Deletion of Mouse Rad9 Causes Abnormal Cellular Responses to DNA Damage, Genomic Instability, and Embryonic Lethality

Abstract

The fission yeast Schizosaccharomyces pombe rad9 gene promotes cell survival through activation of cell cycle checkpoints induced by DNA damage. Mouse embryonic stem cells with a targeted deletion of Mrad9, the mouse ortholog of this gene, were created to evaluate its function in mammals. Mrad9؊/؊ cells demonstrated a marked increase in spontaneous chromosome aberrations and HPRT mutations, indicating a role in the maintenance of genomic integrity. These cells were also extremely sensitive to UV light, gamma rays, and hydroxyurea, and heterozygotes were somewhat sensitive to the last two agents relative to Mrad9 ؉/؉ controls. Mrad9 ؊/؊ cells could initiate but not maintain gamma-ray-induced G 2 delay and retained the ability to delay DNA synthesis rapidly after UV irradiation, suggesting that checkpoint abnormalities contribute little to the radiosensitivity observed. Ectopic expression of Mrad9 or human HRAD9 complemented Mrad9 ؊/؊ cell defects, indicating that the gene has radioresponse and genomic maintenance functions that are evolutionarily conserved. Mrad9 ؉/؊ mice were generated, but heterozygous intercrosses failed to yield Mrad9 ؊/؊ pups, since embryos died at midgestation. Furthermore, Mrad9 ؊/؊ mouse embryo fibroblasts were not viable. These investigations establish Mrad9 as a key mammalian genetic element of pathways that regulate the cellular response to DNA damage, maintenance of genomic integrity, and proper embryonic development.

The effect of Hus1 on ionizing radiation sensitivity is associated with homologous recombination repair but is independent of nonhomologous end-joining

“…However, as shown here, Hus1 deficient cells show increased sensitivity to IR-induced killing when using the clonogenic assay that measures reproductive ability. These results support the model that Hus1 associates with Rad1 and Rad9 as a 9-1-1 complex to respond to IR-induced DNA damage because Rad9 deficient cells are hypersensitive to IR-induced killing (Roos-Mattjus et al, 2003;Hopkins et al, 2004). IRinduced DNA double strands breaks (DSBs) are the most severe threat for cell survival.…”

“…Rad9 is involved in IRinduced DNA damage response and Rad9 deficient cells are hypersensitive to IR-induced killing (Roos-Mattjus et al, 2003;Hopkins et al, 2004). It is reasoned, then, that Hus1 should also affect the sensitivity of mammalian cells to IR-induced killing.…”

The role of DNA exonucleases in protecting genome stability and their impact on ageing

“…hRad9 alone (not as part of the 9-1-1 complex) is also implicated in numerous other pathways; for example in ribonucleotide synthesis via stimulation of the carbamoyl phosphate synthetase activity of CAD ) and in apoptosis by interaction with members of the Bcl-2 family of proteins (Komatsu et al 2000). Mouse rad9 nulls are not viable (Hopkins et al 2004) presumably because Rad9 has so many different roles that there cannot be full complementation of all its activities. The exact role(s) of hRad9 nuclease activity has not yet been elucidated, nor its link with ageing, but in the nematode worm C. elegans, the 9-1-1 complex is implicated in telomere maintenance (Meier et al 2006).…”

Chemopreventive Potential of an Ethyl Acetate Fraction from Curcuma Longa is Associated with Upregulation of p57kip2and Rad9 in the PC-3M Prostate Cancer Cell Line

“…Further, in LNCaP cells, over-expression of p57 kip2 inhibited tumor formation in nude mice and the prostate of p57 kip2 knockout mice developed prostatic intraepithelial neoplasia and adenocarcinoma suggesting that p57 kip2 is an important gene in prostate cancer tumorigenesis, and the p57 kip2 pathway may be a potential target for prostate cancer prevention and therapy, more specifically, during turmeric chemoprevention (Jin et al, 2004(Jin et al, : 2008Williams et al, 2005). The Rad9 gene has many functions that could bear on carcinogenesis, including a role in maintaining genome integrity and regulating cell cycle checkpoints (Lieberman et al, 1996;Bessho & Sancar, 2000;Komatsu et al, 2000;Hopkins et al, 2004;Aiping et al, 2008). Rad9 protein can bind AR and this protein-protein interaction represses the ability of testosterone to induce a conformational change in the receptor, to activate a receptor transcription regulatory function, and subsequently to express downstream target genes critical for proper prostate function Hsu et al, 2005;Lieberman, 2006).…”

“…HRad9 is an evolutionarily conserved human gene important for promoting resistance to DNA damage and regulating cell cycle checkpoints (Lieberman et al, 1996). The encoded protein can induce apoptosis (Komatsu et al, 2000), and regulate genomic stability (Hopkins et al, 2004). It has 3'to 5' exonuclease activity (Bessho et al, 2000), can bind p53 consensus DNA-binding sequences and up-regulate transcription of p21, as well as other downstream genes (Aiping et al, 2008).…”