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

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. In conclusion, our functional and structural analyses provide insight into the molecular mechanisms of polymerization of bacterial RecA as a helical nucleoprotein filament.

Authors:
ORCiD logo; ; ; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
INCT-FBN; National Institute of General Medical Sciences (NIGMS)
OSTI Identifier:
1347027
DOE Contract Number:
GM098885
Resource Type:
Journal Article
Resource Relation:
Journal Name: PLoS ONE; Journal Volume: 11; Journal Issue: 7
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Leite, Wellington C., Galvão, Carolina W., Saab, Sérgio C., Iulek, Jorge, Etto, Rafael M., Steffens, Maria B. R., Chitteni-Pattu, Sindhu, Stanage, Tyler, Keck, James L., Cox, Michael M., and Spies, Maria. Structural and Functional Studies of H. seropedicae RecA Protein – Insights into the Polymerization of RecA Protein as Nucleoprotein Filament. United States: N. p., 2016. Web. doi:10.1371/journal.pone.0159871.
Leite, Wellington C., Galvão, Carolina W., Saab, Sérgio C., Iulek, Jorge, Etto, Rafael M., Steffens, Maria B. R., Chitteni-Pattu, Sindhu, Stanage, Tyler, Keck, James L., Cox, Michael M., & Spies, Maria. Structural and Functional Studies of H. seropedicae RecA Protein – Insights into the Polymerization of RecA Protein as Nucleoprotein Filament. United States. doi:10.1371/journal.pone.0159871.
Leite, Wellington C., Galvão, Carolina W., Saab, Sérgio C., Iulek, Jorge, Etto, Rafael M., Steffens, Maria B. R., Chitteni-Pattu, Sindhu, Stanage, Tyler, Keck, James L., Cox, Michael M., and Spies, Maria. 2016. "Structural and Functional Studies of H. seropedicae RecA Protein – Insights into the Polymerization of RecA Protein as Nucleoprotein Filament". United States. doi:10.1371/journal.pone.0159871.
@article{osti_1347027,
title = {Structural and Functional Studies of H. seropedicae RecA Protein – Insights into the Polymerization of RecA Protein as Nucleoprotein Filament},
author = {Leite, Wellington C. and Galvão, Carolina W. and Saab, Sérgio C. and Iulek, Jorge and Etto, Rafael M. and Steffens, Maria B. R. and Chitteni-Pattu, Sindhu and Stanage, Tyler and Keck, James L. and Cox, Michael M. and Spies, Maria},
abstractNote = {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. In conclusion, our functional and structural analyses provide insight into the molecular mechanisms of polymerization of bacterial RecA as a helical nucleoprotein filament.},
doi = {10.1371/journal.pone.0159871},
journal = {PLoS ONE},
number = 7,
volume = 11,
place = {United States},
year = 2016,
month = 7
}
  • Light scattering has been used to monitor and distinguish between two types of aggregation reactions observed with the recA protein of Escherichia coli. These are (1) the cooperative binding of recA protein to ssDNA in a pathway leading to DNA strand exchange and (2) the formation of free filaments by recA protein in the absence of DNA. Free filament formation requires Mg/sup 2 +/, is very sensitive to ionic strength, and occurs in the absence of single-stranded DNA and RNA. Turbidity measurements indicate that free recA filaments exhibit properties consistent with rigid rods which are 1 micron or more inmore » length. A kinetically distinct nucleation step in free filament formation is observed under some conditions and becomes rate limiting at high pH. Ninety-degree light scattering was employed to measure binding of recA protein to ssDNA under conditions that either favor or block free filament formation. recA protein saturates ssDNA at a stoichiometric ratio of approximately four nucleotide residues per recA monomer. When free filament formation is blocked by various means, the apparent dissociation constant of the recA X ssDNA complex is approximately 10 nM. Under conditions in which free recA filaments form readily, however, the apparent dissociation constant increases to approximately 1 microM. This dramatic decrease in the observed affinity of recA protein for ssDNA under conditions that permit free filament formation does not reflect a change in the intrinsic affinity of recA protein for ssDNA. Instead, it provides evidence that free filament formation and ssDNA binding by recA protein are competing reactions.« less
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