CDK-mediated Yku80 Phosphorylation Regulates the Balance Between Non-homologous End Joining (NHEJ) and Homologous Directed Recombination (HDR).

There are two major pathways for repairing DNA double-strand breaks (DSBs): homologous directed recombination (HDR) and non-homologous end-joining (NHEJ). While NHEJ functions throughout the cell cycle, HDR is only possible during S/G2 phases, suggesting that there are cell cycle-specific mechanisms regulating the balance between the two repair systems. The regulation exerted by CDKs on HDR has been extensively demonstrated, and here we present evidence that the CDK Pho85, in association with the G1 cyclin Pcl1, phosphorylates Yku80 on Ser 623 to regulate NHEJ activity. Cells bearing a non-phosphorylatable version of Yku80 show increased NHEJ and reduced HDR activity. Accordingly, yku80S623A cells present diminished viability upon treatment with the DSB-producer bleomycin, specifically in the G2 phase of the cell cycle. Interestingly, the mutation of the equivalent residue in human Ku80 increases sensitivity to bleomycin in several cancer cell lines, suggesting that this mechanism is conserved in humans. Altogether, our results reveal a new mechanism whereby G1-CDKs mediate the choice between HDR and NHEJ repair pathways, putting the error prone NHEJ on a leash and enabling error free HDR in G2 when homologous sequences are available.

Comprehensive and quantitative analysis of G1 cyclins. A tool for studying the cell cycle.

In eukaryotes, the cell cycle is driven by the actions of several cyclin dependent kinases (CDKs) and an array of regulatory proteins called cyclins, due to the cyclical expression patterns of the latter. In yeast, the accepted pattern of cyclin waves is based on qualitative studies performed by different laboratories using different strain backgrounds, different growing conditions and media, and different kinds of genetic manipulation. Additionally, only the subset of cyclins regulating Cdc28 was included, while the Pho85 cyclins were excluded. We describe a comprehensive, quantitative and accurate blueprint of G1 cyclins in the yeast Saccharomyces cerevisiae that, in addition to validating previous conclusions, yields new findings and establishes an accurate G1 cyclin blueprint. For the purposes of this research, we produced a collection of strains with all G1 cyclins identically tagged using the same and most respectful procedure possible. We report the contribution of each G1 cyclin for a broad array of growing and stress conditions, describe an unknown role for Pcl2 in heat-stress conditions and demonstrate the importance of maintaining the 3'UTR sequence of cyclins untouched during the tagging process.

Intertwined control of the cell cycle and nucleocytoplasmic transport by the cyclin-dependent kinase Pho85 and RanGTPase Gsp1 in Saccharomyces cerevisiae.

Deciphering the molecular mechanisms that connect cell cycle progression and nucleocytoplasmic transport is of particular interest: this intertwined relationship, once understood, may provide useful insight on the diseases resulting from the malfunction of these processes. In the present study we report on findings that indicate a biochemical connection between the cell cycle regulator CDK Pho85 and Ran-GTPase Gsp1, an essential nucleocytoplasmic transport component. When Gsp1 cannot be phosphorylated by Pho85, the cell cycle progression is impaired. Accordingly, a nonphosphorylatable version of Gsp1 abnormally localizes to the nucleus, which impairs the nuclear transport of molecules, including key components of cell cycle progression. Furthermore, our results suggest that the physical interaction of Gsp1 and the Kap95 karyopherin, essential to the release of nuclear cargoes, is altered. Altogether, the present findings point to the involvement of a biochemical mechanism in the interlocked regulation of the cell cycle and nuclear transport.