Mismatch repair-dependent G2 checkpoint induced by low doses of SN1 type methylating agents requires the ATR kinase.
S(N)1-type alkylating agents represent an important class of chemotherapeutics, but the molecular mechanisms underlying their cytotoxicity are unknown. Thus, although these substances modify predominantly purine nitrogen atoms, their toxicity appears to result from the processing of O(6)-methylguanine ((6Me)G)-containing mispairs by the mismatch repair (MMR) system, because cells with defective MMR are highly resistant to killing by these agents. In an attempt to understand the role of the MMR system in the molecular transactions underlying the toxicity of alkylating agents, we studied the response of human MMR-proficient and MMR-deficient cells to low concentrations of the prototypic methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). We now show that MNNG treatment induced a cell cycle arrest that was absolutely dependent on functional MMR. Unusually, the cells arrested only in the second G(2) phase after treatment. Downstream targets of both ATM (Ataxia telangiectasia mutated) and ATR (ATM and Rad3-related) kinases were modified, but only the ablation of ATR, or the inhibition of CHK1, attenuated the arrest. The checkpoint activation was accompanied by the formation of nuclear foci containing the signaling and repair proteins ATR, the S(*)/T(*)Q substrate, gamma-H2AX, and replication protein A (RPA). The persistence of these foci implied that they may represent sites of irreparable damage.
Stojic
Lovorka
L
Institute of Molecular Cancer Research, University of Zurich, CH-8008 Zurich.
Mojas
Nina
N
Cejka
Petr
P
Di Pietro
Massimiliano
M
Ferrari
Stefano
S
Marra
Giancarlo
G
Jiricny
Josef
J
D064007:Ataxia Telangiectasia Mutated Proteins; D020137:Base Pair Mismatch; D002110:Caffeine / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects / Q000235:genetics; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D000071877:Checkpoint Kinase 1; D004247:DNA / Q000378:metabolism; D004249:DNA Damage / Q000502:physiology; D004260:DNA Repair / Q000187:drug effects / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000187:drug effects / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016195:G2 Phase / Q000187:drug effects*; D006657:Histones / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D008745:Methylation; D008769:Methylnitronitrosoguanidine / Q000633:toxicity*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D051716:Replication Protein A; D015398:Signal Transduction; D019311:Staurosporine / Q000031:analogs & derivatives* / Q000494:pharmacology; D025521:Tumor Suppressor Proteins
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; C468783:H2AX protein, human; D006657:Histones; D047428:Protein Kinase Inhibitors; C497167:RPA1 protein, human; D051716:Replication Protein A; D025521:Tumor Suppressor Proteins; D008769:Methylnitronitrosoguanidine; D002110:Caffeine; C054852:7-hydroxystaurosporine; D004247:DNA; D011494:Protein Kinases; C576324:ATM protein, human; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases; D019311:Staurosporine