Sandra Moser1, Arno Alpi2, Ingo Bsing3, Ralf Schnabel3 and Anton Gartner1. DNA damage checkpoints have evolved to ensure genomic stability. Upon DNA damage these checkpoints transiently stop cell cycle progression which allows the repair of damaged DNA or if the damage is too severe the removal of compromised cells by apoptosis.
rad-5 is an evolutionary conserved DNA damage checkpoint gene that in C. elegans has been shown to be required for ?-irradiation induced cell cycle arrest and apoptosis. Genetic observations revealed that
rad-5 does not act in the conserved
mrt-2 hus-1 DNA damage pathway but its exact role in the DNA damage checkpoint pathway is still unknown. In addition to its role as DNA damage checkpoint gene
rad-5 is also required for embryonic development as
rad-5 mutants shifted to 25C show embryonic lethality. . To assess the developmental phenotypes of
rad-5 and to potentially determine whether these are related to
rad-5 function in maintaining genome stability we aim to describe the developmental phenotypes of
rad-5. To determine the precise embryonic defects we performed a lineage analysis for
rad-5 mutants that were shifted to 25C. Interestingly, in contrast to other DNA damage checkpoint genes like
atl-1 or
chk-1 which when knocked out also result into embryonic lethality
rad-5 mutants exhibit a distinct lineage defect at the 12 cell stage but dont show any abnormalities earlier in development {Garcia-Muse et Boulton, 2005, Kalogeropoulos et al., 2004, Brauchle et al., 2003). Surprisingly, a
rad-5 deletion mutant
rad-5 (
tm1528) showed no apparent defect in embryonic development but worms homozygous for
rad-5 (
tm1528) are sterile and show a protruding vulva (pvl) phenotype indicating that
rad-5 might play a role in the vulva cell organization or in the cell lineages giving rise to the vulva. To further pin down these defects we are currently creating
rad-5 (
tm1528) lines carrying GFP makers that stain specific vulva cells. To determine whether the slow growth phenotype of some
rad-5 alleles is rescued by other checkpoint proteins we made the respective doubles and found that only
cep-1 leads to a partial rescue of the slow growth phenotype, whereas atm does not have such a defect whereas
mrt-2 and
hus-1 enhance the slow growth phenotype (Ahmed Alpi et al., 2001). . Finally to start addressing how
rad-5 might function at the molecular level, failing to get new insights from a suppressor screen that only lead to the identification of intragenic suppressors, we are now trying to identify interacting proteins of RAD-5 using immunoprecipitation and to assess
rad-5 localization and potential changes in its localization upon DNA damage by immunofluorescence in worm and tissue culture based systems.