Enhancement of recA protein-promoted DNA strand exchange activity by volume-occupying agents.

We have characterized an enzymatic activity from human cell nuclei which is capable of catalyzing strand exchange between homologous DNA sequences. The strand exchange activity was Mg2+ dependent and required ATP hydrolysis. In addition, it was capable of promoting reannealing of homologous DNA sequences and could form nucleoprotein networks in a fashion reminiscent of purified bacterial RecA protein. Using an in vitro recombination assay, we also showed that the strand exchange activity was biologically important. The factor(s) responsible for the activity has been partially purified.

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

Molecular Cell ◽

10.1016/s1097-2765(03)00188-6 ◽

2003 ◽

Vol 11 (5) ◽

pp. 1337-1347 ◽

Cited By ~ 278

Author(s):

Katsumi Morimatsu ◽

Stephen C Kowalczykowski

Keyword(s):

Reca Protein ◽

Strand Exchange ◽

Dna Strand Exchange ◽

Dna Strand ◽

Gapped Dna

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

Journal of Bacteriology ◽

10.1128/jb.00376-07 ◽

2007 ◽

Vol 189 (12) ◽

pp. 4502-4509 ◽

Cited By ~ 23

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

Molecular and Cellular Biology ◽

10.1128/mcb.25.11.4377-4387.2005 ◽

2005 ◽

Vol 25 (11) ◽

pp. 4377-4387 ◽

Cited By ~ 30

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.

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