Protein phosphatase 2A antagonizes ATM and ATR in a Cdk2- and Cdc7-independent DNA damage checkpoint.
We previously used a soluble cell-free system derived from Xenopus eggs to investigate the role of protein phosphatase 2A (PP2A) in chromosomal DNA replication. We found that immunodepletion of PP2A or inhibition of PP2A by okadaic acid (OA) inhibits initiation of DNA replication by preventing loading of the initiation factor Cdc45 onto prereplication complexes. Evidence was provided that PP2A counteracts an inhibitory protein kinase that phosphorylates and inactivates a crucial Cdc45 loading factor. Here, we report that the inhibitory effect of OA is abolished by caffeine, an inhibitor of the checkpoint kinases ataxia-telangiectasia mutated protein (ATM) and ataxia-telangiectasia related protein (ATR) but not by depletion of ATM or ATR from the extract. Furthermore, we demonstrate that double-strand DNA breaks (DSBs) cause inhibition of Cdc45 loading and initiation of DNA replication and that caffeine, as well as immunodepletion of either ATM or ATR, abolishes this inhibition. Importantly, the DSB-induced inhibition of Cdc45 loading is prevented by addition of the catalytic subunit of PP2A to the extract. These data suggest that DSBs and OA prevent Cdc45 loading through different pathways, both of which involve PP2A, but only the DSB-induced checkpoint implicates ATM and ATR. The inhibitory effect of DSBs on Cdc45 loading does not result from downregulation of cyclin-dependent kinase 2 (Cdk2) or Cdc7 activity and is independent of Chk2. However, it is partially dependent on Chk1, which becomes phosphorylated in response to DSBs. These data suggest that PP2A counteracts ATM and ATR in a DNA damage checkpoint in Xenopus egg extracts.
Department of Pathology 0612, University of California at San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0612, USA.
Paris
P
Petersen
Danny M
DM
Chou
Zhongsheng
Z
You
Tony
T
Hunter
Johannes C
JC
Walter
Gernot
G
Walter
D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D002110:Caffeine / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000187:drug effects / Q000378:metabolism*; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D051357:Cyclin-Dependent Kinase 2 / Q000187:drug effects / Q000378:metabolism*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004249:DNA Damage / Q000502:physiology*; D004261:DNA Replication / Q000187:drug effects; D004268:DNA-Binding Proteins / Q000187:drug effects / Q000378:metabolism*; D005260:Female; D019319:Okadaic Acid / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000187:drug effects / Q000378:metabolism*; D011494:Protein Kinases / Q000187:drug effects / Q000378:metabolism; D054648:Protein Phosphatase 2; D017346:Protein Serine-Threonine Kinases / Q000187:drug effects / Q000378:metabolism*; D025521:Tumor Suppressor Proteins / Q000187:drug effects / Q000378:metabolism*; D029867:Xenopus Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*
C502211:Cdc45 protein, Xenopus; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D025521:Tumor Suppressor Proteins; D029867:Xenopus Proteins; D019319:Okadaic Acid; D002110:Caffeine; D004247:DNA; D011494:Protein Kinases; C576898:Atr protein, Xenopus; C489405:CDC7 protein, Xenopus; D064447:Checkpoint Kinase 2; D064007:Ataxia Telangiectasia Mutated Proteins; D000071877:Checkpoint Kinase 1; C000605875:Chek1 protein, Xenopus; D017346:Protein Serine-Threonine Kinases; D051357:Cyclin-Dependent Kinase 2; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2