Abstract
Developing lymphocytes of jawed vertebrates cleave and combine distinct gene segments to assemble antigen–receptor genes. This process called V(D)J recombination that involves the RAG recombinase binding and cutting recombination signal sequences (RSSs) composed of conserved heptamer and nonamer sequences flanking less well-conserved 12- or 23-bp spacers. Little quantitative information is known about the contributions of individual RSS positions over the course of the RAG–RSS interaction. We employ a single-molecule method known as tethered particle motion to track the formation, lifetime and cleavage of individual RAG–12RSS–23RSS paired complexes (PCs) for numerous synthetic and endogenous 12RSSs. We reveal that single-bp changes, including in the 12RSS spacer, can significantly and selectively alter PC formation or the probability of RAG-mediated cleavage in the PC. We find that some rarely used endogenous gene segments can be mapped directly to poor RAG binding on their adjacent 12RSSs. Finally, we find that while abrogating RSS nicking with Ca2+ leads to substantially shorter PC lifetimes, analysis of the complete lifetime distributions of any 12RSS even on this reduced system reveals that the process of exiting the PC involves unidentified molecular details whose involvement in RAG–RSS dynamics are crucial to quantitatively capture kinetics in V(D)J recombination.
Highlights
Jawed vertebrates call upon developing lymphocytes to undergo a genomic cut-and-paste process known as V(D)J recombination, where disparate gene segments that do not individually code for an antigen–receptor protein are systematically combined to assemble a complete, antigen receptorencoding gene [1]
Due to the depth of insight offered by waiting time distributions generated by the tethered particle motion (TPM) assay, in an attempt to provide some of the first measurements of various recombination-activating gene (RAG)–recombination signal sequences (RSSs) kinetics, we show through our analysis of the paired complexes (PCs) lifetime distributions that regardless of choice of 12RSS or divalent cation, our TPM data consistently disagree with a single-rate model
To explore how RAG–RSS interactions are affected by single bp changes, we examined 40 synthetic RSSs consisting of single bp changes across 21 positions of the V4-57-1 12RSS, with a particular focus on altering the 12 bp spacer that is the least well-understood element in the RSS
Summary
Jawed vertebrates call upon developing lymphocytes to undergo a genomic cut-and-paste process known as V(D)J recombination, where disparate gene segments that do not individually code for an antigen–receptor protein are systematically combined to assemble a complete, antigen receptorencoding gene [1]. The process of V(D)J recombination (schematized in Figure 1) is initiated with the interaction between the recombination-activating gene (RAG) protein complex and two short sequences of DNA neighboring the gene segments, one that is 28 bp and another that is 39 bp in length. These recombination signal sequences (RSSs) are composed of a well-conserved heptamer region immediately adjacent to the gene segment, a more variable 12- (for the 12RSS) or 23-bp (for the 23RSS) spacer sequence and a well-conserved nonamer region.
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