In tight collaboration with the Englert group at the FLI we are using zebrafish as an animal model. We perform mRNA knock-downs by morpholino-nucleotides of individual components of the TRC complex. Cdc45p and RecQL4 knock-downs develop over five to six days, but display craniofacial abnormalities, including a flat head and small eyes, which may serve as a model for hypomorphic human diseases such as DiGeorge syndrome for Cdc45 and Rothmund-Thomson syndrome for RecQL4.
Apparently, the fertilized egg cells contain enough of these two proteins to allow for several rounds of DNA replication. In striking contrast, the TopBP1 knock-downs display a much more severe phenotype that do not allow hatching of the eggs and leads to embryonic lethality after two days. We currently characterize the morphants in detail in terms of development, proliferation capacity and cell death.
Presently, we are producing fish-specific antibodies against these proteins. With these we are planning to measure the amounts of replication factors in developing fish. We are further interested whether the craniofacial abnormalities seen in Cdc45p and RecQL4 knock-downs are due to p53-induced apoptosis. To this end we would like to knock-down p53 as well and study whether we can rescue the observed phenotypes. Moreover, we will use the fish system to investigate whether we can resurrect normal development by injecting mRNAs of the N- or C-terminal fragments of the corresponding replication factors. This should allow us to fine-map which of the amino acids of the corresponding replication proteins are essential for replication and which are not. Collectively, these approaches should contribute to a significant increase of our knowledge on basic aspects of DNA replication in vertebrates and particularly in human beings.
Knock-downs of Cdc45 cause complex developmental defects and increased apoptosis.
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