Thapa, A. | Search bioRxiv

bioRxiv2026

Trans-branching of polyubiquitin chains orchestrates the DNA replication stress response

Polyubiquitin chain geometry dictates functional consequences of ubiquitylation. Although branched polyubiquitin chains are abundant in cells, little is known about their functions. Here we show that branching on the DNA replication factor PCNA, mediated by the ubiquitin-conjugating enzyme UBE2K and involving lysines 63 and 48 of ubiquitin, orchestrates the sequence of events in response to replication stress. By inducing VCP-dependent extraction of PCNA from chromatin, branching promotes re-priming of stalled forks and necessitates a BRCA1-dependent pathway of daughter-strand gap repair. Our study identifies hyper-accumulation of daughter-strand gaps as the mechanistic basis underlying the toxicity of inhibitors of the PCNA-specific isopeptidase, USP1, in BRCA1-deficient cells. Moreover, an unexpected preference of UBE2K to operate in trans suggests a general timing mechanism to organize hierarchies amongst ubiquitin signals.

molecular biology↗

bioRxiv2018

Brief Freezing Steps Lead to Robust Immunofluorescence in Drosophila Larval Brains

Drosophila melanogaster possess complex neuronal networks regulating sophisticated behavioral outputs that aid in studying the molecular mechanisms of neuronal function and neurodegenerative disease. Immunofluorescence (IF) techniques provide a way to visualize the spatiotemporal organization of these networks, permitting observation of their development, functional location, remodeling, and eventually - degradation. However, general immunostaining techniques do not always result in sufficient antibody penetration through the brain, and techniques used to enhance permeability can compromise structural integrity. We have found that freezing larval brains facilitates permeability with no apparent loss of antibody specificity or structural integrity. To demonstrate the advantage of this freezing technique, we compared results to two commonly used permeation methods: Detergent alone (Basic) and proteolytic degradation (Collagenase) techniques.\n\nSummaryHere we compare four different immunofluorescence techniques demonstrating that freezing Drosophila brains results in robust staining of small neurons in the larval brain without compromising structural integrity.

cell biology↗