Checkpoint Responses for Protecting Genome Integrity in S-Phase

描述(申请人提供)：保护基因组完整性对于防止可能导致癌症、衰老相关疾病、神经、免疫和发育疾病、不孕不育、出生缺陷和许多其他疾病的基因突变和染色体重排至关重要。DNA损伤触发检验点激酶ATM和ATR，使染色质两侧DNA损伤中组蛋白H_2AX的C末端磷酸化。这种磷酸-H_2AX，被称为伽马-H_2AX，作为一个信号和蛋白质对接平台来调节DNA修复和细胞周期检查点活动。虽然伽马-H_2AX在电离辐射和其他致裂剂造成的DNA双链断裂中具有众所周知的作用，但在细胞周期的DNA合成(S)阶段，它在停滞或损坏的复制叉处的作用是神秘的。我们最近发现，Gamma-Hax会招募基因组维护因子Brc1来阻止或损坏复制叉子。我们还发现，在缺乏Rqh1的情况下，Brc1与Gamma-H2AX的结合是至关重要的，Rqh1是人类BLM DNA解旋酶的同源基因，在Bloom综合征中突变。在这个项目中，我们建议：(1)发现为什么当Rqh1 DNA解旋酶缺陷时，H_2AX是至关重要的；(2)定义和表征产生对H_2AX和Brc1的关键要求的遗传缺陷；(3)评估Brc1的BRCT5-6域间连接子中的电负性表面的功能。这些研究的影响将是显著提高对S阶段如何保护基因组完整性的理解。

DESCRIPTION (provided by applicant): Protection of genome integrity is critical for preventing genetic mutations and chromosomal rearrangements that can lead to cancer, aging-related disorders, neurological, immunological and developmental diseases, infertility, birth defects and many other disorders. DNA damage triggers the checkpoint kinases ATM and ATR to phosphorylate the C-terminus of histone H2AX in chromatin flanking DNA lesions. This phospho-H2AX, known as gamma-H2AX, serves as a signaling and protein docking platform to regulate DNA repair and cell cycle checkpoint activities. While gamma-H2AX has well known roles at DNA double- strand breaks created by ionizing radiation and other clastogens, its role at stalled or damaged replication forks during the DNA synthesis (S)-phase of the cell cycle is enigmatic. We recently discovered that gamma-HAX recruits the genome maintenance factor Brc1 to stalled or damaged replication forks. We also found that Brc1 binding to gamma-H2AX is crucial in the absence of Rqh1, which is the ortholog of human BLM DNA helicase that is mutated in Bloom's Syndrome. In this project we propose to: (1), discover why ¿H2AX is crucial when Rqh1 DNA helicase is defective; (2), define and characterize the genetic deficiencies that create a critical requirement for ¿H2AX and Brc1; (3), assess the functions of the electronegative surface in the BRCT5-6 interdomain linker of Brc1. The impact of these studies will be to significantly improve the understanding of how genome integrity is protected in S-phase.