ATM is required for the repair of Topotecan-induced replication-associated double-strand breaks.
OBJECTIVE
DNA replication is a promising target for anti-cancer therapies. Therefore, the understanding of replication-associated DNA repair mechanisms is of great interest. One key factor of DNA double-strand break (DSB) repair is the PIK kinase Ataxia-Telangiectasia Mutated (ATM) but it is still unclear whether ATM is involved in the repair of replication-associated DSBs. Here, we focused on the involvement of ATM in homology-directed repair (HDR) of indirect DSBs associated with replication.


METHODS
Experiments were performed using ATM-deficient and -proficient human cells. Replication-associated DSBs were induced with Topotecan (TPT) and compared with γ-irradiation (IR). Cell survival was measured by clonogenic assay. Overall DSB repair and HDR were evaluated by detecting residual γH2AX/53BP1 and Rad51 foci, respectively. Cell cycle distribution was analysed by flow cytometry and protein expression by Western blot.


RESULTS
ATM-deficiency leads to enhanced numbers of residual DSBs, resulting in a pronounced S/G2-block and decreased survival upon TPT-treatment. In common with IR, persisting Rad51 foci were detected following TPT-treatment.


CONCLUSIONS
These results demonstrate that ATM is essentially required for the completion of HR-mediated repair of TPT-induced DSBs formed indirectly at replication forks.
Heinrich-Pette-Institute Leibniz-Institute for Experimental Virology, Hamburg, Germany; Institute of Radiobiology and Molecular Radiation Oncology, Philipps-University of Marburg, Germany. Electronic address: sabrina.koecher@hpi.uni-hamburg.de.
Sabrina
S
Köcher
Anja
A
Spies-Naumann
Malte
M
Kriegs
Jochen
J
Dahm-Daphi
Irena
I
Dornreiter
D064007:Ataxia Telangiectasia Mutated Proteins / Q000502:physiology*; D002453:Cell Cycle / Q000187:drug effects; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D006801:Humans; D059004:Topoisomerase I Inhibitors / Q000494:pharmacology*; D019772:Topotecan / Q000494:pharmacology*
D059004:Topoisomerase I Inhibitors; D019772:Topotecan; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins
ATM; Homology-directed repair; Replication-associated DSBs; SKX; Topotecan