scholarly journals Swi5-Sfr1 protein stimulates Rad51-mediated DNA strand exchange reaction through organization of DNA bases in the presynaptic filament

2013 ◽  
Vol 42 (4) ◽  
pp. 2358-2365 ◽  
Author(s):  
Louise H. Fornander ◽  
Axelle Renodon-Cornière ◽  
Naoyuki Kuwabara ◽  
Kentaro Ito ◽  
Yasuhiro Tsutsui ◽  
...  
Keyword(s):  
Exchange Reaction ◽  
Linear Dichroism ◽  
Strand Exchange ◽  
Filament Axis ◽  
Double Stranded Dna ◽  
Dna Base ◽  
Dna Strand Exchange ◽  
Dna Strand ◽  
Base Orientation

Abstract The Swi5-Sfr1 heterodimer protein stimulates the Rad51-promoted DNA strand exchange reaction, a crucial step in homologous recombination. To clarify how this accessory protein acts on the strand exchange reaction, we have analyzed how the structure of the primary reaction intermediate, the Rad51/single-stranded DNA (ssDNA) complex filament formed in the presence of ATP, is affected by Swi5-Sfr1. Using flow linear dichroism spectroscopy, we observe that the nucleobases of the ssDNA are more perpendicularly aligned to the filament axis in the presence of Swi5-Sfr1, whereas the bases are more randomly oriented in the absence of Swi5-Sfr1. When using a modified version of the natural protein where the N-terminal part of Sfr1 is deleted, which has no affinity for DNA but maintained ability to stimulate the strand exchange reaction, we still observe the improved perpendicular DNA base orientation. This indicates that Swi5-Sfr1 exerts its activating effect through interaction with the Rad51 filament mainly and not with the DNA. We propose that the role of a coplanar alignment of nucleobases induced by Swi5-Sfr1 in the presynaptic Rad51/ssDNA complex is to facilitate the critical matching with an invading double-stranded DNA, hence stimulating the strand exchange reaction.

scholarly journals A novel pairing process promoted by Escherichia coli RecA protein: inverse DNA and RNA strand exchange

2000 ◽  
Vol 14 (6) ◽  
pp. 740-749
Author(s):  
Eugene N. Zaitsev ◽  
Stephen C. Kowalczykowski
Keyword(s):  
Escherichia Coli ◽  
Exchange Reaction ◽  
Reca Protein ◽  
Strand Exchange ◽  
Loop Formation ◽  
Dna And Rna ◽  
Double Stranded Dna ◽  
Dna Strand Exchange ◽  
Dna Strand ◽  
Recombination Reactions

Traditionally, recombination reactions promoted by RecA-like proteins initiate by forming a nucleoprotein filament on a single-stranded DNA (ssDNA), which then pairs with homologous double-stranded DNA (dsDNA). In this paper, we describe a novel pairing process that occurs in an unconventional manner: RecA protein polymerizes along dsDNA to form an active nucleoprotein filament that can pair and exchange strands with homologous ssDNA. Our results demonstrate that this “inverse” reaction is a unique, highly efficient DNA strand exchange reaction that is not due to redistribution of RecA protein from dsDNA to the homologous ssDNA partner. Finally, we demonstrate that the RecA protein–dsDNA filament can also pair and promote strand exchange with ssRNA. This inverse RNA strand exchange reaction is likely responsible for R-loop formation that is required for recombination-dependent DNA replication.

scholarly journals Opposing Roles for Two Molecular Forms of Replication Protein A in Rad51-Rad54-Mediated DNA Recombination in Plasmodium falciparum

mBio ◽  
2013 ◽  
Vol 4 (3) ◽  
Author(s):  
Anusha M. Gopalakrishnan ◽  
Nirbhay Kumar
Keyword(s):  
Dna Damage ◽  
Dna Repair ◽  
Antigenic Variation ◽  
Protein A ◽  
Dna Recombination ◽  
Strand Exchange ◽  
Content Type ◽  
Dna Strand Exchange ◽  
Dna Strand

ABSTRACT The bacterial RecA protein and its eukaryotic homologue Rad51 play a central role in the homologous DNA strand exchange reaction during recombination and DNA repair. Previously, our lab has shown that PfRad51, the Plasmodium falciparum homologue of Rad51, exhibited ATPase activity and promoted DNA strand exchange in vitro. In this study, we evaluated the catalytic functions of PfRad51 in the presence of putative interacting partners, especially P. falciparum homologues of Rad54 and replication protein A. PfRad54 accelerated PfRad51-mediated pairing between single-stranded DNA (ssDNA) and its homologous linear double-stranded DNA (dsDNA) in the presence of 0.5 mM CaCl2. We also present evidence that recombinant PfRPA1L protein serves the function of the bacterial homologue single-stranded binding protein (SSB) in initiating homologous pairing and strand exchange activity. More importantly, the function of PfRPA1L was negatively regulated in a dose-dependent manner by PfRPA1S, another RPA homologue in P. falciparum. Finally, we present in vivo evidence through comet assays for methyl methane sulfonate-induced DNA damage in malaria parasites and accompanying upregulation of PfRad51, PfRad54, PfRPA1L, and PfRPA1S at the level of transcript and protein needed to repair DNA damage. This study provides new insights into the role of putative Rad51-interacting proteins involved in homologous recombination and emphasizes the physiological role of DNA damage repair during the growth of parasites. IMPORTANCE Homologous recombination plays a major role in chromosomal rearrangement, and Rad51 protein, aided by several other proteins, plays a central role in DNA strand exchange reaction during recombination and DNA repair. This study reports on the characterization of the role of P. falciparum Rad51 in homologous strand exchange and DNA repair and evaluates the functional contribution of PfRad54 and PfRPA1 proteins. Data presented here provide mechanistic insights into DNA recombination and DNA damage repair mechanisms in this parasite. The importance of these research findings in future work will be to investigate if Rad51-dependent mechanisms are involved in chromosomal rearrangements during antigenic variation in P. falciparum. A prominent determinant of antigenic variation, the extraordinary ability of the parasite to rapidly change its surface molecules, is associated with var genes, and antigenic variation presents a major challenge to vaccine development.

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