Interaction between human mismatch repair recognition proteins and checkpoint sensor Rad9-Rad1-Hus1

Abstract

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 (hMutSα) and hMSH2/hMSH3 (hMutSβ) are the mismatch recognition factors of the mismatch repair pathway. hMutSα 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 hMutSα and hMutLα. 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 hMutSα binding with G/T-containing DNA. Rad9, Rad1, and Hus1 individual subunits can also stimulate the DNA binding activity of hMutSα. 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.

Clamping down on mammalian meiosis

“…41 Furthermore, human RAD9A and MSH6 colocalize in nuclear foci following DNA methylating agent treatment, and RAD9A focus formation is dependent upon the presence of MSH6. 41 Li and coworkers also demonstrated that RAD9A is important for nucleotide excision repair by maintaining, among others, the protein level of the excision factor DDB2 in human cells, 43 and RAD9A-deficient mouse embryonic stem cells are slow to repair UV-induced DNA damage. 44 These repair-related functions of 9-1-1 are likely to be relevant to meiotic chromosome biology, as many of the DNA repair factors mentioned here have known or predicted roles in meiotic cells.…”

“…40 Multiple interactions have also been reported between 9-1-1 subunits and the DNA mismatch repair factors MutS homolog 2 (MSH2), MSH3, MSH6, and MutL homolog 1 (MLH1), 41,42 and mismatch recognition by the MSH proteins is stimulated by the presence of 9-1-1. 41 Furthermore, human RAD9A and MSH6 colocalize in nuclear foci following DNA methylating agent treatment, and RAD9A focus formation is dependent upon the presence of MSH6.…”

“…40 Multiple interactions have also been reported between 9-1-1 subunits and the DNA mismatch repair factors MutS homolog 2 (MSH2), MSH3, MSH6, and MutL homolog 1 (MLH1), 41,42 and mismatch recognition by the MSH proteins is stimulated by the presence of 9-1-1. 41 Furthermore, human RAD9A and MSH6 colocalize in nuclear foci following DNA methylating agent treatment, and RAD9A focus formation is dependent upon the presence of MSH6. 41 Li and coworkers also demonstrated that RAD9A is important for nucleotide excision repair by maintaining, among others, the protein level of the excision factor DDB2 in human cells, 43 and RAD9A-deficient mouse embryonic stem cells are slow to repair UV-induced DNA damage.…”

HUS1 (HUS1 checkpoint homolog (S. pombe))

“…Thus, the 9-1-1 complex may provide a platform for the assembly and function of the BER machinery (Balakrishnan et al, 2009;Lu et al, 2006). The 9-1-1 complex enhances mismatch repair via direct interaction with mismatch recognition proteins (MSH2/MSH3, MSH2/MSH6, and MLH1/PMS2) (Bai et al, 2010;He et al, 2008). Hus1 interacts with MSH2/MSH3 and MSH2/MSH6, but not with MLH1/PMS2 (Bai et al, 2010;He et al, 2008).…”

“…The 9-1-1 complex enhances mismatch repair via direct interaction with mismatch recognition proteins (MSH2/MSH3, MSH2/MSH6, and MLH1/PMS2) (Bai et al, 2010;He et al, 2008). Hus1 interacts with MSH2/MSH3 and MSH2/MSH6, but not with MLH1/PMS2 (Bai et al, 2010;He et al, 2008). In the NER pathway, interactions between Saccharomyces cerevisiae Rad14 (hXPA homolog) and the checkpoint proteins ScDdc1 (hRad9 homolog) and ScMec3 (hHus1 homolog) have been demonstrated (Giannattasio et al, 2004).…”