A Functional Analysis of the Spacer of V(D)J Recombination Signal Sequences

During lymphocyte development, V(D)J recombination assembles antigen receptor genes from component V, D, and J gene segments. These gene segments are flanked by a recombination signal sequence (RSS), which serves as the binding site for the recombination machinery. The murine Jβ2.6 gene segment is a recombinationally inactive pseudogene, but examination of its RSS reveals no obvious reason for its failure to recombine. Mutagenesis of the Jβ2.6 RSS demonstrates that the sequences of the heptamer, nonamer, and spacer are all important. Strikingly, changes solely in the spacer sequence can result in dramatic differences in the level of recombination. The subsequent analysis of a library of more than 4,000 spacer variants revealed that spacer residues of particular functional importance are correlated with their degree of conservation. Biochemical assays indicate distinct cooperation between the spacer and heptamer/nonamer along each step of the reaction pathway. The results suggest that the spacer serves not only to ensure the appropriate distance between the heptamer and nonamer but also regulates RSS activity by providing additional RAG:RSS interaction surfaces. We conclude that while RSSs are defined by a “digital” requirement for absolutely conserved nucleotides, the quality of RSS function is determined in an “analog” manner by numerous complex interactions between the RAG proteins and the less-well conserved nucleotides in the heptamer, the nonamer, and, importantly, the spacer. Those modulatory effects are accurately predicted by a new computational algorithm for “RSS information content.” The interplay between such binary and multiplicative modes of interactions provides a general model for analyzing protein–DNA interactions in various biological systems.

在淋巴细胞发育过程中，V(D)J重组通过组合V、D、J基因片段组装抗原受体基因。这些基因片段两侧均带有重组信号序列（recombination signal sequence, RSS），该序列可作为重组酶系统的结合位点。小鼠Jβ2.6基因片段属于重组失活假基因，但对其RSS的分析并未发现其无法发生重组的明确原因。针对Jβ2.6 RSS的诱变实验表明，七聚体、九聚体以及间隔序列的序列均对重组过程至关重要。令人意外的是，仅对间隔序列进行改变即可导致重组水平出现显著差异。后续针对包含4000余个间隔序列变体的文库开展分析后发现，具有特定功能重要性的间隔序列残基与它们的保守程度显著相关。生化实验结果表明，在重组反应通路的各个阶段，间隔序列与七聚体/九聚体之间存在独特的协同作用。研究结果显示，间隔序列不仅用于维持七聚体与九聚体之间的合适间距，还可通过提供额外的RAG蛋白与RSS的相互作用界面来调控RSS的活性。我们的研究结论如下：尽管RSS的定义基于对绝对保守核苷酸的“数字化”要求，但RSS的功能质量是以“模拟式”方式决定的，即通过RAG蛋白与七聚体、九聚体以及（尤为关键的）间隔序列中保守性较弱的核苷酸之间的多种复杂相互作用来确定。这类调控效应可通过一种全新的“RSS信息含量”计算算法实现精准预测。这类二元相互作用与倍增式相互作用模式之间的协同互作，为各类生物系统中的蛋白质-DNA相互作用分析提供了通用研究模型。