Zagelbaum Jennifer, Shimazaki Noriko, Esguerra Zitadel Anne, Watanabe Go, Lieber Michael R, Rothenberg Eli
Department of Biochemistry and Molecular Pharmacology, Perlmutter Cancer Center, New York University School of Medicine, New York, NY 10016.
Department of Pathology, University of Southern California (USC) Keck School of Medicine, USC Norris Comprehensive Cancer Center, Los Angeles, CA 90033; Department of Biochemistry & Molecular Biology, University of Southern California (USC) Keck School of Medicine, USC Norris Comprehensive Cancer Center, Los Angeles, CA 90033; Department of Molecular Microbiology & Immunology, University of Southern California (USC) Keck School of Medicine, USC Norris Comprehensive Cancer Center, Los Angeles, CA 90033; Section of Molecular & Computational Biology, Department of Biological Sciences, University of Southern California (USC), Los Angeles, CA 90089.
Proc Natl Acad Sci U S A. 2016 Oct 18;113(42):11853-11858. doi: 10.1073/pnas.1606721113. Epub 2016 Oct 4.
Single-molecule FRET (smFRET) and single-molecule colocalization (smCL) assays have allowed us to observe the recombination-activating gene (RAG) complex reaction mechanism in real time. Our smFRET data have revealed distinct bending modes at recombination signal sequence (RSS)-conserved regions before nicking and synapsis. We show that high mobility group box 1 (HMGB1) acts as a cofactor in stabilizing conformational changes at the 12RSS heptamer and increasing RAG1/2 binding affinity for 23RSS. Using smCL analysis, we have quantitatively measured RAG1/2 dwell time on 12RSS, 23RSS, and non-RSS DNA, confirming a strict RSS molecular specificity that was enhanced in the presence of a partner RSS in solution. Our studies also provide single-molecule determination of rate constants that were previously only possible by indirect methods, allowing us to conclude that RAG binding, bending, and synapsis precede catalysis. Our real-time analysis offers insight into the requirements for RSS-RSS pairing, architecture of the synaptic complex, and dynamics of the paired RSS substrates. We show that the synaptic complex is extremely stable and that heptamer regions of the 12RSS and 23RSS substrates in the synaptic complex are closely associated in a stable conformational state, whereas nonamer regions are perpendicular. Our data provide an enhanced and comprehensive mechanistic description of the structural dynamics and associated enzyme kinetics of variable, diversity, and joining [V(D)J] recombination.
单分子荧光共振能量转移（smFRET）和单分子共定位（smCL）分析使我们能够实时观察重组激活基因（RAG）复合体的反应机制。我们的smFRET数据揭示了在切口和突触形成之前，重组信号序列（RSS）保守区域存在不同的弯曲模式。我们发现高迁移率族蛋白盒1（HMGB1）作为一种辅助因子，可稳定12RSS七聚体的构象变化，并增加RAG1/2对23RSS的结合亲和力。通过smCL分析，我们定量测量了RAG1/2在12RSS、23RSS和非RSS DNA上的停留时间，证实了严格的RSS分子特异性，在溶液中存在配对RSS时这种特异性会增强。我们的研究还提供了单分子速率常数的测定，这些常数以前只能通过间接方法获得，这使我们得出结论，RAG结合、弯曲和突触形成先于催化作用。我们的实时分析深入了解了RSS-RSS配对的要求、突触复合体的结构以及配对RSS底物的动力学。我们表明突触复合体极其稳定，并且突触复合体中12RSS和23RSS底物的七聚体区域以稳定的构象状态紧密相连，而非聚体区域则相互垂直。我们的数据对可变、多样和连接[V(D)J]重组的结构动力学及相关酶动力学提供了更详细和全面的机制描述。
