The genome stability is maintained by coordinated action of DNA repairs and checkpoints, which delay progression through the cell cycle in response to DNA damage. Rad9 is conserved from yeast to human and functions in cell cycle checkpoint controls. Here, a regulatory mechanism for Rad9 function is reported. In this study Rad9 has been found to interact with and be methylated by protein arginine methyltransferase 5 (PRMT5). Arginine methylation of Rad9 plays a critical role in S/M and G2/M cell cycle checkpoints. The activation of the Rad9 downstream checkpoint effector Chk1 is impaired in cells only expressing a mutant Rad9 that cannot be methylated. Additionally, Rad9 methylation is also required for cellular resistance to DNA damaging stresses. In summary, we uncovered that arginine methylation is important for regulation of Rad9 function, and thus is a major element for maintaining genome integrity.

Cellular DNA lesions are efficiently countered by DNA repair in conjunction with delays in cell cycle progression. Previous studies have demonstrated that Rad9, Hus1, and Rad1 can form a heterotrimeric complex (the 9-1-1 complex) that plays dual roles in cell cycle checkpoint activation and DNA repair in eukaryotic cells. Although the 9-1-1 complex has been proposed to form a toroidal structure similar to proliferating cell nuclear antigen (PCNA), which plays essential roles in DNA replication and repair, the structural basis by which it performs different functions has not been elucidated. Here we report the crystal structure of the human 9-1-1 complex at 3.2 Å resolution. The crystal structure, together with biochemical assays, reveals that the interdomain connecting loops (IDC loop) of hRad9, hHus1, and hRad1 are largely divergent, and further cocrystallization study indicates that a PCNA-interacting box (PIP box)-containing peptide derived from hFen1 binds tightly to the interdomain connecting loop of hRad1, providing the molecular basis for the damage repair-specific activity of the 9-1-1 complex in contrast to PCNA. Furthermore, structural comparison with PCNA reveals other unique structural features of the 9-1-1 complex that are proposed to contribute to DNA damage recognition.

Microgravity (MG) and space radiation are two major environmental factors of space environment. Ionizing radiation generates reactive oxygen species (ROS) which plays a key role in radiation-induced DNA damage. Interestingly, simulated microgravity (SMG) also increases ROS production in various cell types. Thus, it is important to detect whether SMG could potentiate ROS production induced by genotoxins including radiation, especially at a minimal level not sufficient to induce detectable ROS. In this study, we treated mouse embryonic stem (MES) cells with H₂O₂ and SMG for 24 h. The concentration of H₂O₂ used was within 30 μmol/L at which intracellular ROS was the same as that in untreated cells. Exposure of cells to SMG for 24 h did not induce significantly higher levels of intracellular ROS than that of control cells either. Simultaneous exposure of cells to both SMG- and H₂O₂-induced ROS and apoptosis in MES cells. Although incubation in medium containing 5 or 30 μmol/L H₂O₂ induced a small enhancement of DNA double-strand breaks (DSBs), the addition of SMG treatment dramatically increased DSB levels. Taken together, SMG can significantly potentiate the effects of H₂O₂ at a low concentration that induce a small or negligible change in cells on ROS, apoptosis, and DNA damage. The results were discussed in relation to the combined effects of space radiation and MG on human body in this study.

Background: Cells are constantly exposed to stresses from cellular metabolites as well as environmental genotoxins. DNA damage caused by these genotoxins can be efficiently fixed by DNA repair in cooperation with cell cycle checkpoints. Unrepaired DNA lesions can lead to cell death, gene mutation and cancer. The Rad1 protein, evolutionarily conserved from yeast to humans, exists in cells as monomer as well as a component in the 9-1-1 protein complex. Rad1 plays crucial roles in DNA repair and cell cycle checkpoint control, but its contribution to carcinogenesis is unknown.Results: To address this question, we constructed mice with a deletion ofMrad1. Matings between heterozygousMrad1mutant mice producedMrad1^+/+andMrad1^+/-but noMrad1^-/-progeny, suggesting theMrad1null is embryonic lethal.Mrad1^+/-mice demonstrated no overt abnormalities up to one and half years of age. DMBA-TPA combinational treatment was used to induce tumors on mouse skin. Tumors were larger, more numerous, and appeared earlier on the skin ofMrad1^+/-mice compared toMrad1^+/+animals. Keratinocytes isolated fromMrad1^+/-mice had significantly more spontaneous DNA double strand breaks, proliferated slower and had slightly enhanced spontaneous apoptosis thanMrad1^+/+control cells.Conclusion: These data suggest thatMrad1is important for preventing tumor development, probably through maintaining genomic integrity. The effects of heterozygous deletion ofMrad1on proliferation and apoptosis of keratinocytes is different from those resulted fromMrad9heterozygous deletion (from our previous study), suggesting thatMrad1also functions independent ofMrad9besides its role in the Mrad9-Mrad1-Mhus1 complex in mouse cells.

Activation-induced cytidine deaminase (AID) is required for the generation of antibody diversity through initiating both somatic hypermutation (SHM) and class switch recombination. A few research groups have successfully used the feature of AID for generating mutant libraries in directed evolution of target proteins in B cells in vitro. B cells, cultured in suspension, are not convenient for transfection and cloning. In this study, we established an AID-based mutant accumulation and sorting system in adherent human cells. Mouse AID gene was first transfected into the human non-small cell lung carcinoma H1299 cells, and a stable cell clone (H1299-AID) was selected. Afterwards, anti-hTNF-α scFv (ATscFv) was transfected into H1299-AID cells and ATscFv was displayed on the surface of H1299-AID cells. By 4-round amplification/flow cytometric sorting for cells with the highest affinities to hTNF-alpha, two ATscFv mutant gene clones were isolated. Compared with the wild type ATscFv, the two mutants were much more efficient in neutralizing cytotoxicity of hTNF-alpha. The results indicate that directed evolution by somatic hypermutation can be carried out in adherent non-B cells, which makes directed evolution in mammalian cells easier and more efficient.