ABSTRACT
A major impediment to the characterization of mtDNA repair mechanisms in comparison to nuclear DNA repair mechanisms is the difficulty of specifically addressing mitochondrial damage. Using a mitochondria-penetrating peptide, we can deliver DNA-damaging agents directly to mitochondria, bypassing the nuclear compartment. Here, we describe the use of an mtDNA-damaging agent in tandem with CRISPR/Cas9 screening for the genome-wide discovery of factors essential for mtDNA damage response. Using mitochondria-targeted doxorubicin (mtDox), we generate mtDNA double-strand breaks (mtDSBs) specifically in this organelle. Combined with an untargeted doxorubicin (Dox) screen, we identify genes with significantly greater essentiality during mitochondrial versus nuclear DNA damage. We characterize the essentiality of our top hit, WRNIP1âobserved here for the first time to respond to mtDNA damage. We further investigate the mitochondrial role of WRNIP1 in innate immune signaling and nuclear genome maintenance, outlining a model that experimentally supports mitochondrial turnover in response to mtDSBs.
Subject(s)
DNA, Mitochondrial , Mitochondria , DNA, Mitochondrial/genetics , Mitochondria/genetics , DNA Repair , DNA Damage , DoxorubicinABSTRACT
A major impediment to the characterization of mtDNA repair mechanisms, in comparison to nuclear DNA repair mechanisms, is the difficulty of specifically addressing mitochondrial damage. Using a mitochondria-penetrating peptide, we can deliver DNA-damaging agents directly to mitochondria, bypassing the nuclear compartment. Here, we describe the use of a mtDNA-damaging agent in tandem with CRISPR/Cas9 screening for the genome-wide discovery of factors essential for mtDNA damage response. Using mitochondria-targeted doxorubicin (mtDox) we generate mtDNA double-strand breaks (mtDSBs) specifically in this organelle. Combined with an untargeted Dox screen, we identify genes with significantly greater essentiality during mitochondrial versus nuclear DNA damage. We characterize the essentially of our top hit - WRNIP1 - observed here for the first time to respond to mtDNA damage. We further investigate the mitochondrial role of WRNIP1 in innate immune signaling and nuclear genome maintenance, outlining a model that experimentally supports mitochondrial turnover in response to mtDSBs.
ABSTRACT
Advances in the field of fluorine chemistry have been applied extensively to the syntheses of 18F-labelled organic compounds and radiopharmaceuticals. However, 18F has sparely been used as a tool to explore inorganic chemistry and can be viewed as a research area worthy of further development. This review highlights the application of 18F in development of inorganic fluorinating agents, mechanistic studies and imaging tools.
