Biophysics of V(D)J recombination and Genome Packaging: In singulo studies on RAG, HMGB1, and TFAM

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.

Everybody Farts: Celebrating the Body and Refuting Medical Paternalism in Joyce's Ulysses

James Joyce’s Ulysses celebrates all facets of daily life in its refusal to censor raw human emotions and emissions. He adopts a critically medical perspective to portray this honest, unfiltered narrative. In doing so, he reveals the ineffectiveness of the physician-patient relationship due to doctors’ paternalistic attitudes that hinder nonjudgmental, open listening of this unfiltered narrative. His exploration of the doctor’s moral scrutiny, cultural prejudices, and authoritative estrangement from the patient underscore the importance in remembering that physicians and patients alike are ultimately just fellow human beings. Wryly, he drives this point to literal nausea, as his narrative proudly asserts the revulsive details of public health, digestion, and death. In his gritty ruminations on the human body’s material reality, Joyce mocks the physician’s highbrow paternalism by forcing him to identify with the farting, vomiting, decaying bodies around him. In celebrating the uncensored human narrative, Joyce challenges physician and patient alike to openly listen to the stories of others.