Rim11, the human GSK-3$\beta$ homolog is involved in replication stress response in yeasts

Abstract

The GSK3-$\beta$ kinase is linked to many kinds of cancer either as a tumor suppressor or as a tumor promoter. Genomic instability is one of the underlying hallmarks for tumor initiation. The link between GSK3-$\beta$ and genomic instability is still unclear. In the perspective of tumor development and progression, mutations are believed to accumulate due to compromised DNA repair. Using yeast genetics as a powerful tool, we discovered Rim11, the human GSK3-$\beta$ homolog as a suppressor of dna2 and rad18 mutants. Dna2 is an essential endonuclease/helicase in Okazaki fragments synthesis, whereas Rad18 is an E3 ubiquitin ligase responsible for activating the post-replication repair (PRR). Overexpression of Rim11 kinase suppressed the lethality of dna2 helicase-dead mutant and the methyl methane sulfonate (MMS) sensitivity of rad18 null mutant. The substrate for Rim11 responsible for the suppression is Ume6, a DNA binding protein. Ume6 interacts with the histone deacetylase complex (HDAC) Sin3/Rpd3, and this interaction and the deacetylase activity of Rpd3 are necessary for the Rim11-dependent suppression of dna2 mutant. Through epistatic analysis we showed that the Rim11-initiated suppression of dna2 and rad18 mutants promotes sister-chromatids recombination (SCR) mediated by the Rad52/Rad59 proteins. In support of this, both dna2 and rad18 mutants could be rescued by the overexpression of Rad52. Moreover, we found that checkpoint arrest is heavily enforced in dna2 mutant. Checkpoint could impose restriction on recombination-mediated repair. As previously reported Rpd3-mediated deacetylation of Rad53 facilitates in suppressing the activation of checkpoint. We asked whether the role of Rpd3 is to suppress checkpoint therefore allowing HR repair. Indeed, the removal of key checkpoint proteins like Rad9 or Rad53

or overexpression of ribonucleotide reductase inhibitor Sml1 allowed cells with dna2 mutation to be viable.

Our data demonstrate that GSK3-$\beta$ homolog Rim11 is directly involved in repair of faults in Okazaki fragment synthesis, by promoting homologous recombination through suppression checkpoint activation, revealing a novel pathway of post-replication repair (PRR) that is distinct from the well-described Rad6-Rad18 pathways. Our finding could explain why GSK3-$\beta$ promote cell proliferation and tumor growth and explain why down regulating GSK3-$\beta$ is beneficial in cancer therapy. The elevated levels of GSK3-$\beta$ could allow tumor cells to easily overcome replicative stress by facilitating DNA repair in cancer.