Nitrosative stress suppresses checkpoint activation after DNA synthesis inhibition.
DNA synthesis is promoted by the dephosphorylation and activation of cyclin-dependent kinase 2 (Cdk2) complexes by Cdc25A. Nitrosative stress suppresses Cdk2 dephosphorylation by Cdc25A in vitro and inhibits Cdc25A protein translation in cells, but the effects on S-phase progression remain unexamined. Herein we report that nitrosative stress catalyzed by inducible nitric oxide (*NO) synthase (iNOS) or the chemical nitrosant S-nitrosocysteine ethyl ester (SNCEE) rapidly inhibited DNA synthesis concomitant with Cdc25A loss. Surprisingly, this inhibition of DNA synthesis was refractory to ectopic expression of Cdc25A or a Cdc25-independent Cdk2 mutant. Nitrosative stress inhibited DNA synthesis without activating checkpoint signaling, thus distinguishing it from S-phase arrest mediated by other reactive *NO-derived species. The apparent lack of checkpoint activation was due to an active suppression because accumulation of pSer345-Chk1, pThr68-Chk2 and gammaH2AX was inhibited by nitrosative stress in cells exposed to DNA damage or replication inhibitors. We speculate that failure to activate the S-phase checkpoint in precancerous cells undergoing nitrosative stress may elevate the risk of transmitting damaged genomes to daughter cells upon cell cycle reentry.
Tomko
Robert J
RJ
Department of Pharmacology and Chemical Biology and University of Pittsburgh Drug Discovery Institute, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA.
Azang-Njaah
Ndang N
NN
Lazo
John S
JS
D002453:Cell Cycle; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D051357:Cyclin-Dependent Kinase 2 / Q000378:metabolism; D004249:DNA Damage; D004261:DNA Replication; D045325:HCT116 Cells; D006801:Humans; D009569:Nitric Oxide / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D016196:S Phase; D020687:cdc25 Phosphatases / Q000378:metabolism
D009569:Nitric Oxide; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C000605867:CHEK1 protein, human; C578634:CHEK2 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases; D051357:Cyclin-Dependent Kinase 2; D020687:cdc25 Phosphatases