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A New Chromatin Remodeling Mechanism

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In eukaryotic cells, Rad51 protein plays an important role in homologous recombination. Rad51 polymerizes single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA). Rad51 assembles into a filamentous structure on ssDNA and thereby catalyzing homologous recombination and this mechanism is studied extensively, but in dsDNA, it is not clear. To determine the role of Rad51 in chromatin accessibility, they used Saccharomyces cerevisiae Rad51 on dsDNA and the effects of nucleosomes on Rad51 polymerization mechanism.

To explore the function of Saccharomyces cerevisiae Rad51 on different chromatinized DNA template they used biochemical and microscopic techniques. This study on remodeling mechanism suggests that Rad51 with Rad54 is a capable remodeler and gives a better understanding of its role in recombination. Rad51 polymerization on linear Nucleosomal templates The strength of remodeling activity was tested using a stronger nucleosome positioning sequence (601). This positioning sequence (601) binds to the histone octamer 150 times stronger than the 5S rDNA gene. The strength of polymerization-induced remodeling mechanism was assessed at the saturation ratio. Rad51]/[bp]=1/3, at this ratio the Rad51 polymerization led to nucleosomal removal. When the ratio was lowered ([Rad51]/[bp] =1/30), more than 10% of the chromatinized 601 was remodeled. This points out that there is a higher level of cooperativity of Rad51 polymerization, even in the presence of nucleosome. About 60%-90% of the molecules were remodeled beyond the saturation ratio. The structure of Rad51 filaments was analyzed at the saturation ratio using negative staining TEM experiments to confirm the eviction of the nucleosome. The measured average length was 183±25 nm and a pitch value of 9.5±0.5 nm which was approximately equal to the previously in agreement with reported values, this excludes the nucleosome present inside the filament. A PAGE remodeling assay was developed based on destabilization of the Rad51 filament by EDTA just after Rad51 polymerization on the 601 mononucleosome substrate. The assay results were compared with TEM experiment results and the assay results were consistent.

Rad51 polymerization on circular nucleosomal templates In the circular chromatinized template the remodeling assay was tested to determine whether the Rad51 filament disrupt the nucleosomes. On the ΦX174 supercoiled plasmid (5386bp) the nucleosomes were assembled. When the Rad51 was added to the chromatinized template, which forms polymers from the nucleation sites and the polymers are separated by constrained clusters of nucleosomes. A reverse remodeling assay was conducted to determine the number and binding of the nucleosomes remaining on the plasmid. In this assay, the Rad51 is completely removed by incubating with a high concentration of EDTA. Once the Rad51 is removed the nucleosomes is left in an isolated array. This indicates that during the initial remodeling step the nucleosomes slide on the DNA. In the next step, nucleosome sliding was induced by incubating it for 20 minutes at 40°C. From this analysis, 31±4 nucleosomes were initially present on the plasmids after the remodeling step it was 23±3 nucleosomes and the remaining nucleosomes were ejected. This assay confirmed the remodeling effect of Rad51 polymerization and its unique way to shift the whole nucleosome arrays along the template.

Remodeling mechanism

The chromatin remodeling depends on the polymerization process and this remodeling process was different from another remodeling process. In this whole, nucleosome array was physically pushed along the DNA and destabilizes them. Polymerization, a powerful mechanism of protein progression which facilitates remodeling events. In the chromatin remodeling, the first protein showed was Rad51 recombinase through an ATP-fueled dynamic polymerization process. This remodeling process is strong among the known remodeling process. The concentration of Rad51 used in strand exchange assay was used in this study.

From the analysis, the nucleosomes can be displaced through Rad51 polymerization and cooperative binding. The Rad51 polymerization on the short dsDNA segment was sufficient to induce an effect on nucleosomes. Rad51 polymerization requires ATP but for “one short” remodeling ATP hydrolysis was not required. To increase the turnover of destabilization of Rad51 on dsDNA it requires ATPase activity on Rad51 and Rad54. Rad51 forms D-Loop with chromatinized DNA template more efficiently in existence of Rad54 when compared to naturally occurring DNA template.

Eukaryotic factors such as Rad51 and Rad54 may be evolved together to deal with several steps of chromatin recombination in vivo. In Rad51 polymerization, all the nucleosomal arrays are moved to the progressing filament and the movement is done by recombinase proteins. In this mechanism, Rad51 destabilizes the nucleosome for a considerable length of the DNA. So, it is a powerful mechanism of chromatin remodeling. These significant features open new possibilities for understanding DNA recombination and reveal new types of ATP-dependent chromatin dynamics

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GradesFixer. (2018, September, 04) A New Chromatin Remodeling Mechanism. Retrived January 29, 2020, from https://gradesfixer.com/free-essay-examples/a-new-chromatin-remodeling-mechanism/
"A New Chromatin Remodeling Mechanism." GradesFixer, 04 Sep. 2018, https://gradesfixer.com/free-essay-examples/a-new-chromatin-remodeling-mechanism/. Accessed 29 January 2020.
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