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

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Figure 2.1: Expression and purification of RAG1/2c from suspension 293-T and 293-E cells.a) 100mL 293-E (blue) or 293-T (red) cells were diluted to 1.0 x 106 cells/mL before (T=0h) and after (T= 48h) transfection the cellular density was determined for cell
Figure 2.2: Size exclusion chromatography-multiangle light-scattering (SEC-MALS) andfunctional analysis of RAG1/2c.c) long DNAs in the presence of 225nM HMGB1,increasing amounts RAG1/2c [5, 10 and50nM], and 300ng of radiolabeled 12/23RSS or 12/12RSS DNA su
Figure 3.1: Schematic of states associated with a single V(D)J recombination event. (A)The DNA molecule (tan) has a 12RSS (maroon) and a 23RSS (yellow)
Figure 3.2: Tethered particle motion (TPM) acquisition and analysis. (A) After we assem-ble DNA-tethered beads we select beads in a field of view (white boxes) and keep thoseto thewith a single DNA molecule coupled to them
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Introduction to Genome Packaging Size exclusion chromatography and multiangle light-scattering Detection of single RAG-RSS complexes in real-time HMGB1 alters the geometry of RAG-RSS complexes Direct observation of paired complex formation The dynamics of 12/23 rule-regulated bead release as a function of TPM: RAG1/2c-RSS, RAG1/2c-HMGB1-RSS binding, hairpin pro- Appendix 1: Supplementary Information DNA bending and TFAM dimerization are required for efficient DNA TPM trajectories and distributions from Fig 5.7B, C and E

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