Duan MR and Smerdon MJ (2010), UV damage in DNA promotes nucleosome unwrapping.

XB-ART-44365

J Biol Chem 2010 Aug 20;28534:26295-303. doi: 10.1074/jbc.M110.140087.

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UV damage in DNA promotes nucleosome unwrapping.

Duan MR , Smerdon MJ .

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The association of DNA with histones in chromatin impedes DNA repair enzymes from accessing DNA lesions. Nucleosomes exist in a dynamic equilibrium in which portions of the DNA molecule spontaneously unwrap, transiently exposing buried DNA sites. Thus, nucleosome dynamics in certain regions of chromatin may provide the exposure time and space needed for efficient repair of buried DNA lesions. We have used FRET and restriction enzyme accessibility to study nucleosome dynamics following DNA damage by UV radiation. We find that FRET efficiency is reduced in a dose-dependent manner, showing that the presence of UV photoproducts enhances spontaneous unwrapping of DNA from histones. Furthermore, this UV-induced shift in unwrapping dynamics is associated with increased restriction enzyme accessibility of histone-bound DNA after UV treatment. Surprisingly, the increased unwrapping dynamics is even observed in nucleosome core particles containing a single UV lesion at a specific site. These results highlight the potential for increased "intrinsic exposure" of nucleosome-associated DNA lesions in chromatin to repair proteins.

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Species referenced: Xenopus laevis
Genes referenced: cpd tff3.7

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	FIGURE 1. FRET system to analyze the dynamic changes of nucleosomal DNA. A, locations of donor (Cy3) and acceptor (Cy5) on a 147-bp DNA. The DNA is the strongly positioned sequence 601. The upper panel shows the locations of Cy3 (green) and Cy5 (red) on the naked DNA. The two dyes are 80 bp apart, and the distance between the two dyes is ∼27 nm, which is far beyond the R0 (6 nm) for the Cy3-Cy5 pair. The lower panel is a schematic illustration of the locations of the dyes on the NCP structure. The distance between the two dyes is ∼3 nm, which falls into the R0, so that robust energy transfer could happen upon the excitation of the donor. The NCP structure was generated from the crystal structure with Protein Data Bank code 1KX5 (39). B, emission spectra of naked 601 DNA (pink) and 601 NCPs (blue) with excitation at 515 nm. C, salt-induced unwrapping of NCPs monitored by FRET. An appropriate volume of 5 m NaCl was added to the NCP sample, and the solution was allowed to equilibrate for 30 min at room temperature. Emission spectra were taken at different salt concentrations as shown. The data were normalized by the Cy5 signals excited at 615 nm.
	FIGURE 2. UV damage does not affect NCP reconstitution with the 601 sequence. A, NCP reconstitution with UV-undamaged and -damaged DNA. The 147-bp 601 DNA containing both UV lesions and labeling were mixed with histone octamer at 2 m NaCl. The reconstitution was performed by stepwise salt dialysis, and the final NaCl concentration was 50 mm. The reconstituted products were resolved in 5% native polyacrylamide gel and stained with SYBR Gold. The 100-bp DNA markers are indicated on the left. B, presence of CPDs and 6-4PPs in UV-damaged DNA. The different UV-damaged DNA were blotted on the nitrocellulose and detected by lesion-specific antibodies. The same membranes were reprobed with 32P-labeled DNA to show equal loading. C, Southern blot of the photoproduct yield of the UV-irradiated DNA fragment. The DNA was treated with or without photolyase prior to the T4 DNA polymerase (pol) digestion. The digested samples were blotted on the nylon membrane and probed with with 32P-labeled DNA. D, quantification data of the photoproduct yield by Southern blots. The CPD signals were calculated by subtracting the total signals with the 6-4PPs signals. Three independent experiments were performed to show error bars.
	FIGURE 3. Incorporation of UV lesions drives nucleosome unwrapping. A, energy transfer shown by gel-based FRET. NCPs reconstituted with undamaged and damaged DNA were resolved on a 5% native polyacrylamide gel and then scanned on Typhoon 9400. The excitation lasers and emission filters are shown on the left. The green laser was excited at 532 nm, and the red laser was excited at 633 nm. 580 BP 30 and 670 BP 30 are the band pass emission filters that pass the band of lights centered at 580 nm or 670 nm. The FRET efficiency was calculated as E = IA/(IA + γID) (26), where γ is 1.0 in this case and is shown in the middle of the panels. B, emission spectra of damaged 601 NCPs following different UV doses. The final NaCl concentration was 50 mm. The samples were excited at 515 nm, and emission spectra from 550 nm to 700 nm were recorded. The data were normalized by Cy5 signals excited at 615 nm. C, relative FRET efficiency (E/E0) for the NCP irradiated at different UV doses. E is the FRET efficiency of UV-damaged NCPs, and E0 is the FRET efficiency of undamaged NCPs, which was set as 1. D, equilibrium constants (Keq) for partial DNA unwrapping as a function of UV damage. The constants were calculated from the data in B as described before (24).
	FIGURE 4. Salt-induced NCP dissociation profiles. The FRET efficiency at the lowest salt concentration (50 mm) was set as 1 for comparison of different UV-damaged NCPs. Values are means ± 1 S.D. for three independent experiments. Error bars for 0 kJ/m2 and 24 kJ/m2 are the only ones shown for clarity. The omitted error bars are all in the same range (≤4.5%) as those shown.
	FIGURE 5. Accessibility of restriction enzyme to UV-damaged NCPs. A, schematic illustration of the restriction enzyme digestion sites on the 601 sequence. The DNA was prepared from an EcoRV digestion of the plasmid pLMG601-23, which contained 23 tandem repeats of the 149-bp 601 sequence (37). B, RsaI digestion of NCPs. The UV doses were reduced corresponding to the increase of the DNA length (from 95 bp to 149 bp). S denotes substrates and P1 and P2 denote digestion products. The digestion products were separated on a 16% native polyacrylamide gel and stained with SYBR Gold. The gels were scanned on a STORM 840 FluorImager. C, HaeIII digestion of NCPs. The experiments were performed the same way as in B. D, quantitative analysis of the digestion of UV-damaged NCPs. The fraction undigested was defined as (counts in S)/(counts in S + P1 + P2).
	FIGURE 6. Incorporation of single UV photoproducts increases nucleosome dynamics. A, schematic illustration of the incorporation of a single CPD or 6-4PP into the 601 sequence. The DFs are 12-mer oligonucleotides with the sequences shown. Solid triangles indicate the positions of the photoproduct. The dark circle represents Cy5, and the open circle represents Cy3. B, FRET changes for the single CPD containing NCPs. The 601 sequence was changed according to the DF and denoted 601.DF1. The left panel shows emission spectra for the CPD containing NCPs compared with undamaged NCPs. The right panel shows the FRET efficiency values determined in each case, where the value for 601.DF1 NCPs was set to 1. The values shown are the mean ± 1 S.D. of three independent experiments. C, the same as B except the NCPs contained a single 6-4PP and the sequence denoted 601.DF2. D, relative FRET efficiency of salt dependence of NCPs containing a single CPD. The FRET efficiency at the lowest salt concentration (50 mm) was set to 1. Values are the mean ± 1 S.D. of three independent experiments. E, salt titration profile of NCPs containing a single 6-4PP. Values were plotted the same as in D.

References [+] :

Anderson, Spontaneous access of proteins to buried nucleosomal DNA target sites occurs via a mechanism that is distinct from nucleosome translocation. 2002, Pubmed