Abstract

BackgroundPds5 is an abundant HEAT-repeat-containing protein that binds to cohesin and mediates sister chromatid cohesion. In vertebrates, Pds5A and Pds5B are known to protect DNA replication fork, as their loss leads to DNA damage. Pds5 interacts directly with Wapl, to remove cohesin during mitosis.AimTo analyze the effects of the loss of Pds5 proteins-mediated DNA damage on the cell cycle checkpoints and to examine the possibility that Pds5 proteins have an overlapping function.MethodsWe first analyzed the cell cycle regulation of Pds5 proteins and defects in S-phase; DNA damage was confirmed after Pds5A/B knockdown. The activation of cell cycle checkpoints and apoptosis were examined by the level of p-Chk1S317, MAD2 localization, and the level of pro-apoptotic markers, respectively.ResultsPds5 proteins dissociated from chromatin in a stepwise manner, and their loss led to activation of pro-apoptotic markers associated with the phosphorylation of Chk1S317 due to DNA damage. Depletion of either Pds5A or Pds5B alone increased Smc3 acetylation in perturbed cell cycle, while depletion of both proteins severely impaired Smc3 acetylation. Moreover, the loss of Pds5A/Pds5B activated the SAC in an ATR-Chk1-dependent manner and stabilized Wapl on chromatin. The depletion of Chk1 rescued the S-phase delay associated with Pds5 depletion and significantly increased mitotic catastrophe.ConclusionPds5A and Pds5B display overlapping functions in facilitating Smc3 acetylation. Somewhat paradoxically, they also have non-redundant functions in terms of cohesin removal due to the activated surveillance mechanism that leads to phosphorylation of Chk1S317.

Highlights

  • Sister chromatid cohesion is mediated by the multi-subunit cohesin complex, which comprises four core proteins Smc1, Smc3, kleisin subunit Scc1 (Rad21 in humans), and Scc3 (SA1 or SA2 in vertebrates), and the regulatory proteins include sororin, wings apart-like (Wapl), and Pds5 (Schmitz et al, 2007; Shintomi and Hirano, 2009; Nasmyth, 2011; Losada, 2014)

  • We report that the depletion of Pds5A or Pds5B or both induced phosphorylation of Chk1 (S317) with concomitant Smc3 acetylation and DNA damage-mediated stalling of DNA replication forks in perturbed and unperturbed cell cycle

  • Data obtained from the time-course experiment showed that Pds5A and Pds5B protein expression levels remained constant throughout the cell cycle (Figure 1B)

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Summary

Introduction

Sister chromatid cohesion is mediated by the multi-subunit cohesin complex, which comprises four core proteins Smc, Smc, kleisin subunit Scc (Rad in humans), and Scc (SA1 or SA2 in vertebrates), and the regulatory proteins include sororin, wings apart-like (Wapl), and Pds (Schmitz et al, 2007; Shintomi and Hirano, 2009; Nasmyth, 2011; Losada, 2014). Sororin competes with Wapl to bind Pds and antagonize Wapl to maintain sister chromatid cohesion during interphase (Nishiyama et al, 2010). Phosphorylation of sororin by Cdk inhibits its binding to Pds (Nishiyama et al, 2013), allowing the binding of Wapl to Pds and the removal of cohesin from the chromosome arms. Centromeric cohesin is protected from degradation by shugoshin 1 (Sgo). Pds interacts directly with Wapl, to remove cohesin during mitosis

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