TY - JOUR
T1 - ATM and the Mre11-Rad50-Nbs1 complex respond to nucleoside analogue-induced stalled replication forks and contribute to drug resistance
AU - Ewald, Brett
AU - Sampath, Deepa
AU - Plunkett, William
PY - 2008/10/1
Y1 - 2008/10/1
N2 - The Mre11-Rad50-Nbs1 complex and autophosphorylated Ser1981-ATM are involved in recognizing and repairing DNA damage, such as double-strand breaks (DSB). However, the role of these factors in response to stalled replication forks is not clear. Nucleoside analogues are agents that are incorporated into DNA duringreplication, which cause stalling of replication forks. The molecular mechanisms that sense these events may signal for DNA repair and contribute to survival but are poorly understood. Cellular responses to both DSBs and stalled replication forks are marked by H2AX phosphorylation on Ser139 (γ-H2AX), which forms nuclear foci at sites of DNA damage. Here, concentrations of the nucleoside analogues 1-β-D- arabinofuranosylcytosine (cytarabine; ara-C), gemcitabine, and troxacitabine, which inhibited DNA synthesis by 90% within 2 hours, were determined for each agent. Using γ-H2AX as a marker for changes in chromatin structure, we show that Mre11, Rad50, Nbs1, and phosphorylated ATM respond to nucleoside analogue-induced stalled replication forks by formingnu clear foci that colocalize with γ-H2AX within 2 hours. Because neither DSBs nor single-strand breaks were detectable after nucleoside analogue exposure, we conclude that this molecular response is not due to the presence of DNA breaks. Deficiencies in ATM, Mre11, or Rad50 led to a 2- to 5-fold increase in clonogenic sensitization to gemcitabine, whereas Nbs1 and H2AX deficiency did not affect reproductive growth. Taken together, these results suggest that ATM, Mre11, and Rad50 are required for survival after replication fork stalling, whereas Nbs1 and H2AX are inconsequential.
AB - The Mre11-Rad50-Nbs1 complex and autophosphorylated Ser1981-ATM are involved in recognizing and repairing DNA damage, such as double-strand breaks (DSB). However, the role of these factors in response to stalled replication forks is not clear. Nucleoside analogues are agents that are incorporated into DNA duringreplication, which cause stalling of replication forks. The molecular mechanisms that sense these events may signal for DNA repair and contribute to survival but are poorly understood. Cellular responses to both DSBs and stalled replication forks are marked by H2AX phosphorylation on Ser139 (γ-H2AX), which forms nuclear foci at sites of DNA damage. Here, concentrations of the nucleoside analogues 1-β-D- arabinofuranosylcytosine (cytarabine; ara-C), gemcitabine, and troxacitabine, which inhibited DNA synthesis by 90% within 2 hours, were determined for each agent. Using γ-H2AX as a marker for changes in chromatin structure, we show that Mre11, Rad50, Nbs1, and phosphorylated ATM respond to nucleoside analogue-induced stalled replication forks by formingnu clear foci that colocalize with γ-H2AX within 2 hours. Because neither DSBs nor single-strand breaks were detectable after nucleoside analogue exposure, we conclude that this molecular response is not due to the presence of DNA breaks. Deficiencies in ATM, Mre11, or Rad50 led to a 2- to 5-fold increase in clonogenic sensitization to gemcitabine, whereas Nbs1 and H2AX deficiency did not affect reproductive growth. Taken together, these results suggest that ATM, Mre11, and Rad50 are required for survival after replication fork stalling, whereas Nbs1 and H2AX are inconsequential.
UR - http://www.scopus.com/inward/record.url?scp=54249084045&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=54249084045&partnerID=8YFLogxK
U2 - 10.1158/0008-5472.CAN-08-0971
DO - 10.1158/0008-5472.CAN-08-0971
M3 - Article
C2 - 18829552
AN - SCOPUS:54249084045
SN - 0008-5472
VL - 68
SP - 7947
EP - 7955
JO - Cancer Research
JF - Cancer Research
IS - 19
ER -