engleski

Using the zebra fi sh as an in vivo model to study the role of P97/Vcp and Sprtn in DNA-Protein Crosslink Repair (DPCR)

DNA-Protein-Crosslinks (DPCs) are DNA lesions which occur when proteins become irreversibly covalently bound to DNA, thereby preventing all DNA transactions, including replication and transcription. DPC repair (DPCR) via proteolysis is a specialized DNA damage repair pathway required for the proteolytic cleavage and degradation of such crosslinked proteins. Several enzymes with a speci fi c function in DPCR have been identi fi ed, such as Sprtn, a protease required to degrade some types of DPCs ; absence or perturbed activity of these enzymes have been linked to ageing, cancer and neurodegenerative diseases. Among them, the P97/VCP AAA-ATPase is a multi-functional segregase which extracts proteins from various cellular compartments and targets them for degradation. It has been recently shown in vitro that P97/VCP is required to unfold a tightly packed DPC protein in order for Sprtn to proteolytically degrade it. We are currently investigating the speci fi c roles of several enzymes in DPCR including the P97/Vcp segregase and Sprtn using the zebra fi sh as a model for in vivo studies. First, we found that the p97 gene is duplicated in zebra fi sh. Interestingly, p97 is ubiquitously expressed whereas its paralog zgc:136908 is expressed only in skeletal muscle tissues, which suggests that p97 is the true ortholog of the ubiquitous mammalian P97/VCP. To study the role of p97 and zgc:136908 in DPCR, we are knocking-down their expression by injecting speci fi c antisense morpholino oligonucleotides into zebra fi sh embryos. DPC levels will be quanti fi ed in P97- de fi cient embryos before and after treatment with formaldehyde (a model DPC inducer). By co-injecting the p97 morpholino with an mRNA encoding either WT or mutated P97, we will create P97-de fi cient zebra fi sh embryos to study the role of P97 speci fi cally in DPCR. To determine the epistasis of p97 and sprtn in vivo, we will co-inject morpholinos targeting p97 and sprtn in zebra fi sh embryos and quantify DPC levels. Taken together, our results will uncover the role of P97 in the orchestration of DPC repair in vivo

DNA, P97/VCP, DNA-Protein-Crosslinks, DPC repair

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