy titration, fluorescence resonance energy transfer and analytical ultracentrifugation methods, we present direct quantitative analysis of the initial steps in primosome assembly, involving PriA, PriB and DnaT proteins and the minimal primosome assembly site (PAS). The obtained results provide a quantitative framework for initiation of primosome formation and elucidation of further steps in the assembly process.