Repair of UV-induced (6-4) Photoproducts in Nucleosome Core DNA

C to T mutation hotspots in skin cancers occur primarily at methylated CpG sites that coincide with sites of UV-induced cyclobutane pyrimidine dimer (CPD) formation. These mutations are proposed to arise from the insertion of A by DNA polymerase opposite the T that results from deamination of the methylC ( m C) within the CPD. Although the frequency of CPD formation and repair is modestly modulated by its rotational position within a nucleosome, the effect of position on the rate of m C deamination in a CPD has not been previously studied. We now report that deamination of a T m C CPD whose sugar phosphate backbone is positioned against the histone core surface decreases by a factor of 4.7, whereas that of a T m C CPD positioned away from the surface increases by a factor of 8.9 when compared with unbound DNA. Because the m Cs undergoing deamination are in similar steric environments, the difference in rate appears to be a consequence of a difference in the flexibility and compression of the two sites due to DNA bending. Considering that formation of the CPD positioned away from the surface is also enhanced by a factor of two, a T m CG site in this position might be expected to have up to an 84-fold higher probability of resulting in a UV-induced m C to T mutation than one positioned against the surface. These results indicate that rotational position may play an important role in the formation of UV-induced C to T mutation hotspots, as well as in the mutagenic mechanism of other DNA lesions.

mentioning

confidence: 59%

Rotational Position of a 5-Methylcytosine-containing Cyclobutane Pyrimidine Dimer in a Nucleosome Greatly Affects Its Deamination Rate

Song

Cannistraro

Taylor

2011

“…Thus, in normal cells the repair synthesis that is enriched in thiol-reactive chromatin at early times may partially reflect damage enriched in open regions of chromatin. Furthermore, the less prevalent UV photoproduct pyrimidine (6 -4)-pyrimidone is repaired more rapidly in human cells (59,60), and some of the patches we detect are from repair of these lesions. However, regardless of where in chromatin these nascent repair patches are located and which UV lesion is removed, our results indicate that sulfhydryl groups are transiently exposed during NER in chromatin of normal human cells and, most likely, reflect nucleosome unfolding in condensed chromatin domains.…”

Section: Discussionmentioning

confidence: 91%

Nucleosome Unfolding during DNA Repair in Normal and Xeroderma Pigmentosum (Group C) Human Cells

Baxter¹,

Smerdon

1998

Self Cite

The fate of nucleosomes during nucleotide excision repair is unclear. We have used organomercurial chromatography to capture accessible thiol groups of proteins at (or near) nascent repair sites in normal and xeroderma pigmentosum (group C) human cells. The reactive groups include cysteine 110 of histone H3, which is exposed in unfolded nucleosomes. Immediately after UV irradiation and a short pulse labeling of repair patches, intact nuclei were digested with restriction enzymes to release ϳ18% of the chromatin into soluble fragments, which are enriched (ϳ4-fold) in a constitutively transcribed gene. Upon organomercurial affinity fractionation, ϳ1.8% of the soluble chromatin remains bound in high salt (0.5 M NaCl) and is released with dithiothreitol. In normal cell chromatin, this fraction is enriched in nascent repair patches (1.5-1.8-fold) over the unbound fraction. This enrichment decreases following short chase periods with a time course similar to the loss of enhanced nuclease sensitivity of these regions (t 1 ⁄2 Ϸ 30 min). Much less enrichment of nascent repair patches is observed in the thiol-reactive fraction from XPC cells, which repair primarily the transcribed strand of active genes. These results suggest that transient nucleosome unfolding occurs during nucleotide excision repair in normal human cells, and this unfolding may require the XPC protein.

“…4 and TABLE ONE) but does not override the free energy that supports the specific rotational setting dictated by the TG motif (TABLE ONE). Thus, highly favorable DNA-histone interactions within the TG motif "force" CTDs to adjust to specific rotational settings, distinguishing the TG motif from bulk genomic DNA sequences where CTDs can affect rotational settings on the nucleosome surface (28). Thus, it appears that the DNA sequences bracketing CTDs in genomic DNA can play a significant role in determining their rotational setting on nucleosomes.…”

Section: Discussionmentioning

confidence: 99%

“…Interestingly, when free genomic DNA containing randomly distributed CPDs is assembled into nucleosomes, the CPDs are found preferentially facing away from the histone surface ( Fig. 1) (28). Thus, there is an energy penalty in burying CPD lesions on the histone surface (29).…”

mentioning

confidence: 99%

Accommodation and Repair of a UV Photoproduct in DNA at Different Rotational Settings on the Nucleosome Surface

Svedružić

Wang²,

Kosmoski³

et al. 2005

Self Cite

Cyclobutane-thymine dimers (CTDs), the most common DNA lesion induced by UV radiation, cause 30°bending and 9°unwinding of the DNA helix. We prepared site-specific CTDs within a short sequence bracketed by strong nucleosome-positioning sequences. The rotational setting of CTDs over one turn of the helix near the dyad center on the histone surface was analyzed by hydroxyl radical footprinting. Surprisingly, the position of CTDs over one turn of the helix does not affect the rotational setting of DNA on the nucleosome surface. Gel-shift analysis indicates that one CTD destabilizes histone-DNA interactions by 0.6 or 1.1 kJ/mol when facing away or toward the histone surface, respectively. Thus, 0.5 kJ/mol energy penalty for a buried CTD is not enough to change the rotational setting of sequences with strong rotational preference. The effect of rotational setting on CTD removal by nucleotide excision repair (NER) was examined using Xenopus oocyte nuclear extracts. The NER rates are only 2-3 times lower in nucleosomes and change by only 1.5-fold when CTDs face away or toward the histone surface. Therefore, in Xenopus nuclear extracts, the rotational orientation of CTDs on nucleosomes has surprisingly little effect on rates of repair. These results indicate that nucleosome dynamics and/or chromatin remodeling may facilitate NER in gaining access to DNA damage in nucleosomes.In eukaryotes, DNA is organized in chromatin structures within the cell nucleus. The chromatin serves a dual purpose, tight DNA packing within the nucleus and organization of dynamic processes such as DNA transcription, replication, and repair. Of these, DNA repair protects genomic integrity from DNA-damaging agents and is vital for cell survival (1). Indeed, occasional failures in DNA repair can lead to aging and to multiple diseases, including cancer (2, 3). When DNA is irradiated with UV light, adjacent pyrimidines can form stable covalent bonds, yielding cis-syn cyclobutane-pyrimidine dimers (CPDs).5 A common UV lesion caused by exposure to direct sunlight, CPDs can lead to skin cancer, one of the most common forms of cancer (4). The CPD lesion can stall DNA replication (5) and transcription (6) and disrupt DNA interactions with other proteins that regulate gene expression (6). Each of these events can lead to genetic instability.The effect of chromatin structure on repair of UV damage was first implicated by the findings of Wilkins and Hart (7). These authors showed that UV-induced lesions in permeable human cells are removed more slowly from DNA that is less accessible to lesion-specific nucleases in chromatin. It was then found that newly repaired DNA in UVirradiated human fibroblasts is initially more accessible to exogenous nucleases than bulk DNA in chromatin (8) and that nucleosome rearrangement occurs in these regions (9). Studies with a yeast mini-chromosome showed that repair of CPDs is faster in nucleosome-free segments of DNA, being highest in the transcribed strand of a selectable gene and correlating with transcription rate in diff...