Methylator-induced, mismatch repair-dependent G2 arrest is activated through Chk1 and Chk2.
SN1 DNA methylating agents such as the nitrosourea N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) elicit a G2/M checkpoint response via a mismatch repair (MMR) system-dependent mechanism; however, the exact nature of the mechanism governing MNNG-induced G2/M arrest and how MMR mechanistically participates in this process are unknown. Here, we show that MNNG exposure results in activation of the cell cycle checkpoint kinases ATM, Chk1, and Chk2, each of which has been implicated in the triggering of the G2/M checkpoint response. We document that MNNG induces a robust, dose-dependent G2 arrest in MMR and ATM-proficient cells, whereas this response is abrogated in MMR-deficient cells and attenuated in ATM-deficient cells treated with moderate doses of MNNG. Pharmacological and RNA interference approaches indicated that Chk1 and Chk2 are both required components for normal MNNG-induced G2 arrest. MNNG-induced nuclear exclusion of the cell cycle regulatory phosphatase Cdc25C occurred in an MMR-dependent manner and was compromised in cells lacking ATM. Finally, both Chk1 and Chk2 interact with the MMR protein MSH2, and this interaction is enhanced after MNNG exposure, supporting the notion that the MMR system functions as a molecular scaffold at the sites of DNA damage that facilitates activation of these kinases.
Adamson
Aaron W
AW
Department of Biochemistry and Molecular Biology and the Stanley S. Scott Cancer Center, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA.
Beardsley
Dillon I
DI
Kim
Wan-Ju
WJ
Gao
Yajuan
Y
Baskaran
R
R
Brown
Kevin D
KD
D020137:Base Pair Mismatch; D002455:Cell Division; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D004249:DNA Damage; D019175:DNA Methylation; D004260:DNA Repair; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005434:Flow Cytometry; D016195:G2 Phase; D006801:Humans; D015151:Immunoblotting; D047468:Immunoprecipitation; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D051718:MutS Homolog 2 Protein; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000235:genetics / Q000502:physiology*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000378:metabolism / Q000502:physiology*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D019311:Staurosporine / Q000031:analogs & derivatives* / Q000494:pharmacology; D013347:Subcellular Fractions; D013997:Time Factors
D004268:DNA-Binding Proteins; D047428:Protein Kinase Inhibitors; D011518:Proto-Oncogene Proteins; D034741:RNA, Small Interfering; D008769:Methylnitronitrosoguanidine; C054852:7-hydroxystaurosporine; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C000605867:CHEK1 protein, human; C578634:CHEK2 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases; C497172:MSH2 protein, human; D051718:MutS Homolog 2 Protein; D019311:Staurosporine