Biology subjects

Harper, J. A.

Publications and source records attributed to Harper, J. A..

3 recordsLinked to original sources

bioRxiv2026

Transcription directs Holliday junction branch migration

During meiosis, genetic diversity arises from the resolution of branched DNA intermediates called Holliday junctions to create crossovers--sites of reciprocal exchange between parental chromosomes. Holliday junctions arise during the repair of Spo11-induced DNA breaks, yet the principles linking break formation, repair, and chromosome architecture remain unclear. Top3, a type IA topoisomerase, acts on Holliday junctions, but its spatiotemporal dynamics are unknown. Here, we map Top3 catalytic activity throughout meiotic prophase with strand specificity and nucleotide resolution. We identify a DNA sequence motif associated with catalysis and a pattern of activity around Spo11 hotspots that requires ongoing repair and Top3s helicase partner, Sgs1. Strikingly, Top3 activity shifts over time, influenced by transcription, cohesin, and the crossover factors Msh5 and Mer3, redistributing toward sites of convergent transcription--known locations of meiotic cohesin association. Upon prophase exit, as crossovers are resolved, Top3 activity subsides. Remarkably, maps of genome-wide recombination reveal that crossover resolution preferentially occurs at these same regions of convergent transcription. Collectively, we propose that Top3 coordinates the transcription-coupled movement of Holliday junctions from Spo11 hotspots towards cohesin-associated axis sites, whereupon crossover resolution occurs to ensure accurate meiotic chromosome segregation.

molecular biology↗

bioRxiv2026

Localised negative feedback shapes genome-wide patterning of meiotic DNA breaks

Genetic diversity within sexually reproducing species arises via the formation and repair of programmed DNA double-strand breaks (DSBs) created by the evolutionarily conserved topoisomerase-like enzyme, Spo11. Because DSBs threaten genome stability, their formation is tightly regulated in both space and time. In S. cerevisiae, Tel1, the orthologue of mammalian Ataxia Telangiectasia Mutated (ATM) kinase, suppresses nearby DSB formation through local inhibition known as DSB interference. However, whether such local inhibition reshapes the genome-wide DSB landscape remains unclear. Here, we develop a quantitative simulation framework to model how Tel1-mediated feedback shapes Spo11-DSB formation across the yeast genome. We demonstrate that innate chromosome-specific DSB patterns, when combined with interference, generate complex, population-level redistribution of DSBs. We define the spatial range over which interference propagates and provide evidence that this regulatory mechanism requires Tel1 recruitment to DSBs via Xrs2 and Tel1 kinase activity. Although the pro-DSB factor Rec114 contributes to DSB regulation, mutation of potential Rec114 phosphorylation sites indicates that it is not an essential target of Tel1. Together, these findings demonstrate how localised negative feedback can drive broad-scale, emergent patterning of a fundamental genome-modifying process, with the potential in meiosis to influence recombination initiation and, consequently, genetic variation across generations.

cell biology↗

bioRxiv2025

The Balance between B55α and Greatwall expression levels predicts sensitivity to Greatwall inhibition in cancer cells

The Greatwall kinase inhibits PP2A-B55 phosphatase activity during mitosis to stabilise critical Cdk1-driven mitotic phosphorylation. Although Greatwall represents a potential oncogene and prospective therapeutic target, our understanding of cellular and molecular consequences of chemical Greatwall inactivation remains limited. To address this, we introduce C-604, a highly selective Greatwall inhibitor, and characterise both immediate and long-term cellular responses to the chemical attenuation of Greatwall activity. We demonstrate that Greatwall inhibition causes systemic destabilisation of the mitotic phosphoproteome, premature mitotic exit and pleiotropic cellular pathologies. Importantly, we demonstrate that the cellular and molecular abnormalities linked to reduced Greatwall activity are specifically dependent on the B55 isoform rather than other B55 variants, underscoring PP2A-B55 phosphatases as key mediators of cytotoxic effects of Greatwall-targeting agents in human cells. Additionally, we show that sensitivity to Greatwall inhibition varies in different cell line models and that dependency on Greatwall activity reflects the balance between Greatwall and B55 expression levels. Our findings highlight Greatwall dependency as a cell-specific vulnerability and propose the B55-to-Greatwall expression ratio as a predictive biomarker of cellular responses to Greatwall-targeted therapeutics.

cell biology↗