Cell cycle checkpoints are surveillance systems which delay or arrest cell division when conditions are inappropriate or inopportune. During mitosis, the replicated chromatin fibers are condensed and compacted into sister chromatids and then the sister chromatids are segregated to the daughter cells. Several checkpoint systems regulate the onset of mitosis in mammalian cells. The DNA damage G2 checkpoint delays mitosis by inhibiting the protein kinase that promotes mitotic entry (mitosis-promoting-factor, MPF) and excluding MPF from the nucleus so that it cannot interact with nuclear substrates. Ataxia telangiectasia-mutated (ATM) and BRCA1 are required for effective DNA damage G2 checkpoint function. DNA topoisomerase II (topoll) is an essential enzyme that is required to separate topologically intertwined daughter chromatids after DNA replication and prior to mitosis. G2 human cells actively delay the onset of mitosis when chromatid decatenation is blocked using the topoll catalytic inhibitor, ICRF-193. Override of the ICRF-193-induced G2 delay caused the appearance of constrictions between chromatid arms in metaphase spreads, suggesting the presence of linkages or catenations due to inhibition of topoll. The active arrest of G2 cells when topoll was inhibited did not require ATM and was not associated with activation of Chkl or significant inhibition of MPF. The topoll-dependent G2 delay that is induced by ICRF- 193 does not, therefore, appear to be a DNA damage G2 checkpoint response. The topoll-dependent G2 delay did require the activity of the AT- and rad3-related (ATR) checkpoint kinase and expression of BRCA11 establishing the G2 delay in ICRF-193-treated cells as an active, checkpoint response. ATR and BRCA1 appear to enforce the topoll-dependent G2 checkpoint through inhibition of polo-like kinase I (Plkl). Plkl phosphorylates cyclin B1 on ser 147, and the Crml nuclear exporter binds at this site to actively transport cyclin B1 out of the nucleus. Plkl-dependent phosphorylation of ser147 blocks Crml interaction with cyclin B1 and causes accumulation of MPF within the interphase nucleus where it can interact with its various substrates to initiate mitosis. ATR- and BRCAl-dependent inhibition of Plkl should cause MPF to be excluded from the nucleus, thereby blocking the onset of mitosis. Studies in this project will test several questions concerning the ICRF-193-induced G2 checkpoint response. Does the ICRF-193-induced G2 delay reflect the activity of a checkpoint that monitors the status of chromatid decatenation following DNA replication? Does ATR sends a signal through BRCA1 to inhibit Plkl when chromatid catenations are sensed? Is defective decatenation checkpoint function associated with genetic instability in cancer?

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA081343-08
Application #
7087919
Study Section
Special Emphasis Panel (ZRG1-PTHB (04))
Program Officer
Pelroy, Richard
Project Start
1999-06-01
Project End
2008-04-30
Budget Start
2006-05-01
Budget End
2007-04-30
Support Year
8
Fiscal Year
2006
Total Cost
$247,885
Indirect Cost
Name
University of North Carolina Chapel Hill
Department
Pathology
Type
Schools of Medicine
DUNS #
608195277
City
Chapel Hill
State
NC
Country
United States
Zip Code
27599
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Bower, J J; Karaca, G F; Zhou, Y et al. (2010) Topoisomerase IIalpha maintains genomic stability through decatenation G(2) checkpoint signaling. Oncogene 29:4787-99
Bower, Jacquelyn J; Zhou, Yingchun; Zhou, Tong et al. (2010) Revised genetic requirements for the decatenation G2 checkpoint: the role of ATM. Cell Cycle 9:1617-28
Chen, Bo; Simpson, Dennis A; Zhou, Yingchun et al. (2009) Human papilloma virus type16 E6 deregulates CHK1 and sensitizes human fibroblasts to environmental carcinogens independently of its effect on p53. Cell Cycle 8:1775-87
Kaufmann, William K (2009) Analysis of the topoisomerase II-dependent decatenation G2 checkpoint and checkpoint kinases in human cells. Methods Mol Biol 582:155-66
Tran, Nancy; Qu, Ping-Ping; Simpson, Dennis A et al. (2009) In silico construction of a protein interaction landscape for nucleotide excision repair. Cell Biochem Biophys 53:101-14
Kaufmann, William K (2007) Initiating the uninitiated: replication of damaged DNA and carcinogenesis. Cell Cycle 6:1460-7
Unsal-Kacmaz, Keziban; Chastain, Paul D; Qu, Ping-Ping et al. (2007) The human Tim/Tipin complex coordinates an Intra-S checkpoint response to UV that slows replication fork displacement. Mol Cell Biol 27:3131-42
Kaufmann, William K (2006) Dangerous entanglements. Trends Mol Med 12:235-7
Kaufmann, William K; Filatov, Leonid; Oglesbee, Stephen E et al. (2006) Radiation clastogenesis and cell cycle checkpoint function as functional markers of breast cancer risk. Carcinogenesis 27:2519-27

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