Dpb11/TopBP1 contributes to genomicstability via homologous recombinationand checkpoint signaling

TY - BOOK

T1 - Dpb11/TopBP1 contributes to genomicstability via homologous recombinationand checkpoint signaling

AU - Germann, Susanne Manuela

N1 - Academic advisor: Dr. Michael Lisby

PY - 2009

Y1 - 2009

N2 - Homologous recombination (HR) is essential for maintaining genome integrity and isa major pathway for repairing (DSBs). DPB11 is an essential gene conserved fromyeast to human (TopBP1), which is involved in initiation of DNA replication andDNA checkpoint signaling. We found that Dpb11 forms foci in response to doublestrand breaks (DSBs) in G1, S and G2 phase in vivo. These foci are dependent onMec3 (9-1-1 complex) as well as Rad24 (clamp loader), but independent of the HRprotein Rad52. Nevertheless, these Dpb11 foci colocalize with Rad52 in S and G1phase, and a single defined DSB is sufficient for recruitment. Also, the chickenhomologue TopBP1 colocalizes with RPA1 as well as Rad51 when DNA damage isinduced.Previously, dpb11 mutants have been shown to be sensitive to DNA-damagingagents that cause DSBs, DNA alkylation and stalled replication forks. Interestingly,we found the point mutants dpb11-PF and dpb11-R being exclusively sensitive toDSB-inducing agents, suggesting a separation of function between DNA repair andreplication. These two mutants interact significantly less with members of the 9-1-1complex or the Rad24 clamp loader in a two-hybrid analysis, suggesting a defect incheckpoint signaling. Importantly, Dpb11 foci are independent of checkpoint kinasesMec1 and Tel1, as well as Rad9, further strengthening the upstream position ofDpb11 in the DNA damage checkpoint response. Moreover, dpb11-PF has a defect inS-phase checkpoint function, albeit to a lesser extent than dpb11-1. Altered rates ofheteroallelic and direct repeat recombination implicate a role for Dpb11 inhomologous recombination. Physical monitoring of mating-type switching as a modelfor DSB repair revealed that the repair kinetics of dpb11-PF are delayed.Finally, we found Dpb11 in budding yeast as well as TopBP1 in chickenDT40 cells colocalizing to structures resembling anaphase bridges when cells are inmitosis, suggesting that these proteins are utilized to protect or process fragile sitesduring anaphase. We conclude that Dpb11/TopBP1 has distinct roles in replication,checkpoint response and recombination processes, thereby substantially contributingto genome stability.

AB - Homologous recombination (HR) is essential for maintaining genome integrity and isa major pathway for repairing (DSBs). DPB11 is an essential gene conserved fromyeast to human (TopBP1), which is involved in initiation of DNA replication andDNA checkpoint signaling. We found that Dpb11 forms foci in response to doublestrand breaks (DSBs) in G1, S and G2 phase in vivo. These foci are dependent onMec3 (9-1-1 complex) as well as Rad24 (clamp loader), but independent of the HRprotein Rad52. Nevertheless, these Dpb11 foci colocalize with Rad52 in S and G1phase, and a single defined DSB is sufficient for recruitment. Also, the chickenhomologue TopBP1 colocalizes with RPA1 as well as Rad51 when DNA damage isinduced.Previously, dpb11 mutants have been shown to be sensitive to DNA-damagingagents that cause DSBs, DNA alkylation and stalled replication forks. Interestingly,we found the point mutants dpb11-PF and dpb11-R being exclusively sensitive toDSB-inducing agents, suggesting a separation of function between DNA repair andreplication. These two mutants interact significantly less with members of the 9-1-1complex or the Rad24 clamp loader in a two-hybrid analysis, suggesting a defect incheckpoint signaling. Importantly, Dpb11 foci are independent of checkpoint kinasesMec1 and Tel1, as well as Rad9, further strengthening the upstream position ofDpb11 in the DNA damage checkpoint response. Moreover, dpb11-PF has a defect inS-phase checkpoint function, albeit to a lesser extent than dpb11-1. Altered rates ofheteroallelic and direct repeat recombination implicate a role for Dpb11 inhomologous recombination. Physical monitoring of mating-type switching as a modelfor DSB repair revealed that the repair kinetics of dpb11-PF are delayed.Finally, we found Dpb11 in budding yeast as well as TopBP1 in chickenDT40 cells colocalizing to structures resembling anaphase bridges when cells are inmitosis, suggesting that these proteins are utilized to protect or process fragile sitesduring anaphase. We conclude that Dpb11/TopBP1 has distinct roles in replication,checkpoint response and recombination processes, thereby substantially contributingto genome stability.

M3 - Ph.D. thesis

BT - Dpb11/TopBP1 contributes to genomicstability via homologous recombinationand checkpoint signaling

PB - Museum Tusculanum

CY - Biologisk Institut

ER -