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Biophysics of V(D)J recombination an...

Lovely, Geoffrey.

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

紀錄類型:	書目-電子資源 : Monograph/item
正題名/作者:	Biophysics of V(D)J recombination and Genome Packaging In singulo studies on RAG, HMGB1, and TFAM./
作者:	Lovely, Geoffrey.
面頁冊數:	145 p.
附註:	Source: Dissertation Abstracts International, Volume: 76-05(E), Section: B.
Contained By:	Dissertation Abstracts International76-05B(E).
標題:	Biophysics. -
電子資源:	http://pqdd.sinica.edu.tw/twdaoapp/servlet/advanced?query=3668985
ISBN:	9781321443967

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

Biophysics of V(D)J recombination and Genome Packaging In singulo studies on RAG, HMGB1, and TFAM. - 145 p.

Source: Dissertation Abstracts International, Volume: 76-05(E), Section: B.

Thesis (Ph.D.)--California Institute of Technology, 2015.

This item is not available from ProQuest Dissertations & Theses.

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. Interestingly, we find the synaptic complex has a mean lifetime of roughly 400s and is highly reversible; only 28% of observed synapses result in cleavage for consensus RSS binding sites. 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.

ISBN: 9781321443967Subjects--Topical Terms:

518360
Biophysics.

Biophysics of V(D)J recombination and Genome Packaging In singulo studies on RAG, HMGB1, and TFAM.
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520 $a 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. Interestingly, we find the synaptic complex has a mean lifetime of roughly 400s and is highly reversible; only 28% of observed synapses result in cleavage for consensus RSS binding sites. 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.
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