Department of Immunobiology, Yale University School of Medicine, 300 Cedar Street, New Haven, CT 06511, USA.
Nucleic Acids Res. 2013 Mar 1;41(5):3289-301. doi: 10.1093/nar/gks1461. Epub 2013 Jan 15.
During V(D)J recombination, recombination activating gene (RAG)1 and RAG2 bind and cleave recombination signal sequences (RSSs), aided by the ubiquitous DNA-binding/-bending proteins high-mobility group box protein (HMGB)1 or HMGB2. HMGB1/2 play a critical, although poorly understood, role in vitro in the assembly of functional RAG-RSS complexes, into which HMGB1/2 stably incorporate. The mechanism of HMGB1/2 recruitment is unknown, although an interaction with RAG1 has been suggested. Here, we report data demonstrating only a weak HMGB1-RAG1 interaction in the absence of DNA in several assays, including fluorescence anisotropy experiments using a novel Alexa488-labeled HMGB1 protein. Addition of DNA to RAG1 and HMGB1 in fluorescence anisotropy experiments, however, results in a substantial increase in complex formation, indicating a synergistic binding effect. Pulldown experiments confirmed these results, as HMGB1 was recruited to a RAG1-DNA complex in a RAG1 concentration-dependent manner and, interestingly, without strict RSS sequence specificity. Our finding that HMGB1 binds more tightly to a RAG1-DNA complex over RAG1 or DNA alone provides an explanation for the stable integration of this typically transient architectural protein in the V(D)J recombinase complex throughout recombination. These findings also have implications for the order of events during RAG-DNA complex assembly and for the stabilization of sequence-specific and non-specific RAG1-DNA interactions.
在 V(D)J 重组过程中，重组激活基因 (RAG)1 和 RAG2 结合并切割重组信号序列 (RSS)，这一过程得到普遍存在的 DNA 结合/弯曲蛋白高迁移率族蛋白 (HMGB)1 或 HMGB2 的辅助。HMGB1/2 在体外组装功能性 RAG-RSS 复合物中起着至关重要但尚未完全理解的作用，HMGB1/2 稳定地掺入其中。HMGB1/2 的募集机制尚不清楚，尽管有人提出与 RAG1 相互作用。在这里，我们报告的数据表明，在几种测定中，包括使用新型 Alexa488 标记的 HMGB1 蛋白的荧光各向异性实验，在没有 DNA 的情况下，HMGB1 与 RAG1 只有微弱的相互作用。然而，在荧光各向异性实验中向 RAG1 和 HMGB1 添加 DNA 会导致复合物形成的大量增加，表明存在协同结合效应。下拉实验证实了这些结果，因为 HMGB1 以 RAG1 浓度依赖性的方式被募集到 RAG1-DNA 复合物中，而且有趣的是，没有严格的 RSS 序列特异性。我们发现 HMGB1 与 RAG1-DNA 复合物的结合比 RAG1 或 DNA 本身更紧密，这为这种通常短暂的结构蛋白在整个重组过程中稳定整合到 V(D)J 重组酶复合物中提供了一个解释。这些发现还对 RAG-DNA 复合物组装过程中的事件顺序以及稳定序列特异性和非特异性 RAG1-DNA 相互作用具有影响。
