Genetic Evidence for Roles of Yeast Mitotic Cyclins at Single-Stranded Gaps Created by DNA Replication

Paused/stalled replication forks are major threats to genome integrity; unraveling the complex pathways that contribute to fork stability/restart is crucial. Experimentally, fork stalling is induced by growth in presence of hydroxyurea (HU), which depletes the pool of deoxynucleoside triphosphates (dNTPs) and slows down replication progression in yeast. Here, I report an epistasis analysis, based on sensitivity to HU, between CLB2, the principal mitotic cyclin gene in S. cerevisiae, and genes involved in fork stability and recombination. clb2 cells are not sensitive to HU, but the strong synergistic effect of clb2 with most genes tested indicates, unexpectedly, that CLB2 plays an important role in DNA replication, in the stability and restart of stalled forks, in pathways dependent and independent of homologous recombination. Results indicate that CLB2 functions in parallel to SGS1 helicase-EXO1 exonuclease to allow proper Rad51 recombination, but also regulates a combined Sgs1-Exo1 activity in a Mec1- and a Rad53- checkpoint protein kinase dependent pathway. Data argue that Mec1 regulates Clb2 to prevent a deleterious Sgs1-Exo1 activity at paused/stalled forks, while Rad53 checkpoint activation regulates Clb2 to allow a necessary Sgs1-Exo1 activity at stalled/collapsed forks. Altogether, this study indicates that Clb2 regulates the activity of numerous nucleases at single-stranded gaps created by DNA replication. A model is proposed for the function and regulation of Clb2 at stalled forks. These data open new perspectives on the role of mitotic cyclins at the end of S phase.

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