ABSTRACT
DNA replication stress-induced fork arrest represents a significant threat to genomic integrity. One major mechanism of replication restart involves repriming downstream of the arrested fork by PRIMPOL, leaving behind a single-stranded DNA (ssDNA) gap. Accumulation of nascent strand ssDNA gaps has emerged as a possible determinant of the cellular hypersensitivity to genotoxic agents in certain genetic backgrounds such as BRCA deficiency, but how gaps are converted into cytotoxic structures is still unclear. Here, we investigate the processing of PRIMPOL-dependent ssDNA gaps upon replication stress induced by hydroxyurea and cisplatin. We show that gaps generated in PRIMPOL-overexpressing cells are expanded in the 3'-5' direction by the MRE11 exonuclease, and in the 5'-3' direction by the EXO1 exonuclease. This bidirectional exonucleolytic gap expansion ultimately promotes their conversion into DSBs. We moreover identify the de-ubiquitinating enzyme USP1 as a critical regulator of PRIMPOL-generated ssDNA gaps. USP1 promotes gap accumulation during S-phase, and their expansion by the MRE11 and EXO1 nucleases. This activity of USP1 is linked to its role in de-ubiquitinating PCNA, suggesting that PCNA ubiquitination prevents gap accumulation during replication. Finally, we show that USP1 depletion suppresses DSB formation in PRIMPOL-overexpressing cells, highlighting an unexpected role for USP1 in promoting genomic instability under these conditions.
Subject(s)
DNA Replication , DNA-Directed DNA Polymerase , Ubiquitin-Specific Proteases , DNA/genetics , DNA Damage , DNA, Single-Stranded/genetics , DNA-Directed DNA Polymerase/genetics , DNA-Directed DNA Polymerase/metabolism , Proliferating Cell Nuclear Antigen/genetics , Humans , Ubiquitin-Specific Proteases/metabolismABSTRACT
Current treatments of bacterial biofilms are limited by the poor penetration of antibiotics through their physical barrier as well as significant off-target toxicity of antibiotics and the induction of antibiotic resistance. Here we report a microneedle patch-mediated treatment for the effective elimination of biofilms by penetrating the biofilm and specifically delivering antibiotics to regions of active growth. We fabricated patches with self-dissolvable microneedles and needle tips loaded with chloramphenicol (CAM)-bearing and gelatinase-sensitive gelatin nanoparticles (CAM@GNPs). During the microneedle patch-mediated treatment, arrays of 225 microneedles simultaneously penetrate the biofilm matrix. Once inside, the microneedles dissolve and uniformly release CAM@GNPs into the surrounding area. In response to the gelatinase produced by the active bacterial community, the CAM@GNPs disassemble and release CAM into these active regions of the biofilm. Moreover, CAM@GNPs exhibited minimal off-target toxicity compared to direct CAM administration, which in turn favors wound healing. Importantly, we found that our microneedle-mediated treatment is more effective in treating Vibrio vulnificus biofilms than drug in free solution. We believe this new treatment strategy can be used to improve the delivery of a wide range of antimicrobial agents to biofilm-contaminated sites.
