XPF/ERCC1复合物在抗体类别转换中介导DNA双链断裂修复的机制研究

Class switch recombination (CSR) in mature B cells is a type of antibody gene rearrangement that replaces the constant region of an originally expressed IgM class of antibody with a different one for different downstream effector functions without altering its antigen recognition specificity. On the molecular level, CSR involves generation of site-specific DNA double-strand breaks (DSBs) in the immunoglobulin heavy chain switch regions that are subsequently recognized by ATM-dependent DNA damage response (DDR) and fused by non-homologous end joining (NHEJ) mechanisms. Previous reports showed that in classical NHEJ-deficient B cells, CSR can still occur with a relative high frequency, implicating the existence of alternative NHEJ pathways for the completion of CSR, but the molecules and how they function to mediate alternative NHEJ remain largely unknown. Through an innovative next-generation DNA sequencing method, we found that extensive resection and elevated microhomology usage take place in NHEJ-deficient cells, but the enzyme(s) to process such resection-generated long single strand DNA before ligation of broken ends remains elusive. Based on previous studies, we propose that structure-specific DNA endonuclease XPF/ERCC1 complex functions to remove these long single strand DNA flaps for alternative NHEJ in classical NHEJ-deficient cells. We plan to generate mutant B cells defective in both classical NHEJ factors and ERCC1/XPF complex components, and to employ conventional molecular immunology techniques and high-throughput sequencing to investigate the potential roles of ERCC1/XPF complex in mediating alternative-NHEJ. The completion of this study will not only shed new light on the mechanisms of how different DSB end joining pathways facilitate CSR and antibody maturation, but also provide new candidates for the clinical diagnosis and treatment of primary antibody deficiencies.

B细胞抗体类别转换(CSR)通过在重链基因特定位点诱导双链断裂(DSB)后以非同源末端连接(NHEJ)完成修复，以实现不同的抗体下游效应功能，CSR效能低下与原发性抗体缺陷病密切相关。研究表明NHEJ途径缺失的B细胞仍可以一定活性进行类别转换，提示胞内存在替代性连接途径，但对其机制知之甚少。我们发现NHEJ突变体中存在大量DSB末端切除产生单链DNA以促进微同源序列退火，但不清楚退火产生的非同源尾端如何被切除以便连接。结合前期工作，我们猜想XPF/ERCC1核酸内切酶复合物可能参与这些非同源尾端的切除，促进以替代性连接途径完成CSR。本项目计划在小鼠成熟B细胞系中构建NHEJ因子和XPF/ERCC1缺失的突变体，通过分子免疫学和高通量测序等方法系统研究这一复合物在CSR过程中的作用。这一工作可以进一步阐释不同末端连接途径如何介导修复及抗体成熟，并为原发性抗体缺陷病的诊治提供新的潜在靶点。

B细胞抗体类别转换(CSR)是发生在生发中心成熟B细胞中的一种分子事件，对于B细胞生成具有不同下游效应功能的抗体以发挥体液免疫功能具有及其重要的意义，其功能异常往往导致原发性免疫缺陷病和B细胞淋巴瘤的发生。在CSR过程中，抗体重链基因特定位点诱导双链断裂(DSB)，然后以非同源末端连接(NHEJ)完成修复，然而，研究表明NHEJ途径缺失的B细胞仍可以一定活性进行类别转换，提示胞内存在替代性连接途径，但对其机制知之甚少。我们发现NHEJ突变体中存在大量DSB末端切除产生单链DNA以促进微同源序列退火，猜想XPF/ERCC1核酸内切酶复合物可能参与这些非同源尾端的切除，促进以替代性连接途径完成CSR。为此，我们在小鼠成熟B细胞系中构建NHEJ因子和XPF/ERCC1缺失的突变体，通过分子免疫学和高通量测序等方法系统研究这一复合物在CSR过程中的作用。我们发现，XPF-ERCC1核酸内切酶通过切除3'末端促进A-EJ而非c-NHEJ介导的CSR；XPF-ERCC1缺失后3'单链DNA受到Trex1核酸外切酶部分降解。同时，我们验证了核酸酶Artemis促进c-NHEJ而非A-EJ介导的CSR。最后，XPF-ERCC1进一步促进A-EJ途径下激活B细胞中c-Myc基因与抗体基因座IgH之间的染色体易位，这一染色体易位常见于人成熟B细胞淋巴瘤中，对于生发中心来源的成熟B细胞淋巴瘤的发生具有很强的推动作用。总之，这一工作不仅揭示了不同末端连接途径介导DNA修复及抗体成熟的新机制，而且为原发性免疫缺陷和B细胞淋巴瘤的诊治提供了新的潜在靶点。