Paula Boucher Deming, Ph.D.

Dissertation research performed under the guidence of Dr. William K. Kaufmann

ABSTRACT
    Topoisomerase II separates intertwined sister chromatids following DNA replication and prior to mitosis through a process known as decatenation. It was hypothesized that chromatid catenation is actively monitored in human cells, with progression from G2 to mitosis being inhibited when chromatid decatenation is insufficient. To test this hypothesis, human cell lines containing inherited or engineered mutations in G2 checkpoint proteins were examined for their ability to undergo mitotic delay following treatment with a topoisomerase II inhibitor [ICRF-193] that prevents decatenation without producing DNA damage. Ataxia telangiectasia (AT) cells, defective in DNA damage checkpoint responses, displayed a normal mitotic delay response following incubation with ICRF-193. However, human cell lines expressing an ataxia-telangiectasia mutated and rad3-related (ATR) kinase-inactive allele (ATRki) or containing a mutation in BRCA1 did not undergo an ICRF-193-induced mitotic delay. The defect in the BRCA1-mutant cells was corrected by expression of wildtype BRCA1. Treatment of cells with ICRF-193 was not associated with the phosphorylation of the effector kinases, Chk1 or Chk2, nor with an inhibition of cyclin B1/Cdk1 kinase activity. However, both over-expression of a mutant cyclin B1 containing a dominant nuclear localization signal and inhibition of Crm1a-mediated nuclear export reversed ICRF-193-induced mitotic delay. Human fibroblasts that were blocked in G2 with ICRF-193 to inhibit decatenation displayed a severe inhibition in Plk1 activity. Cells overexpressing the ATRki allele failed to inhibit Plk1 activity following treatment with ICRF-193. The inhibition in Plk1 activity was required for decatenation checkpoint function, as overexpression of constitutively active Plk1 alleles ablated ICRF-193-induced mitotic delay. The mitotic kinase/s that phosphorylate cyclin B1 in vitro and in vivo were inactive in human fibroblasts that were arrested in G2 by ICRF-193. In combination, it appears that ATR enforces the decatentation checkpoint by signaling to inhibit Plk1 activity, which in turn prevents serine phosphorylation of cyclin B1 and nuclear accumulation of cyclin B1/Cdk1 complexes. Bypass of the decatenation checkpoint in human fibroblasts produced a high incidence of chromosomal aberrations. The chromosomes from untreated fibroblasts over-expressing ATRki displayed similar chromosomal aberrations. Taken together, these observations emphasize the importance of ATR and the decatenation checkpoint in preserving genomic stability.