Essential global role of CDC14 in DNA synthesis revealed by chromosome underreplication unrecognized by checkpoints in cdc14 mutants

Abstract

The CDC14 family of multifunctional evolutionarily conserved phosphatases includes major regulators of mitosis in eukaryotes and of DNA damage response in humans. The CDC14 function is also crucial for accurate chromosome segregation, which is exemplified by its absolute requirement in yeast for the anaphase segregation of nucleolar organizers; however the nature of this essential pathway is not understood. Upon investigation of the rDNA nondisjunction phenomenon, it was found that cdc14 mutants fail to complete replication of this locus. Moreover, other late-replicating genomic regions (10% of the genome) are also underreplicated in cdc14 mutants undergoing anaphase. This selective genome-wide replication defect is due to dosage insufficiency of replication factors in the nucleus, which stems from two defects, both contingent on the reduced CDC14 function in the preceding mitosis. First, a constitutive nuclear import defect results in a drastic dosage decrease for those replication proteins that are regulated by nuclear transport. Particularly, essential RPA subunits display both lower mRNA and protein levels, as well as abnormal cytoplasmic localization. Second, the reduced transcription of MBF and SBF-controlled genes in G1 leads to the reduction in protein levels of many proteins involved in DNA replication. The failure to complete replication of late replicons is the primary reason for chromosome nondisjunction upon CDC14 dysfunction. As the genome-wide slow-down of DNA replication does not trigger checkpoints [Lengronne A, Schwob E (2002) Mol Cell 9:1067-1078], CDC14 mutations pose an overwhelming challenge to genome stability, both generating chromosome damage and undermining the checkpoint control mechanisms.S uccessful chromosome segregation in mitosis requires the coordinated work of cellular regulatory circuits, which transmit cell cycle signals to chromatin and spindle proteins. One of such factors is the Cdc14 protein, an evolutionary conserved protein phosphatase (1-3). While in human cells a CDC14 ortholog has been recently shown to play a key role in DNA damage response (4), studies on S. cerevisiae were mostly focused on Cdc14p roles in anaphase regulation and in the exit from mitosis. The scope of Cdc14p activity in budding yeast is believed to be limited to anaphase, because Cdc14p is sequestered in the nucleolus (5) in apparently inactive form (6) at other cell cycle stages. Therefore, while Cdc14 can potentially dephosphorylate many substrates (7,8), the most studied physiological pathways are the anaphase pathways (FEAR and MEN), which are both dependent on the two sequential bursts of Cdc14 release (1, 9). The cdc14 mutations cause a mitotic exit block, but also display defects in nucleolar (10) and telomeric (11) segregation. The mechanisms of chromosome segregation defects (11-15) in cdc14 mutants are generally poorly understood. While condensin mutations phenocopy the cdc14 rDNA nondisjunction (11, 16) and Cdc14p is required for condensin loading to rDNA (14), it is unlikely that conden...

The maternal nucleolus plays a key role in centromere satellite maintenance during the oocyte to embryo transition

“…Cdc14 is a cell cycle regulatory protein that localises to nucleoli throughout most of the cell cycle. These yeast mutants exhibit heterochromatin under-replication but this does not trigger cell cycle checkpoints and does not alter replication-associated gene expression immediately; rather, this defect is manifested during the next cell cycle (Dulev et al, 2009). Essentially the same situation was observed in our system, including the changes in replication-associated transcripts at the 2-cell stage.…”

Cdc14p resets the competency of replication licensing by dephosphorylating multiple initiation proteins during mitotic exit in budding yeast

“…By contrast, a previous study has discounted Cdc14p as the physiological phosphatase that dephosphorylates initiation proteins to promote pre-RC formation and DNA replication (Noton and Diffley, 2000). A recent study has reported genome-wide under-replication in cdc14-1 cells, which was attributed to dosage insufficiency of some replication proteins; however, the possible function of Cdc14p in promoting replication licensing by dephosphorylating pre-RC components was not addressed (Dulev et al, 2009). Therefore, the essential role of Cdc14p in replication licensing remains controversial.…”

Haploinsufficiency of an RB–E2F1–Condensin II Complex Leads to Aberrant Replication and Aneuploidy

“…Misshapen kinetochores can lead to merotelic microtubule attachments, lagging anaphase chromosomes, and ultimately gains and losses of whole chromosomes in daughter cells ( 45 ). Alternatively, centromere fusions or recombination with other chromosomes ( 7,8 ) may lead to gains or losses of whole chromosome arms, and these are the most common segmental chromosome changes in human cancer ( 46 ). Finally, lagging chromosomes created by these mechanisms are more likely to become incorporated into micronuclei and undergo chromothripsis in subsequent cell cycles ( 47 ).…”

“…Unrepaired errors can be passed on to daughter cells and contribute to cancer ( 1 ). In general, damaged DNA signals the cell cycle to arrest, and Replication stress can lead to fork stalling and chromosomal aberrations ( 7 ). It can create short gaps in sequence, or unresolved replication intermediates ( 3 , 7 , 8 ).…”