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Biophysics of V(D)J Recombination and Genome Packaging: In Singulo Studies on RAG, HMGB1, and TFAM


Lovely, Geoffrey A. (2014) Biophysics of V(D)J Recombination and Genome Packaging: In Singulo Studies on RAG, HMGB1, and TFAM. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/Z9W9573H.


The recombination-activating gene products, RAG1 and RAG2, initiate V(D)J recombination during lymphocyte development by cleaving DNA adjacent to conserved recombination signal sequences (RSSs). The reaction involves DNA binding, synapsis, and cleavage at two RSSs located on the same DNA molecule and results in the assembly of antigen receptor genes. Since their discovery full-length, RAG1 and RAG2 have been difficult to purify, and core derivatives are shown to be most active when purified from adherent 293-T cells. However, the protein yield from adherent 293-T cells is limited. Here we develop a human suspension cell purification and change the expression vector to boost RAG production 6-fold. We use these purified RAG proteins to investigate V(D)J recombination on a mechanistic single molecule level. As a result, we are able to measure the binding statistics (dwell times and binding energies) of the initial RAG binding events with or without its co-factor high mobility group box protein 1 (HMGB1), and to characterize synapse formation at the single-molecule level yielding insights into the distribution of dwell times in the paired complex and the propensity for cleavage upon forming the synapse. We then go on to investigate HMGB1 further by measuring it compact single DNA molecules. We observed concentration dependent DNA compaction, differential DNA compaction depending on the divalent cation type, and found that at a particular HMGB1 concentration the percentage of DNA compacted is conserved across DNA lengths. Lastly, we investigate another HMGB protein called TFAM, which is essential for packaging the mitochondrial genome. We present crystal structures of TFAM bound to the heavy strand promoter 1 (HSP1) and to nonspecific DNA. We show TFAM dimerization is dispensable for DNA bending and transcriptional activation, but is required for mtDNA compaction. We propose that TFAM dimerization enhances mtDNA compaction by promoting looping of mtDNA.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:V(D)J recombination, Genome Packaging, Recombination Activating Genes, High Mobility Group Box Proteins, Transcription Factor A Mitochondrial
Degree Grantor:California Institute of Technology
Division:Biology and Biological Engineering
Major Option:Biochemistry and Molecular Biophysics
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Baltimore, David L. (co-advisor)
  • Phillips, Robert B. (co-advisor)
Thesis Committee:
  • Mayo, Stephen L. (chair)
  • Fraser, Scott E.
  • Cai, Long
  • Baltimore, David L.
  • Phillips , Robert B.
Defense Date:2 May 2014
Non-Caltech Author Email:galovely23 (AT)
Funding AgencyGrant Number
Betty and Gordon Moore FoundationUNSPECIFIED
Alfred P Sloan FoundationUNSPECIFIED
Benjamin Rosen FoundationUNSPECIFIED
Record Number:CaltechTHESIS:06072014-140700155
Persistent URL:
Related URLs:
URLURL TypeDescription adapted for ch. 5
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:8504
Deposited By: Geoffrey Lovely
Deposited On:19 Dec 2014 19:17
Last Modified:08 Nov 2023 00:41

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