Interaction between human mismatch repair recognition proteins and checkpoint sensor Rad9-Rad1-Hus1.
In eukaryotic cells, the cell cycle checkpoint proteins Rad9, Rad1, and Hus1 form the 9-1-1 complex which is structurally similar to the proliferating cell nuclear antigen (PCNA) sliding clamp. hMSH2/hMSH6 (hMutS alpha) and hMSH2/hMSH3 (hMutS beta) are the mismatch recognition factors of the mismatch repair pathway. hMutS alpha has been shown to physically and functionally interact with PCNA. Moreover, DNA methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment induces the G2/M cell cycle arrest that is dependent on the presence of hMutS alpha and hMutL alpha. In this study, we show that each subunit of the human 9-1-1 complex physically interacts with hMSH2, hMSH3, and hMSH6. The 9-1-1 complex from both humans and Schizosaccharomyces pombe can stimulate hMutS alpha binding with G/T-containing DNA. Rad9, Rad1, and Hus1 individual subunits can also stimulate the DNA binding activity of hMutS alpha. Human Rad9 and hMSH6 colocalize to nuclear foci of HeLa cells after exposure to MNNG. However, Rad9 does not form foci in MSH6 defective cells following MNNG treatment. In Rad9 knockdown untreated cells, the majority of the MSH6 is in cytoplasm. Following MNNG treatment, Rad9 knockdown cells has abnormal nuclear morphology and MSH6 is distributed around nuclear envelop. Our findings suggest that the 9-1-1 complex is a component of the mismatch repair involved in MNNG-induced damage response.
Bai
Haibo
H
Department of Biochemistry and Molecular Biology, University of Maryland, Baltimore, MD 21201, USA.
Madabushi
Amrita
A
Guan
Xin
X
Lu
A-Lien
AL
D020137:Base Pair Mismatch; D001483:Base Sequence; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002467:Cell Nucleus / Q000378:metabolism; D019175:DNA Methylation / Q000187:drug effects; D053843:DNA Mismatch Repair; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005092:Exonucleases / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008769:Methylnitronitrosoguanidine / Q000494:pharmacology; D008969:Molecular Sequence Data; D021122:Protein Subunits / Q000378:metabolism; D021381:Protein Transport
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C549605:HUS1 protein, human; C462949:HUS1B protein, human; D021122:Protein Subunits; D008769:Methylnitronitrosoguanidine; C069930:rad9 protein; D005092:Exonucleases; C113574:Rad1 protein, human