scholarly journals Single-Molecule Insights into ATP-Dependent Conformational Dynamics of Nucleoprotein Filaments of Deinococcus radiodurans RecA

2020 ◽  
Vol 21 (19) ◽  
pp. 7389
Author(s):  
Aleksandr Alekseev ◽  
Galina Cherevatenko ◽  
Maksim Serdakov ◽  
Georgii Pobegalov ◽  
Alexander Yakimov ◽  
...  
Keyword(s):  
Dna Repair ◽  
Single Molecule ◽  
Deinococcus Radiodurans ◽  
Atp Hydrolysis ◽  
Conformational Dynamics ◽  
Strand Exchange ◽  
Single Stranded Dna ◽  
Dna Strand Exchange ◽  
High Conservation ◽  
Dna Strand

Deinococcus radiodurans (Dr) has one of the most robust DNA repair systems, which is capable of withstanding extreme doses of ionizing radiation and other sources of DNA damage. DrRecA, a central enzyme of recombinational DNA repair, is essential for extreme radioresistance. In the presence of ATP, DrRecA forms nucleoprotein filaments on DNA, similar to other bacterial RecA and eukaryotic DNA strand exchange proteins. However, DrRecA catalyzes DNA strand exchange in a unique reverse pathway. Here, we study the dynamics of DrRecA filaments formed on individual molecules of duplex and single-stranded DNA, and we follow conformational transitions triggered by ATP hydrolysis. Our results reveal that ATP hydrolysis promotes rapid DrRecA dissociation from duplex DNA, whereas on single-stranded DNA, DrRecA filaments interconvert between stretched and compressed conformations, which is a behavior shared by E. coli RecA and human Rad51. This indicates a high conservation of conformational switching in nucleoprotein filaments and suggests that additional factors might contribute to an inverse pathway of DrRecA strand exchange.

scholarly journals RecA Protein from the Extremely Radioresistant Bacterium Deinococcus radiodurans: Expression, Purification, and Characterization

2002 ◽  
Vol 184 (6) ◽  
pp. 1649-1660 ◽  
Author(s):  
Jong-Il Kim ◽  
Ajay K. Sharma ◽  
Stephen N. Abbott ◽  
Elizabeth A. Wood ◽  
David W. Dwyer ◽  
...  
Keyword(s):  
Deinococcus Radiodurans ◽  
Atp Hydrolysis ◽  
Reca Protein ◽  
Strand Exchange ◽  
Exchange Reactions ◽  
Ssdna Binding ◽  
E Coli ◽  
Dna Strand Exchange ◽  
Ssdna Binding Protein ◽  
Dna Strand

ABSTRACT The RecA protein of Deinococcus radiodurans (RecADr) is essential for the extreme radiation resistance of this organism. The RecADr protein has been cloned and expressed in Escherichia coli and purified from this host. In some respects, the RecADr protein and the E. coli RecA (RecAEc) proteins are close functional homologues. RecADr forms filaments on single-stranded DNA (ssDNA) that are similar to those formed by the RecAEc. The RecADr protein hydrolyzes ATP and dATP and promotes DNA strand exchange reactions. DNA strand exchange is greatly facilitated by the E. coli SSB protein. As is the case with the E. coli RecA protein, the use of dATP as a cofactor permits more facile displacement of bound SSB protein from ssDNA. However, there are important differences as well. The RecADr protein promotes ATP- and dATP-dependent reactions with distinctly different pH profiles. Although dATP is hydrolyzed at approximately the same rate at pHs 7.5 and 8.1, dATP supports an efficient DNA strand exchange only at pH 8.1. At both pHs, ATP supports efficient DNA strand exchange through heterologous insertions but dATP does not. Thus, dATP enhances the binding of RecADr protein to ssDNA and the displacement of ssDNA binding protein, but the hydrolysis of dATP is poorly coupled to DNA strand exchange. The RecADr protein thus may offer new insights into the role of ATP hydrolysis in the DNA strand exchange reactions promoted by the bacterial RecA proteins. In addition, the RecADr protein binds much better to duplex DNA than the RecAEc protein, binding preferentially to double-stranded DNA (dsDNA) even when ssDNA is present in the solutions. This may be of significance in the pathways for dsDNA break repair in Deinococcus.

scholarly journals Fission Yeast Rad51 and Dmc1, Two Efficient DNA Recombinases Forming Helical Nucleoprotein Filaments

2005 ◽  
Vol 25 (11) ◽  
pp. 4377-4387 ◽  
Author(s):  
Synthia Sauvageau ◽  
Alicja Z. Stasiak ◽  
Isabelle Banville ◽  
Mickaël Ploquin ◽  
Andrzej Stasiak ◽  
...  
Keyword(s):  
Reca Protein ◽  
Strand Break ◽  
Strand Exchange ◽  
Exchange Reactions ◽  
Single Stranded Dna ◽  
Strand Breaks ◽  
Lower Eukaryotes ◽  
Dna Double Strand Break ◽  
Dna Strand Exchange ◽  
Dna Strand

ABSTRACT Homologous recombination is important for the repair of double-strand breaks during meiosis. Eukaryotic cells require two homologs of Escherichia coli RecA protein, Rad51 and Dmc1, for meiotic recombination. To date, it is not clear, at the biochemical level, why two homologs of RecA are necessary during meiosis. To gain insight into this, we purified Schizosaccharomyces pombe Rad51 and Dmc1 to homogeneity. Purified Rad51 and Dmc1 form homo-oligomers, bind single-stranded DNA preferentially, and exhibit DNA-stimulated ATPase activity. Both Rad51 and Dmc1 promote the renaturation of complementary single-stranded DNA. Importantly, Rad51 and Dmc1 proteins catalyze ATP-dependent strand exchange reactions with homologous duplex DNA. Electron microscopy reveals that both S. pombe Rad51 and Dmc1 form nucleoprotein filaments. Rad51 formed helical nucleoprotein filaments on single-stranded DNA, whereas Dmc1 was found in two forms, as helical filaments and also as stacked rings. These results demonstrate that Rad51 and Dmc1 are both efficient recombinases in lower eukaryotes and reveal closer functional and structural similarities between the meiotic recombinase Dmc1 and Rad51. The DNA strand exchange activity of both Rad51 and Dmc1 is most likely critical for proper meiotic DNA double-strand break repair in lower eukaryotes.

scholarly journals Single-molecule analysis reveals the molecular bearing mechanism of DNA strand exchange by a serine recombinase

2011 ◽  
Vol 108 (18) ◽  
pp. 7419-7424 ◽  
Author(s):  
H. Bai ◽  
M. Sun ◽  
P. Ghosh ◽  
G. F. Hatfull ◽  
N. D. F. Grindley ◽  
...  
Keyword(s):  
Single Molecule ◽  
Strand Exchange ◽  
Serine Recombinase ◽  
Dna Strand Exchange ◽  
Dna Strand ◽  
Single Molecule Analysis

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.

High-affinity and undissociated capillary electrophoresis for DNA strand exchange analysis

2020 ◽  
Vol 56 (54) ◽  
pp. 7403-7406
Author(s):  
Fangzhi Yu ◽  
Zheng Yuan ◽  
Dapeng Zhang ◽  
Yan Liu ◽  
Qiang Zhao ◽  
...  
Keyword(s):  
Capillary Electrophoresis ◽  
Strand Exchange ◽  
Exchange Reactions ◽  
Single Stranded Dna ◽  
High Affinity ◽  
Dna Strand Exchange ◽  
Dna Strand

Undissociated binding of SSB to single-stranded DNA facilitates fast gel-free capillary electrophoresis analysis of fundamentally important DNA strand exchange reactions.

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