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.
"Although, such open chromatin conformation is beneficial at early stages, upon completion of DNA repair, chromatin environment needs to be restored to its original state so that epigenetic marks and transcriptional status can be maintained . Although it has been demonstrated that UV damage per se results in histone eviction , local chromatin de-condensation is more commonly achieved by posttranslational modifications of core histones and the incorporation of histone variants. Alternatively, nucleosome sliding or disassembly can take place, usually modulated by ATP-dependent chromatin remodelers. "
[Show abstract][Hide abstract] ABSTRACT: Nucleotide excision repair (NER) is a key component of the DNA damage response (DDR) and it is essential to safeguard genome integrity against genotoxic insults. The regulation of NER is primarily mediated by protein post-translational modifications (PTMs). The NER machinery removes a wide spectrum of DNA helix distorting lesions, including those induced by solar radiation, through two sub-pathways: global genome nucleotide excision repair (GG-NER) and transcription coupled nucleotide excision repair (TC-NER). Severe clinical consequences associated with inherited NER defects, including premature ageing, neurodegeneration and extreme cancer-susceptibility, underscore the biological relevance of NER. In the last two decades most of the core NER machinery has been elaborately described, shifting attention to molecular mechanisms that either facilitate NER in the context of chromatin or promote the timely and accurate interplay between NER factors and various post-translational modifications. In this review, we summarize and discuss the latest findings in NER. In particular, we focus on emerging factors and novel molecular mechanisms by which NER is regulated.
Experimental Cell Research 08/2014; 329(1). DOI:10.1016/j.yexcr.2014.08.010 · 3.25 Impact Factor
"CPD adducts are bulky and can block the polymerase during PCR amplification. Therefore, we removed CPDs by photoreactivation as previously described (23,24). Immunoprecipitated DNA was mixed with 30 uM photolyase in repair buffer (50 mm Tris-HCl, pH7.5, 100 mm NaCl, 1 mm ethylenediaminetetraacetic acid and 10 mm DTT) and irradiated at 365 nm using a Spectroline UV lamp (model ENF-240 C, Spectronics Corp., Westbury, NY) for 30 min through a Pyrex filter. "
[Show abstract][Hide abstract] ABSTRACT: Repair of DNA lesions must occur within the chromatin landscape and is associated with alterations in histone modifications and nucleosome rearrangement. To directly associate these chromatin features with DNA damage and repair, it is necessary to be able to map DNA adducts. We have developed a cyclobutane pyrimidine dimer (CPD)-specific immunoprecipitation method and mapped ultraviolet damage hotspots across human chromosomes 1 and 6. CPD hotspots occur almost equally in genic and intergenic regions. However, these hotspots are significantly more prevalent adjacent to repeat elements, especially Alu repeats. Nucleosome mapping studies indicate that nucleosomes are consistently positioned at Alu elements where CPD hotspots form, but by 2 h post-irradiation, these same regions are significantly depleted of nucleosomes. These results indicate that nucleosomes associated with hotspots of CPD formation are readily rearranged, potentially making them accessible to DNA repair machinery. Our results represent the first chromosome scale map of ultraviolet-induced DNA lesions in the human genome, and reveal the sequence features and dynamic chromatin changes associated with CPD hotspots.
Nucleic Acids Research 10/2013; 42(2). DOI:10.1093/nar/gkt912 · 9.11 Impact Factor
"Nucleosome is the fundamental unit of chromatin in eukaryotes, consisting of approximately 147 bp of DNA coiling around a histone octamer , . Nucleosome positioning has been shown to be involved in gene transcription , demarcation of exon borders , , mRNA splicing , DNA replication , and DNA repair . Nucleosomes exert these functions mainly through regulating the accessibility of DNA. "
[Show abstract][Hide abstract] ABSTRACT: Nucleosome occupancy results in complex sequence variation rate heterogeneity by either increasing mutation rate or inhibiting DNA repair in yeast, fish, and human. H2A.Z nucleosome is extensively involved in gene transcription activation and regulation. To test whether H2A.Z nucleosome has the similar impact on sequence variability in the genome, we profiled the H2A.Z nucleosome occupancy and sequence variation rate at gene ends and splicing sites. Consistent with previous studies, H2A.Z nucleosome positioning helps to demarcate the borders of exons. Nucleosome occupancy is anticorrelated with sequence divergence rate in the regions flanking transcription start sites and splicing sites. However, there is no rate heterogeneity between the linker DNA and H2A.Z nucleosomal DNA regardless of nucleosome occupancy, fuzziness, positioning in promoter, coding, and intergenic regions, young or old genes. But the rate at intergenic nucleosomes and the flanking linker regions is higher than that at the genic counterparts. Further analyses found that the high sequence divergence rate in the promoter regions that are usually nucleosome depleted regions may be likely resulted from the high mutation rate in the enriched tandem repeats. Interestingly, within nucleosomes spanning splicing sites, sequence variability of nucleosomal DNA significantly increases from the end within exons to the other end protruding into introns. The relaxed functional constraint in introns contributes to the high rate of nucleosomal DNA residing in introns while the strict functional constraint in exons maintains the low rate of nucleosomal DNA residing in exons. Taken together, H2A.Z nucleosome occupancy has no effect on sequence variability of genome, which is likely determined by local sequence composition and the concomitant selection pressure.
PLoS ONE 03/2013; 8(3):e58295. DOI:10.1371/journal.pone.0058295 · 3.23 Impact Factor
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