title: DNA Damage Response Checkpoint Activation Drives KP1019 Dependent Pre-Anaphase Cell Cycle Delay in S. cerevisiae.
abstract: Careful regulation of the cell cycle is required for proper replication, cell division, and DNA repair. DNA damage--including that induced by many anticancer drugs--results in cell cycle delay or arrest, which can allow time for repair of DNA lesions. Although its molecular mechanism of action remains a matter of debate, the anticancer ruthenium complex KP1019 has been shown to bind DNA in biophysical assays and to damage DNA of colorectal and ovarian cancer cells in vitro. KP1019 has also been shown to induce mutations and induce cell cycle arrest in Saccharomyces cerevisiae, suggesting that budding yeast can serve as an appropriate model for characterizing the cellular response to the drug. Here we use a transcriptomic approach to verify that KP1019 induces the DNA damage response (DDR) and find that KP1019 dependent expression of HUG1 requires the Dun1 checkpoint; both consistent with KP1019 DDR in budding yeast. We observe a robust KP1019 dependent delay in cell cycle progression as measured by increase in large budded cells, 2C DNA content, and accumulation of Pds1 which functions to inhibit anaphase. Importantly, we also find that deletion of RAD9, a gene required for the DDR, blocks drug-dependent changes in cell cycle progression, thereby establishing a causal link between the DDR and phenotypes induced by KP1019. Interestingly, yeast treated with KP1019 not only delay in G2/M, but also exhibit abnormal nuclear position, wherein the nucleus spans the bud neck. This morphology correlates with short, misaligned spindles and is dependent on the dynein heavy chain gene DYN1. We find that KP1019 creates an environment where cells respond to DNA damage through nuclear (transcriptional changes) and cytoplasmic (motor protein activity) events.
authors.affiliation: Department of Biology, Rhodes College, Memphis, Tennessee, United States of America.
authors.forename: Lindsey A
authors.initials: LA
authors.lastname: Bierle
authors.affiliation: Program in Biochemistry and Molecular Biology, Rhodes College, Memphis, Tennessee, United States of America.
authors.forename: Kira L
authors.initials: KL
authors.lastname: Reich
authors.affiliation: Program in Biochemistry and Molecular Biology, Rhodes College, Memphis, Tennessee, United States of America.
authors.forename: Braden E
authors.initials: BE
authors.lastname: Taylor
authors.affiliation: Program in Biochemistry and Molecular Biology, Rhodes College, Memphis, Tennessee, United States of America.
authors.forename: Eliot B
authors.initials: EB
authors.lastname: Blatt
authors.affiliation: Department of Biology, Birmingham-Southern College, Birmingham, Alabama, United States of America.
authors.forename: Sydney M
authors.initials: SM
authors.lastname: Middleton
authors.affiliation: Program in Biochemistry and Molecular Biology, Rhodes College, Memphis, Tennessee, United States of America.
authors.forename: Shawnecca D
authors.initials: SD
authors.lastname: Burke
authors.affiliation: Department of Chemistry, Birmingham Southern College, Birmingham, Alabama, United States of America.
authors.forename: Laura K
authors.initials: LK
authors.lastname: Stultz
authors.affiliation: Department of Biology, Birmingham-Southern College, Birmingham, Alabama, United States of America.
authors.forename: Pamela K
authors.initials: PK
authors.lastname: Hanson
authors.affiliation: Department of Pathology, St. Jude Children's Research Hospital, Memphis, Tennessee, United States of America.
authors.forename: Janet F
authors.initials: JF
authors.lastname: Partridge
authors.affiliation: Department of Biology, Rhodes College, Memphis, Tennessee, United States of America; Program in Biochemistry and Molecular Biology, Rhodes College, Memphis, Tennessee, United States of America.
authors.forename: Mary E
authors.initials: ME
authors.lastname: Miller
mesh_terms: D000705:Anaphase / Q000187:drug effects*; D015415:Biomarkers / Q000378:metabolism*; D015153:Blotting, Western; D002453:Cell Cycle / Q000187:drug effects*; D004249:DNA Damage / Q000187:drug effects*; D004260:DNA Repair / Q000187:drug effects*; D005434:Flow Cytometry; D020869:Gene Expression Profiling; D007191:Indazoles / Q000494:pharmacology*; D009942:Organometallic Compounds / Q000494:pharmacology*; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017975:Ruthenium Compounds; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D008941:Spindle Apparatus / Q000187:drug effects
chemical_list: D015415:Biomarkers; D007191:Indazoles; D009942:Organometallic Compounds; D012333:RNA, Messenger; D017975:Ruthenium Compounds; D029701:Saccharomyces cerevisiae Proteins; C499292:indazolium trans-(tetrachlorobis(1H-indazole)ruthenate (III))