Abstract
The nucleus of mammalian cells is compartmentalized by nuclear bodies such as nuclear speckles, however, involvement of nuclear bodies, especially nuclear speckles, in DNA repair has not been actively investigated. Here, our focused screen for nuclear speckle factors involved in homologous recombination (HR), which is a faithful DNA double-strand break (DSB) repair mechanism, identified transcription-related nuclear speckle factors as potential HR regulators. Among the top hits, we provide evidence showing that USP42, which is a hitherto unidentified nuclear speckles protein, promotes HR by facilitating BRCA1 recruitment to DSB sites and DNA-end resection. We further showed that USP42 localization to nuclear speckles is required for efficient HR. Furthermore, we established that USP42 interacts with DHX9, which possesses DNA–RNA helicase activity, and is required for efficient resolution of DSB-induced R-loop. In conclusion, our data propose a model in which USP42 facilitates BRCA1 loading to DSB sites, resolution of DSB-induced R-loop and preferential DSB repair by HR, indicating the importance of nuclear speckle-mediated regulation of DSB repair.
Highlights
Introduction GenomicDNA of mammalian cells does not distribute uniformly in the nucleus
To investigate whether nuclear speckles spatially contribute to proper double-strand break (DSB) responses, especially homologous recombination (HR), camptothecin (CPT)-induced phosphorylation of RPA2 on Ser[4] and Ser[8], which is thought to be an indicative of the early HR process, was examined in the presence of tubercidin, which induces the dispersion of some nuclear speckle factors, including SRSF1, SC35, and poly(A)+ RNA (Fig. 1a)[29]
To evaluate the effect of nuclear speckle factors on HR, we investigated the frequency of non-crossover gene conversion-mediated HR with a Direct-Repeat GFP (DR-GFP) assay by knocking down each nuclear speckle factors
Summary
Introduction GenomicDNA of mammalian cells does not distribute uniformly in the nucleus. The nucleus of mammalian cells contains various nuclear bodies, such as promyelocytic leukaemia bodies, Cajal bodies, speckles, paraspeckles, and nuclear speckles, which are membraneless structures that highly compartmentalize the nucleus[1,2]. These nuclear bodies play important roles in expressing genomic functions, including stress responses, messenger RNA (mRNA) splicing, and transcription[3], the involvement of these nuclear bodies in the regulation of DNA repair remains elusive. DSBs are repaired mainly by two mutually exclusive pathways: non-homologous end-joining (NHEJ) and homologous recombination (HR). We investigated the potential regulatory mechanism of HR mediated by nuclear speckles, which are functionally associated with transcription
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