Defects in DNA damage signaling and cell cycle checkpoints in a mouse model of Rhno1 deletion

In response to DNA damage or DNA replication stress, cells activate signaling pathways dependent on the kinase, ATR (Ataxia Telangiectasia and Rad3-Related). ATR signaling leads to induction of cell cycle checkpoints, a pause in DNA replication, and upregulation of DNA repair activities. In response to replication stress, ATR is activated by TOPBP1 (Topoisomerase II beta-Binding Protein 1) associated with the 9-1-1 (Rad9-Hus1-Rad1) complex. The three proteins that make up the 9-1-1 complex form a ring encircling DNA at damage sites and help localize TOPBP1 and ATR to signal the presence of damage or replication stress. RHNO1 (Rad9, Hus1, and Rad1-associated Nuclear Orphan 1) was identified as a protein that binds to components of the 9-1-1 complex to promote ATR signaling. Previous studies in cell lines have revealed that RHNO1 activity is required for maintenance of the G₂M cell cycle checkpoint after ionizing radiation treatment, and for DNA repair in mitotic cells. In this study, we report a loss-of-function mouse model, in which Rhno1 is deleted in B lymphocytes, allowing us to test the function of RHNO1 in primary cells. We find that RHNO1 is broadly expressed in mouse tissues but is dispensable for B cell growth under normal conditions. RHNO1-deficient B cells nevertheless show altered checkpoint responses and reduced ability to repair DNA damage in M phase. Whereas initial ATR activation after ionizing radiation treatment appears normal in RHNO1-deficient cells, ATR/CHK1 signaling is reduced at later timepoints. Joining of DNA breaks during class switch recombination, which is dependent on nonhomologous end-joining, is not significantly affected by loss of RHNO1. These results demonstrate that RHNO1, unlike other proteins required for ATR-CHK1 signaling, is not essential for growth of primary cells, but has specific roles in regulating responses to cell stress.