Conserved Conformation of RecA Protein after Executing the DNA Strand-Exchange Reaction. A Site-Specific Linear Dichroism Structure Study†

Biochemistry ◽  
2006 ◽  
Vol 45 (37) ◽  
pp. 11172-11178 ◽  
Author(s):  
Karolin Frykholm ◽  
Katsumi Morimatsu ◽  
Bengt Nordén
Keyword(s):  
Exchange Reaction ◽  
Reca Protein ◽  
Linear Dichroism ◽  
Structure Study ◽  
Strand Exchange ◽  
Site Specific ◽  
Dna Strand Exchange ◽  
Dna Strand ◽  
A Site

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 RecFOR Proteins Load RecA Protein onto Gapped DNA to Accelerate DNA Strand Exchange

2003 ◽  
Vol 11 (5) ◽  
pp. 1337-1347 ◽  
Author(s):  
Katsumi Morimatsu ◽  
Stephen C Kowalczykowski
Keyword(s):  
Reca Protein ◽  
Strand Exchange ◽  
Dna Strand Exchange ◽  
Dna Strand ◽  
Gapped Dna

scholarly journals DNA Helicase Activity of PcrA Is Not Required for the Displacement of RecA Protein from DNA or Inhibition of RecA-Mediated Strand Exchange

2007 ◽  
Vol 189 (12) ◽  
pp. 4502-4509 ◽  
Author(s):  
Syam P. Anand ◽  
Haocheng Zheng ◽  
Piero R. Bianco ◽  
Sanford H. Leuba ◽  
Saleem A. Khan
Keyword(s):  
Dna Recombination ◽  
Reca Protein ◽  
Resonance Energy ◽  
Dna Helicase ◽  
Strand Exchange ◽  
Single Pair ◽  
Helicase Activity ◽  
Dna Strand Exchange ◽  
Dna Strand

ABSTRACT PcrA is a conserved DNA helicase present in all gram-positive bacteria. Bacteria lacking PcrA show high levels of recombination. Lethality induced by PcrA depletion can be overcome by suppressor mutations in the recombination genes recFOR. RecFOR proteins load RecA onto single-stranded DNA during recombination. Here we test whether an essential function of PcrA is to interfere with RecA-mediated DNA recombination in vitro. We demonstrate that PcrA can inhibit the RecA-mediated DNA strand exchange reaction in vitro. Furthermore, PcrA displaced RecA from RecA nucleoprotein filaments. Interestingly, helicase mutants of PcrA also displaced RecA from DNA and inhibited RecA-mediated DNA strand exchange. Employing a novel single-pair fluorescence resonance energy transfer-based assay, we demonstrate a lengthening of double-stranded DNA upon polymerization of RecA and show that PcrA and its helicase mutants can reverse this process. Our results show that the displacement of RecA from DNA by PcrA is not dependent on its translocase activity. Further, our results show that the helicase activity of PcrA, although not essential, might play a facilitatory role in the RecA displacement reaction.

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