scholarly journals Blocked RecA Protein-mediated DNA Strand Exchange Reactions Are Reversed by the RuvA and RuvB Proteins

1995 ◽  
Vol 270 (33) ◽  
pp. 19473-19480 ◽  
Author(s):  
Lisa E. Iype ◽  
Ross B. Inman ◽  
Michael M. Cox
Keyword(s):  
Reca Protein ◽  
Strand Exchange ◽  
Exchange Reactions ◽  
Dna Strand Exchange ◽  
Dna Strand

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 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 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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