Single Molecule Imaging of DNA–Protein Interactions Using DNA Curtains

Crickard, J. Brooks

doi:10.1007/978-1-0716-2847-8_10

Cited by 7 publications

(6 citation statements)

References 22 publications

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“…This indicates that they are defective in hydrolysis but still interact with Rad51. We next used DNA curtains (60, 61) to characterize the WT-Rad54, Rad54R272Q, and Rad54R272A activity. This single molecule approach allows us to measure the DNA binding, speed, and translocation distance of Rad54 molecules ( Supplemental Figure 5A ).…”

Section: Resultsmentioning

confidence: 99%

“…DNA substrates for DNA curtains experiments were made by attaching a biotinylated oligo to one end of the 50 kb Lambda phage genome and an oligo with a digoxygenin moiety on the other. This allowed double tethering of the DNA between chrome barriers and chrome pedestals, as previously described (60, 61). Flow cells were attached to a microfluidic system, and sample delivery was controlled using a syringe pump (KD Scientific).…”

Section: Methodsmentioning

confidence: 99%

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The translocation activity of Rad54 reduces crossover outcomes during homologous recombination

Sridalla,

Woodhouse,

et al. 2024

Preprint

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Homologous recombination (HR) is a template-based DNA double-strand break repair pathway that requires the selection of an appropriate DNA template for repair during the homology search stage of HR. Failure to execute the homology search quickly and efficiently can result in complex intermediates that generate genomic rearrangements, a hallmark of human cancers. Rad54 is an ATP dependent DNA motor protein that functions during the homology search by regulating the recombinase Rad51. How this regulation reduces genomic rearrangements is currently unknown. To better understand how Rad54 can prevent genomic rearrangements, we evaluated several amino acid mutations in Rad54 that were found in the COSMIC database. COSMIC is a collection of amino acid mutations identified in human cancers. These substitutions led to reduced Rad54 function and the discovery of a conserved motif in Rad54. Through genetic, biochemical, and single-molecule approaches, we show that disruption of this motif leads to failure in stabilizing early strand invasion intermediates, causing loss-of-heterozygosity rearrangements. Our study also suggests that the translocation rate of Rad54 is a determinant in balancing genetic exchange. This mechanism is likely fundamental to eukaryotic biology.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

The translocation activity of Rad54 reduces crossover outcomes during homologous recombination

Sridalla,

Woodhouse,

et al. 2024

Preprint

Self Cite

View full text Add to dashboard Cite

show abstract

“…DNA substrates for DNA curtains experiments were made by attaching a biotinylated oligo to one end of the 50 kb Lambda phage genome and an oligo with a digoxygenin moiety on the other. This allowed double tethering of the DNA between chrome barriers and chrome pedestals, as previously described ( 55 , 56 ). Flow cells were attached to a microfluidic system, and sample delivery was controlled using a syringe pump (KD Scientific).…”

Section: Methodsmentioning

confidence: 99%

The translocation activity of Rad54 reduces crossover outcomes during homologous recombination

Sridalla,

Woodhouse,

et al. 2024

Nucleic Acids Research

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Homologous recombination (HR) is a template-based DNA double-strand break repair pathway that requires the selection of an appropriate DNA sequence to facilitate repair. Selection occurs during a homology search that must be executed rapidly and with high fidelity. Failure to efficiently perform the homology search can result in complex intermediates that generate genomic rearrangements, a hallmark of human cancers. Rad54 is an ATP dependent DNA motor protein that functions during the homology search by regulating the recombinase Rad51. How this regulation reduces genomic exchanges is currently unknown. To better understand how Rad54 can reduce these outcomes, we evaluated several amino acid mutations in Rad54 that were identified in the COSMIC database. COSMIC is a collection of amino acid mutations identified in human cancers. These substitutions led to reduced Rad54 function and the discovery of a conserved motif in Rad54. Through genetic, biochemical and single-molecule approaches, we show that disruption of this motif leads to failure in stabilizing early strand invasion intermediates, causing increased crossovers between homologous chromosomes. Our study also suggests that the translocation rate of Rad54 is a determinant in balancing genetic exchange. The latch domain's conservation implies an interaction likely fundamental to eukaryotic biology.

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“…DNA substrates for DNA curtains experiments were made by attaching a biotinylated oligo to one end of the 50 kb Lambda phage genome, and an oligo with a digoxygenin moiety on the other. This allowed double tethering of the DNA between chrome barriers and chrome pedestals, as previously described (Collins et al, 2014;Crickard, 2023). Flow cells were attached to a microfluidic system and sample delivery was controlled using a syringe pump (KD Scientific).…”

Section: Single Molecule Experimentsmentioning

confidence: 99%

Rad53 regulates the lifetime of Rdh54 at homologous recombination intermediates

Ferlez

Dau

et al. 2023

Preprint

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Rdh54 is a conserved DNA translocase that participates in homologous recombination (HR), DNA checkpoint adaptation, and chromosome segregation. Saccharomyces cerevisiae Rdh54 is a known target of the Mec1/Rad53 signaling axis, which globally protects genome integrity during DNA metabolism. While phosphorylation of DNA repair proteins by Mec1/Rad53 is critical for HR progression little is known about how specific post translational modifications alter HR reactions. Phosphorylation of Rdh54 is linked to protection of genomic integrity but the consequences of modification remain poorly understood. Here, we demonstrate that phosphorylation of the Rdh54 C-terminus by the effector kinase Rad53 down regulates Rdh54 activity resulting in a 50% decrease in translocation velocity on dsDNA as revealed by single molecule imaging. Genetic assays reveal that loss of phosphorylation reduces interhomolog gene conversion, increases break induced replication, and decreases crossover outcomes. Our data highlight Rad53 as a key regulator of HR intermediates through activation and attenuation of Rdh54 motor function.

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Single Molecule Imaging of DNA–Protein Interactions Using DNA Curtains

Cited by 7 publications

References 22 publications

The translocation activity of Rad54 reduces crossover outcomes during homologous recombination

The translocation activity of Rad54 reduces crossover outcomes during homologous recombination

The translocation activity of Rad54 reduces crossover outcomes during homologous recombination

Rad53 regulates the lifetime of Rdh54 at homologous recombination intermediates

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