Structural and Functional Studies of H. seropedicae RecA Protein - Insights into the Polymerization of RecA Protein as Nucleoprotein Filament.

Wellington C Leite,Tyler Stanage,Carolina W Galvão,Rafael M Etto,Michael M Cox,Jorge Iulek,Maria B R Steffens,Sindhu Chitteni-Pattu,James L Keck,Sérgio C Saab,Maria Spies

doi:10.1371/journal.pone.0159871

Abstract

The bacterial RecA protein plays a role in the complex system of DNA damage repair. Here, we report the functional and structural characterization of the Herbaspirillum seropedicae RecA protein (HsRecA). HsRecA protein is more efficient at displacing SSB protein from ssDNA than Escherichia coli RecA protein. HsRecA also promotes DNA strand exchange more efficiently. The three dimensional structure of HsRecA-ADP/ATP complex has been solved to 1.7 Å resolution. HsRecA protein contains a small N-terminal domain, a central core ATPase domain and a large C-terminal domain, that are similar to homologous bacterial RecA proteins. Comparative structural analysis showed that the N-terminal polymerization motif of archaeal and eukaryotic RecA family proteins are also present in bacterial RecAs. Reconstruction of electrostatic potential from the hexameric structure of HsRecA-ADP/ATP revealed a high positive charge along the inner side, where ssDNA is bound inside the filament. The properties of this surface may explain the greater capacity of HsRecA protein to bind ssDNA, forming a contiguous nucleoprotein filament, displace SSB and promote DNA exchange relative to EcRecA. Our functional and structural analyses provide insight into the molecular mechanisms of polymerization of bacterial RecA as a helical nucleoprotein filament.

Highlights

The bacterial RecA protein plays a role in the complex system of DNA damage repair
RecA protein catalyzes strand exchange reaction between single-strand DNA and homologous double-strand DNA molecules, and induces the expression of DNA repair proteins in response to DNA damage through a regulatory network known as the SOS response [1,2,3,4,5,6,7,8]
To investigate how Herbaspirillum seropedicae RecA protein (HsRecA) dynamically interacts with single-strand DNA (ssDNA) the DNA-dependent ATPase activity of HsRecA was measured in a coupled spectrophotometric assay using circular ssDNA M13mp18, ATP, an ATP regeneration system in the presence or absence of E. coli single-stranded binding protein (SSB)

Summary

Introduction

The bacterial RecA protein plays a role in the complex system of DNA damage repair. RecA protein catalyzes strand exchange reaction between single-strand DNA (ssDNA) and homologous double-strand DNA (dsDNA) molecules, and induces the expression of DNA repair proteins in response to DNA damage through a regulatory network known as the SOS response [1,2,3,4,5,6,7,8].The Escherichia coli RecA protein (EcRecA) monomers bind onto DNA producing a righthanded helical nucleoprotein filament [9,10]. The bacterial RecA protein plays a role in the complex system of DNA damage repair. RecA protein catalyzes strand exchange reaction between single-strand DNA (ssDNA) and homologous double-strand DNA (dsDNA) molecules, and induces the expression of DNA repair proteins in response to DNA damage through a regulatory network known as the SOS response [1,2,3,4,5,6,7,8]. The Escherichia coli RecA protein (EcRecA) monomers bind onto DNA producing a righthanded helical nucleoprotein filament [9,10]. The EcRecA-ssDNA nucleoprotein filament represents the structural intermediate responsible for homology pairing to a donor dsDNA, and the RecA-dsDNA structure is an end product after the strand exchange reaction [11]

Methods

Results

Conclusion