Rad54 Oligomers Translocate and Cross-bridge Double-stranded DNA to Stimulate Synapsis

Bianco, Piero R.; Bradfield, Justin J.; Castanza, Lauren R.; Donnelly, Andrea N.

doi:10.1016/j.jmb.2007.09.052

Cited by 17 publications

(46 citation statements)

References 49 publications

(104 reference statements)

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“…These studies found that Rdh54 could translocates at a rate of approximately 80 bp s −1 . The motion was highly heterogeneous and included transient pauses and translocation direction reversals, in agreement with similar studies on Rad54 7,57. In addition, DNA loop release events were observed occasionally.…”

Section: Discussionsupporting

confidence: 88%

“…Single molecule assays of various functions of the recombinase RecA and the eukaryotic homolog Rad51 are presented in Sections 2.2 and 2.3 5,45-56. Observation of the eukaryotic Rad54 and Rdh54 translocation and DNA remodeling activity is discussed in Section 2.4 6,7,57,58. Finally, Section 2.5 summarizes experiments on the HJ specific motor complex RuvAB 59-62…”

Section: Discussionmentioning

confidence: 99%

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Single molecule studies of homologous recombination

Finkelstein

Greene

2008

Mol. BioSyst.

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Single molecule methods offer an unprecedented opportunity to examine complex macromolecular reactions that are obfuscated by ensemble averaging. The application of single molecule techniques to study DNA processing enzymes has revealed new mechanistic details that are unobtainable from bulk biochemical studies. Homologous DNA recombination is a multi-step pathway that is facilitated by numerous enzymes that must precisely and rapidly manipulate diverse DNA substrates to repair potentially lethal breaks in the DNA duplex. In this review, we present an overview of single molecule assays that have been developed to study key aspects of homologous recombination and discuss the unique information gleaned from these experiments.

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Section: Discussionsupporting

confidence: 88%

Section: Discussionmentioning

confidence: 99%

Single molecule studies of homologous recombination

Finkelstein

Greene

2008

Mol. BioSyst.

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“…Reactions were initiated by adding Dmc1 or Rad51 to the ssDNA and incubating the sample for 15 min, followed by incubation with Hop2-Mnd1 (5 min) and dsDNA (10 min). Reaction processing was performed as described previously (23) (Fig. 1 A).…”

Section: Coaggregate Assaymentioning

confidence: 99%

Hop2-Mnd1 Condenses DNA to Stimulate the Synapsis Phase of DNA Strand Exchange

Pezza

Camerini‐Otero

Bianco

2010

Biophysical Journal

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Hop2-Mnd1 is a meiotic recombination mediator that stimulates DNA strand invasion by both Dmc1 and Rad51. To understand the biochemical mechanism of this stimulation, we directly visualized the heterodimer acting on single molecules of duplex DNA using optical tweezers and video fluorescence microscopy. The results show that the Hop2-Mnd1 heterodimer efficiently condenses double-stranded DNA via formation of a bright spot or DNA condensate. The condensation of DNA is Hop2-Mnd1 concentration-dependent, reversible, and specific to the heterodimer, as neither Hop2 nor Mnd1 acting alone can facilitate this reaction. The results also show that the rate-limiting nucleation step of DNA condensation is overcome in the presence of divalent metal ions, with the following order of preference: Mn(2+)>Mg(2+)>Ca(2+). Hop2-Mnd1/Dmc1/single-stranded DNA nucleoprotein filaments also condense double-stranded DNA in a heterodimer concentration-dependent manner. Of importance, the concentration dependence parallels that seen in DNA strand exchange. We propose that rapid DNA condensation is a key factor in stimulating synapsis, whereas decondensation may facilitate the invasion step and/or the ensuing branch migration process.

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“…Rad54 can displace Rad51 protein from dsDNA, reposition nucleosomes, and stimulate DNA strand exchange. Recent single-molecule studies have peered into the behavior of both of these two motor proteins [23-26]. …”

Section: Mechanistic Insights Into Enzyme Function From Single-molecumentioning

confidence: 99%

Visualizing protein–DNA interactions at the single-molecule level

Hilario

Kowalczykowski

2010

Current Opinion in Chemical Biology

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SummaryRecent advancements in single-molecule methods have allowed researchers to directly observe proteins acting on their DNA targets in real-time. Single-molecule imaging of protein-DNA interactions permits detection of the dynamic behavior of individual complexes that otherwise would be obscured in ensemble experiments. The kinetics of these processes can be monitored directly, permitting identification of unique sub-populations or novel reaction intermediates. Innovative techniques have been developed to isolate and manipulate individual DNA or protein molecules, and to visualize their interactions. The actions of proteins that have been visualized include: duplex DNA unwinding, DNA degradation, DNA packaging, translocation on DNA, sliding, superhelical twisting, and DNA bending, extension, and condensation. These single-molecule studies have provided new insights into nearly all aspects of DNA metabolism. Here we focus primarily on recent advances in fluorescence imaging and mechanical detection of individual protein-DNA complexes, with emphasis on selected proteins involved in DNA recombination: DNA helicases, DNA translocases, and DNA strand exchange proteins.

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Rad54 Oligomers Translocate and Cross-bridge Double-stranded DNA to Stimulate Synapsis

Cited by 17 publications

References 49 publications

Single molecule studies of homologous recombination

Single molecule studies of homologous recombination

Hop2-Mnd1 Condenses DNA to Stimulate the Synapsis Phase of DNA Strand Exchange

Visualizing protein–DNA interactions at the single-molecule level

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