Biology subjects

Woodhouse, M. V.

Publications and source records attributed to Woodhouse, M. V..

3 recordsLinked to original sources

bioRxiv2025

The Eukaryotic homology search complex distorts donor DNA structure to probe for homology

Homologous recombination (HR) is a DNA double-strand break repair pathway that facilitates genetic exchange and protects damaged replication forks during DNA synthesis. As a template-based repair process, the successful repair of a double-strand break depends on locating suitable homology from a donor DNA sequence elsewhere in the genome. In eukaryotes, Rad51 catalyzes the homology search in coordination with the ATP-dependent motor protein Rad54. The mechanism by which these two proteins regulate forces on dsDNA substrates during homology search remains unknown. Here, we have utilized single-molecule magnetic tweezers and optical trapping methods to monitor remodeling of the DNA template during the homology search. We find that the activity of Rad51 and Rad54 remodels the donor DNA substrate to control the association and dissociation of Rad51-ssDNA filaments in the absence of DNA homology. This mechanism occurs through the application of both linear (tension) and rotational (torsion) forces on the donor DNA. Finally, failure of Rad54 to act processively disrupts target selection in vivo. This study provides a basic understanding of how motorized homology search manipulates the donor DNA during the search for a suitable repair template. Significance StatementHomologous recombination (HR) is a double-strand DNA break repair pathway that utilizes a template-based target search process to locate a suitable homologous DNA sequence in the genome, thereby initiating DNA repair. Called the homology search, in eukaryotes, this process is carried out by the RecA family member Rad51. During the homology search, Rad51 collaborates with the motor protein Rad54 to identify and interrogate homologous DNA sequences within the genome. In this study, we have measured the forces applied by the combination of Rad51 and Rad54 to the donor DNA duplex. These measurements reveal a coordinated effort by these motor proteins to remodel donor DNA to probe for homology, shedding new light on how template-based homology searches interrogate the DNA strands.

biochemistry↗

bioRxiv2024

Human Topoisomerase IIα Promotes Chromatin Condensation Via a Phase Transition

Topoisomerase II (topo II) enzymes are essential enzymes known to resolve topological entanglements during DNA processing. Curiously, while yeast expresses a single topo II, humans express two topo II isozymes, topo II and topo II{beta}, which share a similar catalytic domain but differ in their intrinsically disordered C-terminal domains (CTDs). During mitosis, topo II and condensin I constitute the most abundant chromosome scaffolding proteins essential for chromosome condensation. However, how topo II enables this function is poorly understood. Here, we discovered a new and functionally distinct role for human topo II - it condenses DNA and chromatin at a low topo II concentration (100 pM or less) during a polymer-collapse phase transition. The removal of the topo II CTDs effectively abolishes its condensation ability, indicating that the condensation is mediated by the CTDs. Although topo II{beta} can also perform condensation, it is about 4-fold less effective. During the condensation, topo II-DNA condensates form along DNA, working against a DNA tension of up to 1.5 pN, greater than that previously reported for yeast condensin. In addition, this condensation does not require ATP and thus is independent of topo IIs catalytic activity. We also found that condensation and catalysis can concurrently proceed with minimal mutual interference. Our findings suggest topo II may directly participate in chromosome condensation during mitosis.

biophysics↗

bioRxiv2024

The translocation activity of Rad54 reduces crossover outcomes during homologous recombination

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.

biochemistry↗