Dpb11/TopBP1 plays distinct roles in DNA replication, checkpoint response and homologous recombination.
DPB11/TopBP1 is an essential evolutionarily conserved gene involved in initiation of DNA replication and checkpoint signaling. Here, we show that Saccharomyces cerevisiae Dpb11 forms nuclear foci that localize to sites of DNA damage in G1, S and G2 phase, a recruitment that is conserved for its homologue TopBP1 in Gallus gallus. Damage-induced Dpb11 foci are distinct from Sld3 replication initiation foci. Further, Dpb11 foci are dependent on the checkpoint proteins Mec3 (9-1-1 complex) and Rad24, and require the C-terminal domain of Dpb11. Dpb11 foci are independent of the checkpoint kinases Mec1 and Tel1, and of the checkpoint mediator Rad9. In a site-directed mutagenesis screen, we identify a separation-of-function mutant, dpb11-PF, that is sensitive to DSB-inducing agents yet remains proficient for DNA replication and the S-phase checkpoint at the permissive temperature. The dpb11-PF mutant displays altered rates of heteroallelic and direct-repeat recombination, sensitivity to DSB-inducing drugs as well as delayed kinetics of mating-type switching with a defect in the DNA synthesis step thus implicating Dpb11 in homologous recombination. We conclude that Dpb11/TopBP1 plays distinct roles in replication, checkpoint response and recombination processes, thereby contributing to chromosomal stability.
Department of Biology, University of Copenhagen, Denmark.
Susanne M
SM
Germann
Vibe H
VH
Oestergaard
Caroline
C
Haas
Pauline
P
Salis
Akira
A
Motegi
Michael
M
Lisby
D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics / Q000502:physiology*; D002645:Chickens / Q000235:genetics; D043171:Chromosomal Instability; D053903:DNA Breaks, Double-Stranded; D004249:DNA Damage; D004261:DNA Replication; D016195:G2 Phase; D018816:Genes, cdc; D047908:Intracellular Signaling Peptides and Proteins / Q000235:genetics / Q000502:physiology; D016297:Mutagenesis, Site-Directed; D011995:Recombination, Genetic; D016196:S Phase; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000502:physiology*
D018797:Cell Cycle Proteins; C097185:DPB11 protein, S cerevisiae; D047908:Intracellular Signaling Peptides and Proteins; C099938:MEC3 protein, S cerevisiae; C088385:RAD24 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C069930:rad9 protein