The Markey Scholars Conference Proceedings (2004) / Chapter Skim
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7 Transcription, Chromatin Assembly, and Homologous Recombination in Chromatin
7 Transcription, Chromatin Assembly, and Homologous Recombination in Chromatin
Pages 50-72
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From page 50... ...
This picture displays the periodic nature of chromatin, as the unfolded chromatin filament resembles beads on a string. Figure 3 shows a schematic diagram of chromatin.
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PRESENTED PAPERS 51 FIGURE 1 DNA -- the molecule of life. SOURCE: http://www.ornl.gov/TechResources/Human_Genome/ FIGURE 2 Eukaryotic DNA package into chromatin.
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. Crystal structure of the nucleosome core particle at 2.8 Å resolution.
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One key DNA element that is involved in transcription is the core promoter, which contains the information that is needed for the RNA polymerase II transcriptional machinery to initiate transcription. The core promoter is typically about 40 bp in length and encompasses the transcription start site.
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The TATA box is, by far, the most widely studied core promoter motif, and it is sometimes incorrectly thought that all core promoters contain a TATA box. But, in humans, it is estimated that perhaps only about 30 percent of core promoters have a TATA box.
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From page 55... ...
We therefore felt that it would be important to understand the core promoter elements that are involved in the transcription of the large proportion of genes that lack a TATA box. These studies led Tom Burke to the discovery of a downstream core promoter motif called the DPE, which is located about 30 nucleotides downstream of the transcription start site.
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. The downstream promoter element DPE appears to be as widely used as the TATA box in Drosophila core promoters.
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. Enhancer-promoter specificity mediated by DPE or TATA core promoter motifs.
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Thus, some enhancers function specifically with DPE or TATA elements in the core promoter. More generally, these results indicate that the core promoter is much more than a sequence that directs the proper site of initiation by RNA polymerase II.
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. The DPE is a downstream core promoter element that is located around +30 relative to the transcription start site.
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To this end, Mike Bulger, Takashi Ito, and Jessica Tyler had fractionated, purified, and cloned the factors that mediate the ATP-dependent assembly of periodic nucleosome arrays. These studies led to the discovery of the ATP-utilizing motor protein involved in chromatin assembly, which is termed ACF, as well as one novel histone chaperone, ASF1, and three previouslyknown histone chaperones, CAF-1, NAP-1, and nucleoplasmin (Figure 15)
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. Transcriptional analysis of chromatin assembled with purified ACF and dNAP1 reveals that acetyl CoA is required for preinitiation complex assembly.
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Genes Dev.
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To study how ACF works, Dmitry Fyodorov performed templatecommitment experiments as well as other related mechanistic analyses. His results suggest that ACF is a processive, ATP-driven motor that translocates along DNA and mediates chromatin assembly (Figure 19)
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This work was carried out by Vassili Alexiadis. These experiments were inspired by two things -- first, homologous recombination is a fundamental and important biological process; and second, the Rad54 protein, which is involved in homologous recombination, is a member of the Snf2-like family of ATPases, which comprises the ATPase subunits of all known chromatin remodeling complexes (Figure 20)
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. D Loop FIGURE 21 Initial steps of homologous recombination at a double-strand break.
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He therefore tested the effect of relaxation of superhelical tension upon D loop formation by Rad51 and Rad54. These experiments revealed that relaxation of naked DNA by topoisomerase I results in a greater than 100-fold decrease Rad51 Rad54 ATP D Loop FIGURE 22 Catalysis of strand pairing by Rad51 and Rad54.
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From page 67... ...
To this end, he assembled relaxed DNA into chromatin by using purified recombinant ACF, purified recombinant NAP-1, purified core histones, relaxed plasmid DNA, and ATP in the presence of purified topoisomerase I These reactions were performed in the absence (no chromatin assembly)
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. In addition, these findings may be applicable to studies of homologous recombination in vivo, such as for targeted knockouts and gene therapy.
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2B. Rad51+Rad54 Naked SD DNA Chromatin Naked SD DNA Chromatin Topo I + + ++ Topo I + + Relaxed Chromatin Supercoiled D loop Relaxed DNA 1 2 3 4 5 Agarose Gel + 5 µM Chloroquine Free oligo Ethidium Fluorescence Relative <1 Activity 100 300 220 FIGURE 26 Chromatin facilitates D loop formation in the absence of superhelical tension.
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Genes Dev.
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From page 71... ...
as an activator of DPE-dependent but not TATA-dependent transcription, and Jenny Butler identified the DPE-specific transcriptional enhancers. Rohinton Kamakaka and Mike Bulger developed the S-190 chromatin assembly extract, Mike Bulger and Takashi Ito performed the fractionation of the S-190 and the purification of ACF, Jessica Tyler identified and purified the ASF1/RCAF assembly factor, Dmitry Fyodorov and Mark Levenstein established the purified recombinant chromatin assembly system, and Dmitry carried out the experiments suggesting that ACF is a processive DNA-translocating enzyme as well as the genetic analysis of ACF.
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72 THE MARKEY SCHOLARS CONFERENCE PROCEEDINGS DPE ACF Tom Burke Rohinton Kamakaka Alan Kutach Mike Bulger Patricia Willy Takashi Ito Jenny Butler Jessica Tyler Tammy Juven-Gershon Mark Levenstein Tom Boulay Dmitry Fyodorov Chin Yan Lim Karl Haushalter Emily Dong Rad54 Protein biochemistry Vassili Alexiadis Ryuji Kobayashi (MD Anderson) Section of Molecular Biology UC San Diego FIGURE 29 Coworkers and collaborators (past and present)
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