Chk1-dependent S-M checkpoint delay in vertebrate cells is linked to maintenance of viable replication structures.
We investigated mitotic delay during replication arrest (the S-M checkpoint) in DT40 B-lymphoma cells deficient in the Chk1 or Chk2 kinase. We show here that cells lacking Chk1, but not those lacking Chk2, enter mitosis with incompletely replicated DNA when DNA synthesis is blocked, but only after an initial delay. This initial delay persists when S-M checkpoint failure is induced in Chk2-/- cells with the Chk1 inhibitor UCN-01, indicating that it does not depend on Chk1 or Chk2 activity. Surprisingly, dephosphorylation of tyrosine 15 did not accompany Cdc2 activation during premature entry to mitosis in Chk1-/- cells, although mitotic phosphorylation of cyclin B2 did occur. Previous studies have shown that Chk1 is required to stabilize stalled replication forks during replication arrest, and strikingly, premature mitosis occurs only in Chk1-deficient cells which have lost the capacity to synthesize DNA as a result of progressive replication fork inactivation. These results suggest that Chk1 maintains the S-M checkpoint indirectly by preserving the viability of replication structures and that it is the continued presence of such structures, rather than the activation of Chk1 per se, which delays mitosis until DNA replication is complete.
Zachos
George
G
Beatson Institute for Cancer Research, Garscube Estate, Switchback Rd., Glasgow G61 1BD, United Kingdom.
Rainey
Michael D
MD
Gillespie
David A F
DA
D000818:Animals; D000970:Antineoplastic Agents / Q000378:metabolism; D016590:Aphidicolin / Q000378:metabolism; D016203:CDC2 Protein Kinase / Q000378:metabolism; D045744:Cell Line, Tumor; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D002645:Chickens; D004261:DNA Replication; D018390:Gene Targeting; D008938:Mitosis / Q000502:physiology*; D015739:Nocodazole / Q000378:metabolism; D009690:Nucleic Acid Conformation; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000378:metabolism; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D019311:Staurosporine / Q000031:analogs & derivatives* / Q000378:metabolism
D000970:Antineoplastic Agents; D047428:Protein Kinase Inhibitors; D016590:Aphidicolin; C054852:7-hydroxystaurosporine; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases; D016203:CDC2 Protein Kinase; D019311:Staurosporine; D015739:Nocodazole