title: DDK dependent regulation of TOP2A at centromeres revealed by a chemical genetics approach.
abstract: In eukaryotic cells the CDC7/DBF4 kinase, also known as DBF4-dependent kinase (DDK), is required for the firing of DNA replication origins. CDC7 is also involved in replication stress responses and its depletion sensitises cells to drugs that affect fork progression, including Topoisomerase 2 poisons. Although CDC7 is an important regulator of cell division, relatively few substrates and bona-fide CDC7 phosphorylation sites have been identified to date in human cells. In this study, we have generated an active recombinant CDC7/DBF4 kinase that can utilize bulky ATP analogues. By performing in vitro kinase assays using benzyl-thio-ATP, we have identified TOP2A as a primary CDC7 substrate in nuclear extracts, and serine 1213 and serine 1525 as in vitro phosphorylation sites. We show that CDC7/DBF4 and TOP2A interact in cells, that this interaction mainly occurs early in S-phase, and that it is compromised after treatment with CDC7 inhibitors. We further provide evidence that human DBF4 localises at centromeres, to which TOP2A is progressively recruited during S-phase. Importantly, we found that CDC7/DBF4 down-regulation, as well S1213A/S1525A TOP2A mutations can advance the timing of centromeric TOP2A recruitment in S-phase. Our results indicate that TOP2A is a novel DDK target and have important implications for centromere biology.
authors.affiliation: Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Ireland.
authors.forename: Kevin Z L
authors.initials: KZ
authors.lastname: Wu
authors.affiliation: Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Ireland.
authors.forename: Guan-Nan
authors.initials: GN
authors.lastname: Wang
authors.affiliation: Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Ireland.
authors.forename: Jennifer
authors.initials: J
authors.lastname: Fitzgerald
authors.affiliation: Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Ireland.
authors.forename: Huong
authors.initials: H
authors.lastname: Quachthithu
authors.affiliation: Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Ireland.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Rainey
authors.affiliation: IFOM-FIRC Institute of Molecular Oncology, Milan 20139, Italy.
authors.forename: Angela
authors.initials: A
authors.lastname: Cattaneo
authors.affiliation: IFOM-FIRC Institute of Molecular Oncology, Milan 20139, Italy.
authors.forename: Angela
authors.initials: A
authors.lastname: Bachi
authors.affiliation: Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway, Ireland corrado.santocanale@nuigalway.ie.
authors.forename: Corrado
authors.initials: C
authors.lastname: Santocanale
mesh_terms: D000951:Antigens, Neoplasm / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D002503:Centromere / Q000235:genetics* / Q000378:metabolism*; D004261:DNA Replication; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation; D000075223:Poly-ADP-Ribose Binding Proteins; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D018741:Replication Origin; D016196:S Phase
chemical_list: D000951:Antigens, Neoplasm; D018797:Cell Cycle Proteins; C485678:DBF4 protein, human; D004268:DNA-Binding Proteins; D000075223:Poly-ADP-Ribose Binding Proteins; C068619:CDC7 protein, human; D017346:Protein Serine-Threonine Kinases; D004250:DNA Topoisomerases, Type II; C000618811:TOP2A protein, human