DNA-PROTEIN INTERACTIONS IN LYMPHOCYTE DIFFERENTIATION

B和T淋巴细胞的抗原受体在离散的DNA中编码 在发育过程中通过位点特异性DNA连接的片段 重新安排抗原受体基因组装，或V（D）J重排，是 脊椎动物中唯一已知的位点特异性DNA重组的例子。 异常的V（D）J重组可能参与产生 细胞原癌基因和抗原之间的染色体易位 受体位点，在淋巴恶性肿瘤的比例高。 对V（D）J重组及其调控的理解将继续 这个项目的长期目标。 两种蛋白质，RAG-1和RAG-2，是激活所必需的和足够的 V（D）J重排。我们过去的研究提供了以下信息 证据表明RAG-2蛋白的表达和V（D）J重组是 细胞周期调控：（1）RAG-2蛋白的表达受到限制 通过转录后机制转化为G 0/G1;（2）RAG-2被磷酸化 通过细胞周期蛋白依赖性激酶（cdk）在体外的特定位点;（3） 该位点在体内的磷酸化与快速降解有关 RAG-2的突变;（4）V（D）J位点特异性双链DNA断裂 重组信号序列也优先在G 0/G1中积累。 我们的工作假设是RAG-2被一个或多个 cdk’s调节RAG-2的积累，进而调节V（D）J 重组其他观察表明， RAG-2磷酸化和降解之间的关系可能反映了一种更普遍的 机制 在下一个资助期内，我们希望研究RAG-2表达是如何影响 偶联到细胞周期和这种调节的关系， V（D）J重组的时机。为此，我们提出以下建议： 具体目标：（1）确定RAG-2的结构决定因素 不稳定性和细胞周期调控;（2）确定相对 蛋白质合成和降解对RAG-2调节的贡献 （3）评估细胞周期中RAG-2的磷酸化; 细胞周期，并测试RAG-2降解是否是通过 磷酸化;（4）确定细胞周期依赖性 V（D）J重组中间体的积累是 调节RAG-2表达;（5）检查 培养细胞和转基因小鼠中的非程序性RAG-2表达;以及 (6)开发一种用于分析RAG-2在酵母中降解的系统， 在基因水平上研究这一过程的长期目标。

The antigen receptors of B and T lymphocytes are encoded in discrete DNA segments that are joined during development by site-specific DNA rearrangements. Antigen receptor gene assembly, or V(D)J rearrangement, is the only known example of site-specific DNA recombination in vertebrates. Aberrant V(D)J recombination is likely to be involved in generating the chromosomal translocations between cellular protooncogenes and antigen receptor loci that are seen in a high proportion of lymphoid malignancies. An understanding of V(D)J recombination and its regulation continues to be a long-term goal of this project. Two proteins, RAG-1 and RAG-2, are necessary and sufficient for activation of V(D)J rearrangement. Our past studies have provided the following evidence that expression of the RAG-2 protein and V(D)J recombination are regulated in the cell cycle: (1) expression of RAG-2 protein is restricted to G0/G1 by a posttranscriptional mechanism; (2) RAG-2 is phosphorylated by a cyclin-dependent kinase (cdk) at a specific site in vitro; (3) phosphorylation of this site in vivo is associated with rapid degradation of RAG-2; and (4) site-specific double-strand DNA breaks at V(D)J recombination signal sequences also accumulate preferentially in G0/G1. Our working hypothesis is that phosphorylation of RAG-2 by one or more cdk's regulates accumulation of RAG-2, in turn regulating V(D)J recombination. Additional observations suggest that the association between RAG-2 phosphorylation and degradation may reflect a more general mechanism. In the next funding period, we wish to examine how RAG-2 expression is coupled to the cell cycle and the relationship of this regulation to the timing of V(D)J recombination. To this end, we propose the following specific aims: (1) to define the structural determinants of RAG-2 instability and cell cycle regulation; (2) to determine the relative contributions of protein synthesis and degradation to regulation of RAG-2 accumulation in the cell cycle; (3) to assess phosphorylation of RAG-2 in the cell cycle and to test whether RAG-2 degradation is targeted by phosphorylation; (4) to determine whether cell cycle-dependent accumulation of V(D)J recombination intermediates is a consequence of regulated RAG-2 expression; (5) to examine the physiologic consequences of unscheduled RAG-2 expression in cultured cells and in transgenic mice; and (6) to develop a system for analysis of RAG-2 degradation in yeast, with the long-term goal of examining this process at the genetic level.