David Plesner Mathiasen:
The subnuclear organization of repair proteins is essential in the regulation of the DNA damage response (DDR). We present a high throughput screening method based on fluorescence microscopy to identify protein relocalization in response to DNA damage. Furthermore we present data on a novel one round PCR based N-terminal tagging strategy we used to try to create a complete GFP (Green fluorescent protein) collection.
In S. cerevisiae the genetic loci that, when mutant, confers X-‐ray, but not UV (ultraviolet light), sensitivity, were numbered RAD50 and upwards. Prior to this work all but one mutant had been mapped physically to specific genes. In this work we map RAD56 to the NAT3 gene and discover that the original rad56-1 mutant has a 1 bp deletion leading to a truncation of the Nat3 protein. Nat3 is part of the NatB N-terminal acetylation complex. We present data identifying the potential downstream targets of Nat3, which includes Rad52.
The Exonuclease1 (Exo1) is an exonuclease present in S/G2 phase that is proposed to travel with the replication fork during DNA replication, to process stalled replication forks, in the absence of checkpoint activation. In this work we report that Exo1 is degraded at the metaphase to anaphase transition and that the degradation is mediated by the Anaphase promoting complex (APC) through 4 D-Box sequences in the C-‐terminal end of Exo1. In a high throughput screen we identified 148 gene deletions that affect Exo1 regulation and degradation. We created a non-degradable Exo1 protein (Exo1-ND), present also in anaphase and G1, which induced anaphase bridge formation, recombination rates and genomic instability. We report Exo1-ND to be epistatic with top2-1, suggesting that Exo1 process catenated DNA structures directly in anaphase and replication termination sites, or blocks the recruitment of Top2, thereby inducing anaphase bridge formation.