The Role of Nej1-Lif1 in Double Strand Break Repair Pathway Choice and Non-Homologous End-Joining
Abstract
DNA Double strand breaks (DSBs) are the most deleterious form of DNA damage. Improper repair of DSBs can result in genomic instability and failed repair of even a single DSB results in cell death. Homologous recombination (HR) and Non-homologous end-joining (NHEJ) are the two major pathways of DSB repair and both are highly conserved from yeast to mammals. The choice between these pathways is largely based on 5’ to 3’ resection of DNA at the break site. Resection inhibition favours NHEJ, while extensive resection facilitates HR. The MRX complex, which plays a role in both HR and NHEJ in yeast, promotes the initiation of end-resection at the broken ends, while YKu70/80 rapidly binds DSB ends to prevent the initiation of 5’resection. Nej1 and Lif1 are two NHEJ proteins shown previously to promote the ligation of DSB ends by stimulating Dnl4 ligase activity. The Nej1-Lif1 interaction is also required for additional functions in NHEJ, except these functions were previously uncharacterized.
Using nej1-F335A and nej1-V338A mutants that are unable to interact with Lif1, I show that the Nej1-Lif1 interaction plays a role in preventing end-resection initiation at broken ends in a manner epistatic with YKu70/80. Specifically, I demonstrate that Nej1-Lif1 prevents the Sgs1-Dna2 dependent initiation of end-resection to promote NHEJ pathway choice. I also show that the Nej1-Lif1 interaction plays a role in preventing genomic stability during DSB repair in a redundant manner with the end-bridging function of the Rad50 subunit of the MRX complex.
Finally, the effect of Rad50 and Nej1 mutants on the nuclear periphery re-location of persistent DSB ends to either Mps3 or the nuclear pore complex (NPC) were studied. I show that cells lacking RAD50 absolutely require the NPC to survive even endogenous DNA damage, while cells harboring nej1Δ, yku70Δ, nej1-F335A or nej1-V338A mutants preferentially require Mps3 to promote cell survival and prevent genomic instability during the repair of persistent DSBs.
Description
Keywords
Genetics, Biology--Molecular, Biochemistry
Citation
Sorenson, K. (2017). The Role of Nej1-Lif1 in Double Strand Break Repair Pathway Choice and Non-Homologous End-Joining (Doctoral thesis, University of Calgary, Calgary, Canada). Retrieved from https://prism.ucalgary.ca. doi:10.11575/PRISM/28337