RESUMEN
The Rad51/RecA family of recombinases perform a critical function in typical repair of double-strand breaks (DSBs): strand invasion of a resected DSB end into a homologous double-stranded DNA (dsDNA) template sequence to initiate repair. However, repair of a DSB using single stranded DNA (ssDNA) as a template, a common method of CRISPR/Cas9-mediated gene editing, is Rad51-independent. We have analyzed the genetic requirements for these Rad51-independent events in Saccharomyces cerevisiae by creating a DSB with the site-specific HO endonuclease and repairing the DSB with 80-nt single-stranded oligonucleotides (ssODNs), and confirmed these results by Cas9-mediated DSBs in combination with a bacterial retron system that produces ssDNA templates in vivo. We show that single strand template repair (SSTR), is dependent on Rad52, Rad59, Srs2 and the Mre11-Rad50-Xrs2 (MRX) complex, but unlike other Rad51-independent recombination events, independent of Rdh54. We show that Rad59 acts to alleviate the inhibition of Rad51 on Rad52's strand annealing activity both in SSTR and in single strand annealing (SSA). Gene editing is Rad51-dependent when double-stranded oligonucleotides of the same size and sequence are introduced as templates. The assimilation of mismatches during gene editing is dependent on the activity of Msh2, which acts very differently on the 3' side of the ssODN which can anneal directly to the resected DSB end compared to the 5' end. In addition DNA polymerase Polδ's 3' to 5' proofreading activity frequently excises a mismatch very close to the 3' end of the template. We further report that SSTR is accompanied by as much as a 600-fold increase in mutations in regions adjacent to the sequences directly undergoing repair. These DNA polymerase ζ-dependent mutations may compromise the accuracy of gene editing.
Asunto(s)
Sistemas CRISPR-Cas/genética , Reparación del ADN/genética , ADN de Cadena Simple/genética , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Endonucleasas/genética , Proteínas de Saccharomyces cerevisiae/genética , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , ADN Polimerasa Dirigida por ADN/genética , Endodesoxirribonucleasas/genética , Exodesoxirribonucleasas/genética , Oligonucleótidos/genética , Recombinasa Rad51/genética , Proteína Recombinante y Reparadora de ADN Rad52/genética , Rec A Recombinasas/genética , Saccharomyces cerevisiae/genética , ADN Polimerasa thetaRESUMEN
Doxorubicin (DXR) is a widely used chemotherapy drug that can induce severe intestinal mucositis. While the influence of gut bacteria on DXR-induced damage has been documented, the role of eukaryotic commensals remains unexplored. We discovered Tritrichomonas muris ( Tmu ) in one of our mouse colonies exhibiting abnormal tuft cell hyperplasia, prompting an investigation into its impact on DXR-induced intestinal injury. Mice from Tmu -colonized and Tmu -excluded facilities were injected with DXR, and tissue morphology and gene expression were evaluated at acute injury (6 h) and peak regeneration (120 h) phases. Contrary to previous reports, DXR did not significantly alter villus height, crypt depth, or crypt density in any mice. However, we did observe apoptosis, measured by cleaved caspase 3 (CC3) staining, in intestinal crypts at 6 h post-DXR that was significantly higher in mice colonized by Tmu . Interestingly, while DXR did not alter the expression of active and facultative intestinal stem cell (ISC) marker genes in control mice, it significantly reduced their expression in Tmu + mice. Tmu , but not DXR, is also associated with increased inflammation and expression of the type 2 cytokines IL-5 and IL-13. However, pre-treatment of intestinal organoids with these cytokines is not sufficient to drive elevated DXR-induced apoptosis. These findings highlight the significant influence of commensal microbiota, particularly eukaryotic organisms like Tmu , on intestinal biology and response to chemotherapy, underscoring the complexity of gut microbiota interactions in drug-induced mucositis. NEW & NOTEWORTHY: Our study found that the eukaryotic commensal Tritrichomonas muris ( Tmu ) significantly increases DXR-induced intestinal apoptosis in mice, despite no changes in tissue morphology. Tmu also reduces intestinal stem cell gene expression post-DXR injury, and elevates inflammation and type 2 cytokine expression in the absence of injury. In vitro organoid assays suggest that type 2 cytokines alone are insufficient to promote increased DXR-associated apoptosis. These findings emphasize the complex role of gut microbiota in drug-induced intestinal damage.