A Caltech Library Service

Sequence-Dependent Dynamics of Synthetic and Endogenous RSSs in V(D)J Recombination

Hirokawa, Soichi and Chure, Griffin and Belliveau, Nathan M. and Lovely, Geoffrey A. and Anaya, Michael and Schatz, David G. and Baltimore, David and Phillips, Rob (2020) Sequence-Dependent Dynamics of Synthetic and Endogenous RSSs in V(D)J Recombination. Nucleic Acids Research, 48 (12). pp. 6726-6739. ISSN 0305-1048. PMCID PMC7337519. doi:10.1093/nar/gkaa418.

[img] PDF - Published Version
Creative Commons Attribution.

[img] PDF - Submitted Version
Creative Commons Attribution.

[img] Archive (ZIP) (Supplementary data) - Supplemental Material
Creative Commons Attribution.


Use this Persistent URL to link to this item:


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.

Item Type:Article
Related URLs:
URLURL TypeDescription Paper Image Data CentralArticle
Hirokawa, Soichi0000-0001-5584-2676
Chure, Griffin0000-0002-2216-2057
Belliveau, Nathan M.0000-0002-1536-1963
Anaya, Michael0000-0002-6944-3614
Schatz, David G.0000-0002-5669-1176
Baltimore, David0000-0001-8723-8190
Phillips, Rob0000-0003-3082-2809
Additional Information:© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Received: 31 January 2020; Revision received: 20 April 2020; Accepted: 07 May 2020; Published: 25 May 2020. We thank members of the David G. Schatz, David Baltimore, and Rob Phillips labs for useful discussions and Caltech’s Protein Expression Center for supplying resources and equipment for protein purification. We also thank Miyo Aoki-Ota, Justin Bois, Zev Bryant, Stephanie Johnson, David Nemazee, Eddy Rubin, Charlie Starr and Haojie Zhuang for discussions. Funding: National Institutes of Health (NIH) [R01 GM085286, 1R35 GM118043 (Maximizing Investigators' Research Award, MIRA) to R.P.; R01 AI032524 to D.G.S.]; Caltech Center for Environmental Microbial Interactions (CEMI) (to S.H., R.P.); Foundational Questions Institute (FQXi) [FQXi-RFP-1816 to R.P.]; Sackler Foundation (to D.B.). Funding for open access charge: National Institute of General Medical Sciences [1R35 GM118043], Maximizing Investigators' Research Award (MIRA). The authors declare no conflict of interest.
Group:Caltech Center for Environmental Microbial Interactions (CEMI)
Funding AgencyGrant Number
NIHR01 GM085286
NIH1R35 GM118043
NIHR01 AI032524
Caltech Center for Environmental Microbial Interactions (CEMI)UNSPECIFIED
Foundational Questions Institute (FQXI)FQXi-RFP-1816
Raymond and Beverly Sackler FoundationUNSPECIFIED
Issue or Number:12
PubMed Central ID:PMC7337519
Record Number:CaltechAUTHORS:20191007-074542895
Persistent URL:
Official Citation:Soichi Hirokawa, Griffin Chure, Nathan M Belliveau, Geoffrey A Lovely, Michael Anaya, David G Schatz, David Baltimore, Rob Phillips, Sequence-dependent dynamics of synthetic and endogenous RSSs in V(D)J recombination, Nucleic Acids Research, 48(12): 6726–6739,
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:99107
Deposited By: Tony Diaz
Deposited On:07 Oct 2019 14:57
Last Modified:01 Jun 2023 23:24

Repository Staff Only: item control page