Woodhouse, M. V. | bioRxiv Science

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↗