Targeted DNA cleavage at switch regions in immunoglobulin class switch recombinat

免疫球蛋白（IG）类别转换重组（CSR）是一个过程， 所述B细胞将IG重链的恒定结构域交换为最佳的 清除病原体。这种独特的DNA重组是由DNA酶- 长开关区，需要B细胞特异性因子激活诱导的胞苷 脱氨酶（AID）以及其他普遍表达的DNA修复因子。它 已知CSR是由AID催化的胞苷脱氨基作用引发的， 开关区域的尿嘧啶。然而，开关区域 AID指导顺式作用和尿嘧啶修复途径的相互作用， 最终导致DNA双链断裂的原因仍然不清楚。的 本申请的目的是鉴定DNA中的顺式作用DNA序列， 转换区和反式作用蛋白因子，负责靶向 在类别转换重组期间在限定的基因组基因座处的DNA切割。我们 开发了一种基于细胞的类别转换试验，用于研究 内源染色体基因座上的开关区序列。该测定 基于我们最近在CH 12 F3中高效基因靶向方面的成功 细胞，一种能够在体外产生鲁棒的奎宁诱导的CSR的小鼠B细胞系。我们 设计了一种有效的敲入策略， 开关区域突变的数量。CH 12 F3中的高效基因打靶 细胞还允许通过反向遗传方法研究基因功能， CSR的细胞模型。我们现在可以解决几个重要的问题， 以前在动物模型中很难解决的问题。我们 提出了三个具体目标：（1）确定类所需的短序列基序 开关重组;（2）识别类所需的长序列组织 （3）鉴定开关区域的DNA切割活性。 该项目的完成将使人们更全面地了解 开关区序列的功能和核酸酶的鉴定 参与CSR中的DNA切割。这些发现将提供机制 对涉及类别转换重组的各种人类疾病的洞察。

Immunoglobulin (Ig) class switch recombination (CSR) is a process by which B cells exchange the constant domain of the Ig heavy chain for the optimal clearance of pathogens. This unique DNA recombination is directed by kilobase- long switch regions and requires B cell-specific factor activation-induced cytidine deaminase (AID) as well as other ubiquitously expressed DNA repair factors. It is known that CSR is initiated by AID-catalyzed cytidine deamination resulting in uracils in the switch regions. However, the mechanism by which switch region directs AID actions in-cis and the interplays of uracil repair pathways that ultimately lead to DNA double strand breaks remain poorly defined. The objectives of this application are to identify cis-acting DNA sequences in the switch region and trans-acting protein factors that are responsible for targeted DNA cleavage at defined genomic loci during class switch recombination. We have developed a cell-based class switch assay for studying the function of switch region sequences at the endogenous chromosomal locus. This assay was based on our recent success in highly efficient gene targeting in CH12F3 cells, a mouse B cell line capable of robust cytokine-induced CSR in vitro. We have designed an efficient knock-in strategy to allow assessment of a large number of switch region mutations. Highly efficient gene targeting in CH12F3 cells also allows study of gene function by the reverse genetic approaches in a cellular model for CSR. We are now in position to address several important yet unanswered questions previously difficult to address in animal models. We propose three specific aims: (1) Identify short sequence motifs required for class switch recombination; (2) Identify long sequence organizations required for class switch recombination; (3) Identify the DNA cleavage activity at switch regions. The completion of this project will lead to a more complete understanding of the function of switch region sequences and the identification of the nucleases involved in DNA cleavage in CSR. These findings will provide mechanistic insight to a variety of human diseases involving class switch recombination.