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Molecular and Cellular Biology, January 2005, p. 563-574, Vol. 25, No. 2
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.2.563-574.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Chk1-Dependent S-M Checkpoint Delay in Vertebrate Cells Is Linked to Maintenance of Viable Replication Structures

George Zachos,1 Michael D. Rainey,1 and David A. F. Gillespie1,2*

Beatson Institute for Cancer Research,1 Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, United Kingdom2

Received 27 July 2004/ Returned for modification 3 September 2004/ Accepted 15 October 2004

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.

* Corresponding author. Mailing address: Beatson Institute for Cancer Research, Garscube Estate, Switchback Rd., Glasgow G61 1BD, United Kingdom. Phone: 0141-330-3966. Fax: 0141-942-6521. E-mail: d.gillespie{at}beatson.gla.ac.uk.

Molecular and Cellular Biology, January 2005, p. 563-574, Vol. 25, No. 2
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.2.563-574.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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