Cdc45-induced alterations in replication fork stability enhance cell sensitivity to camptothecin
Authors: Cynthia Lancaster, Clifford Toleman, Robert van Waardenburg, Mary-Ann Bjornsti
Journal: Cancer Research (2007)

AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA

3209 

Topoisomerase I (Top1) functions in DNA replication and transcription by catalyzing changes in DNA topology through a mechanism of transient DNA strand breakage and rejoining. Top1 is also the target of camptothecin (CPT), FDA approved analogs of which are effective new agents in the treatment of human cancers. CPT poisons Top1 by reversibly stabilizing a covalent enzyme-DNA complex. During S-phase, the collision of replication forks with CPT-Top1-DNA adducts produces DNA lesions that signal cell cycle arrest and cell death. Yet, little is known of the interactions with the replication machinery required for the formation of Top1-DNA damage. In yeast genetic screens, we isolated temperature sensitive mutants with enhanced sensitivity to Top1-DNA damage. Our characterization of yeast cdc45-10 and dpb11-10 mutants revealed functional links between the initiation of DNA replication and replication fork progression in mediating cell sensitivity to CPT. Cdc45 and Dpb11 are essential proteins, with well-defined human orthologs, CDC45 and TOPBP1, respectively. Cdc45 functions in pre-replicative complex (pre-RC) formation at origins of replication in G1 phase, and, in concert with Mcm2-7, forms the replicative helicase as forks progress along the DNA in S-phase. Here we show that a decrease in active Cdc45 enhanced yeast and human cell sensitivity to CPT-induced Top1 damage. 2-D gel analyses of origin firing and fork progression in cdc45-10 mutant cells determined that when pre-RCs, formed at 26°C, were shifted to 36°C to complete S-phase, origin firing was delayed relative to wild-type cells, but replication fork progression was unaffected. However, when pre-RCs were formed at 36°C, the replication forks formed in S-phase were extremely unstable. In the presence of CPT induced Top1-DNA adducts, such adverse effects on fork stability would increase the potential for fork collapse and the formation of lethal DNA lesions. To further investigate Cdc45 function, a gene dosage screen isolated suppressors of cdc45-10 sensitivity to Top1 poisons. Increased gene dosage of the SUMO E3 ligase, Siz1, partially restored the CPT resistance of cdc45-10 cells; however, it did not restore the viability of a double cdc45-10 , dpb11-10 mutant. These findings led us to ask if mutating the unique SUMO consensus Cdc45SUMO affected its role in DNA replication. Although cell sensitivity to CPT was unaffected, Cdc45SUMO failed to complement the synthetic lethal phenotype of the cdc45-10, dpb11-10 mutant. These data suggest that SUMO modification of Cdc45 is important for the common essential function with Dpb11. Indeed, SUMO-induced alterations in Cdc45 association with the replication machinery are being investigated in ChIPs. Together, our findings indicate the SUMO-dependent functional interactions of Cdc45 with Dpb11 are distinct from Cdc45’s function in maintaining replication fork stability in the presence of CPT.