Genetic and Cytological Analysis of the Spindle Assembly Checkpoint Component BUB-3 in DNA Damage Response

The spindle assembly checkpoint and the DNA damage response have been studied for decades as independent pathways exerting separate functions. However, crosstalk between these cellular processes has emerged. The extent of overlap between these pathways is still unknown, especially in multicellular organisms. We performed a forward genetic screen in Caenorhabditis elegans to uncover new genes involved in the repair of DNA damage induced by ionizing radiation. We isolated a mutation named gt2000, which confers hypersensitivity to ionizing radiation. We showed that gt2000 introduces a premature stop codon in the bub-3 gene. BUB-3 is a key component of the spindle assembly checkpoint. Irradiated bub-3(gt2000) larvae are developmentally retarded and form abnormal vulvae, consistent with the idea that BUB-3 acts during development. Moreover, bub-3(gt2000) embryos derived from irradiated adults show increased levels of lethality, suggesting that BUB-3 is also active in the germline. We showed that san-1 (the Caenorhabditis elegans homologue of MAD3) deletion alleles confer hypersensitivity to ionizing radiation, consistent with the notion that the spindle assembly checkpoint pathway might be involved in the DNA damage response. bub-3(gt2000) mutants are moderately sensitive to the crosslinking drug cisplatin but not to UV light or methyl methanesulfonate. This is consistent with a role in dealing with DNA double-strand breaks and not with base damage. bub-3 and san-1 mutants did not exhibit a mutator phenotype, which suggests that these genes are not required to preserve genome stability in the absence of exogenous DNA damaging agents. Double mutant analysis revealed that bub-3 acts independently of the three major pathways involved in the repair of DNA double-strand breaks. Finally, the cdc-20 gain-of-function mutant fzy-1(av15), which is refractory to the cell cycle delay conferred by the spindle checkpoint, showed phenotypes similar to bub-3 and san-1 mutants. We speculate that BUB-3 is involved in DNA damage response through regulation of cell cycle timing.

Name dc:type.qualificationname

Doctor of Philosophy

Level dc:type.qualificationlevel

Doctoral Thesis

Grantor dc:publisher.institution

University of Dundee

Year dc:date.issued

2017

Author dc:creator

• Bertolini, Simone

Advisor dc:contributor.advisor

• Gartner, Anton

Language dc:language

eng

Identifier

oai:discovery.dundee.ac.uk:studenttheses/70bea8fe-f480-4aa5-817b-6f456e9b92f0

OAI identifier oai:identifier

oai:discovery.dundee.ac.uk:studenttheses/70bea8fe-f480-4aa5-817b-6f456e9b92f0