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<p>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 G<sub>2</sub>M 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 <i>Rhno1</i> 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.</p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2025年12月19日

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  • Joonyoung Her,
  • Adithi Santhosh,
  • Yanira Gonzalez-Rodriguez,
  • Niphat Jirapongwattana,
  • Channabasavaiah B. Gurumurthy,
  • Adam R. Karpf,
  • Samuel F. Bunting

摘要

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.