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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 (2019) Sequence-Dependent Dynamics of Synthetic and Endogenous RSSs in V(D)J Recombination. . (Unpublished) https://resolver.caltech.edu/CaltechAUTHORS:20191007-074542895

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Abstract

Developing lymphocytes in the immune system of jawed vertebrates assemble antigen-receptor genes by undergoing large-scale reorganization of spatially separated V, D, and J gene segments through a process known as V(D)J recombination. The RAG protein initiates this process by 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 quantify the formation, stability, and cleavage of the RAG-12RSS-23RSS paired complex (PC) for numerous synthetic and endogenous 12RSSs. We thoroughly investigate the sequence space around a RSS by making 40 different single-bp changes and characterizing the reaction dynamics. We reveal that single-bp changes affect RAG function based on their position: loss of cleavage function (first three positions of the heptamer); reduced propensity for forming the PC (the nonamer and last four bp of the heptamer); or variable effects on PC formation (spacer). We find that the rare usage of some endogenous gene segments can be mapped directly to their adjacent 12RSSs to which RAG binds weakly. The 12RSS, however, cannot explain the high-frequency usage of other gene segments. Finally, we find that RSS nicking, while not required for PC formation, substantially stabilizes the PC. Our findings provide detailed insights into the contribution of individual RSS positions to steps of the RAG-RSS re-action that previously have been difficult to assess quantitatively.


Item Type:Report or Paper (Discussion Paper)
Related URLs:
URLURL TypeDescription
https://doi.org/10.1101/791954DOIDiscussion Paper
https://doi.org/10.22002/D1.1288DOIPreprocessed Image Data
ORCID:
AuthorORCID
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 copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY 4.0 International license. bioRxiv preprint first posted online Oct. 4, 2019. Data and code availability: All data and code are publicly available. Raw image files can be obtained upon request. Preprocessed image data can be downloaded from CaltechDATA research data repository under the DOI:10.22002/D1.1288. Processed data files, Matlab, and Python code used in this work can be downloaded either from the paper website or on the dedicated GitHub repository (DOI:10.5281/zenodo.346571). 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 Helen Beilinson, Justin Bois, Zev Bryant, Heun Jin Lee, Stephanie Johnson, Eddy Rubin, Charlie Starr, Yuhang Zhang, and Haojie Zhuang for discussions. This work was supported by R01GM085286 and 1R35 GM118043 Maximizing Investigators’ Research Award (MIRA) (to R.P.). S.H. was also supported by the Caltech Center for Environmental Microbial Interactions (CEMI) (R.P.), the Foundational Questions Institute (FQXI) (R.P.), and the Sackler Foundation (D.B.). S.H., G.A.L., D.G.S., D.B., R.P. designed research. S.H. performed research. S.H., N.M.B., G.A.L., M.A., D.B., R.P. provided new reagents and analytical tools. S.H., G.C., N.M.B., G.A.L., D.G.S., D.B., R.P. analyzed data. S.H., G.C., N.M.B., G.A.L., D.G.S., D.B., R.P. wrote paper. The authors declare no conflict of interest.
Group:Caltech Center for Environmental Microbial Interactions (CEMI)
Funders:
Funding AgencyGrant Number
NIHR01GM085286
NIH1R35 GM118043
Caltech Center for Environmental Microbial Interactions (CEMI)UNSPECIFIED
Foundational Questions Institute (FQXI)UNSPECIFIED
Raymond and Beverly Sackler FoundationUNSPECIFIED
Record Number:CaltechAUTHORS:20191007-074542895
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20191007-074542895
Official Citation:Sequence-Dependent Dynamics of Synthetic and Endogenous RSSs in V(D)J Recombination. Soichi Hirokawa, Griffin Chure, Nathan M. Belliveau, Geoffrey A. Lovely, Michael Anaya, David G. Schatz, David Baltimore, Rob Phillips. bioRxiv 791954; doi: https://doi.org/10.1101/791954
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:99107
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:07 Oct 2019 14:57
Last Modified:07 Oct 2019 14:57

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