Project description:DNA‒protein crosslinks (DPCs) challenge faithful DNA replication and fluid passage of genomic information. Our study unveils the cullin ubiquitin ligase Rtt101 as a novel DPC repair factor. Genetic analyses demonstrate that Rtt101 is essential for resistance to a wide range of DPC types. Using an inducible in vivo DPC system, we reveal the significant impact of Rtt101 on DPC removal, including topoisomerase 1 crosslinks. ChIP-sequencing and ChEC-sequencing specifically highlight the association of Rtt101 with replisomes as well as colocalization with DPCs. Our findings establish Rtt101 as a novel main contributor to DPC repair throughout the yeast cell cycle.
Project description:Covalent DNA-protein crosslinks (DPC) are toxic DNA lesions that require repair by global-genome and replication-coupled pathways. How cells respond when RNA polymerases stall at DPCs during transcription is unknown. DPC-seq is new method for the genome-wide mapping of covalent DNA-protein adducts.
Project description:To identify genes expressed predominantly in the ventral skin dermis of pregnant mice, we performed DNA microarray analysis by using isolated dermal tissues from ventral skin at 0 and 15 dpc, PP2-injected ventral skin at 15 dpc, and dorsal skin at 15 dpc.
Project description:Small vesicles, known as exosomes, are secreted from various cell types. Exosomes secreted by mesenchymal stem cells have therapeutic effects against a variety of diseases, and may be able to partially replace stem cell therapies. Previously, we established and characterized human leukocyte antigen (HLA) haplotype homo (HHH) dental pulp cell (DPC) lines from human wisdom teeth. In this report, we purified the exosomes secreted from HHH-DPCs and evaluated their therapeutic potential in a periodontitis model. The exosomes purified from HHH-DPCs showed homogeneous and spherical membrane structures, and showed low but significant expression of HLA class I molecules. The exosomes further promoted proliferation and migration in DPCs. A comparison of miRNAs revealed that the HHH-DPC exosomes contained higher levels of multiple Let-7 family miRNAs compared to HHH-induced pluripotent stem cell (iPSC)-derived exosomes. Finally, the HHH-DPC exosomes showed preventive effects in a mouse model of periodontitis induced by lipopolysaccharides (LPS). In summary, HHH-DPC exosomes expressed HLA molecules which may induce an immune response in HLA-mismatched transplantations. However, they successfully stimulated the proliferation and migration of cells and showed suppressive effects on LPS-induced periodontitis. Therefore, HHH-DPC exosomes show great potential for applications in periodontal treatments.
Project description:Central nervous system (CNS) trauma and neurodegenerative disorders trigger a cascade of cellular and molecular events resulting in neuronal apoptosis and regenerative failure. The pathogenic mechanisms and gene expression changes associated with these detrimental events can be effectively studied using a rodent optic nerve crush (ONC) model. The purpose of this study was to use a mouse ONC model to: (a) evaluate changes in retina gene expression, (b) identify neurodegenerative pathogenic pathways and (c) discover potential new therapeutic targets. Affymetrix Mouse Gene 1.0 ST arrays were utlized to detail the global gene expression profile following optic nerve crush (ONC) in the retina of BALB/cJ mice at six different days post crush (dpc) (naive, 3 dpc, 7 dpc, 14 dpc, 21 dpc and 28 dpc) to understand the pathogenic responses in relation to neuronal loss and regenerative failure.
Project description:Central nervous system (CNS) trauma and neurodegenerative disorders trigger a cascade of cellular and molecular events resulting in neuronal apoptosis and regenerative failure. The pathogenic mechanisms and gene expression changes associated with these detrimental events can be effectively studied using a rodent optic nerve crush (ONC) model. The purpose of this study was to use a mouse ONC model to: (a) evaluate changes in optic nerve (ON) gene expression, (b) identify neurodegenerative pathogenic pathways and (c) discover potential new therapeutic targets. Affymetrix Mouse Gene 1.0 ST arrays were utlized to detail the global gene expression profile following optic nerve crush (ONC) in the ON of BALB/cJ mice at six different days post crush (dpc) (naive, 3 dpc, 7 dpc, 14 dpc, 21 dpc and 28 dpc) to understand the pathogenic responses in relation to neuronal loss and regenerative failure.
Project description:iPSC were obtained from DPC from TRPC6-mut patient, a idiopathic autistic patient and a control. Original DPC and iPSC obtained were submited to expression analysis in order to check if the expression pattern obtained for the iPSC cells were closer related to embyonic cells than to the original DPC
Project description:DNA-protein crosslinks (DPCs) are bulky DNA lesions that interfere with DNA metabolism and therefore threaten genomic integrity. Recent studies implicate the metalloprotease SPRTN in S-phase removal of DPCs, but how SPRTN activity is coupled to DNA replication is unknown. Using Xenopus egg extracts that recapitulate replication-coupled DPC proteolysis, we show that DPC degradation not only depends on SPRTN but also the proteasome, which act as independent DPC proteases. Proteasome recruitment requires DPC polyubiquitylation, which is triggered by single-stranded DNA, a byproduct of DNA replication. In contrast, SPRTN-mediated DPC degradation is independent of DPC polyubiquitylation but requires polymerase extension of a nascent strand to the lesion. Thus, SPRTN and proteasome activities are coupled to DNA replication by distinct mechanisms that together promote replication across immovable protein barriers.
Project description:Naturally occurring and drug-induced DNA-protein crosslinks (DPCs) interfere with key DNA transactions if not timely repaired. The unique family of DPC-specific proteases Wss1/SPRTN targets DPC protein moieties for degradation, including topoisomerase-1 trapped in covalent crosslinks (Top1ccs). Here we describe that the efficient DPC disassembly requires Ddi1, another conserved predicted protease in Saccharomyces cerevisiae. We found Ddi1 in a genetic screen of the tdp1wss1 mutant defective in Top1cc processing. Ddi1 is recruited to a persistent Top1cc-like DPC lesion in an S-phase dependent manner to assist eviction of crosslinked protein from DNA. Loss of Ddi1 or its putative protease activity hypersensitize cells to DPC trapping agents independently from Wss1 and 26S proteasome, implying its broader role in DPC repair. Among potential Ddi1 targets we found the core component of RNAP II and show that its genotoxin-induced degradation is impaired in ddi1. Together, we propose that the Ddi1 protease contributes to DPC proteolysis. Yeast strains with WSS1 and DDI1 deletions were compared when either complemented with DDI1-WT or ddi1-D220A after hydroxyurea treatment by SILAC MS.