Nucleotide Excision Repair of the 5 S Ribosomal RNA Gene Assembled into a Nucleosome

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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...

Section: Ctd Repair Rates With Nuclear Extracts Fromsupporting

confidence: 91%

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

Svedružić

Wang²,

Kosmoski³

et al. 2005

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“…With Xenopus nuclear extracts, the nucleotide excision repair rates of rotationally positioned CPDs were only 2-3 times lower in nucleosomes and no more than 1.5-fold greater when a CPD was positioned away from the histone surface when compared with against the surface (41). In an earlier study with a 5S rRNA gene, no correlation was observed with rotational positioning of the CPD (45).…”

mentioning

confidence: 78%

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

Song

Cannistraro

Taylor

2011

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.

“…: 509-335-1948; Fax: 509-335-9688; E-mail: reevesr@wsu.edu. 5 The abbreviations used are: NER, nucleotide excision repair; HMG, high mobility group; CPD, cis-syn cyclobutane pyrimidine dimer; nt, nucleotide; PRRII, positive regulatory region II; IL-2R␣, interleukin-2 receptor ␣-subunit; EMSA, electrophoretic mobility shift assay; HA, hemagglutinin; T4 endo V, T4 endonuclease V. …”

mentioning

confidence: 99%

“…In most organisms, DNA helix-distorting bulky lesions are repaired by nucleotide excision repair (NER) 5 involving the excision and replacement of 24 -32 nt of the damaged DNA strand (1). Many factors, such as (a) the type of DNA damage, (b) the DNA sequence surrounding the lesion, (c) the position in chromatin, and (d) the interactions with DNAbinding proteins, are known to affect the efficiency of NER (2)(3)(4).…”

mentioning

confidence: 99%

“…Many factors, such as (a) the type of DNA damage, (b) the DNA sequence surrounding the lesion, (c) the position in chromatin, and (d) the interactions with DNAbinding proteins, are known to affect the efficiency of NER (2)(3)(4). For instance, inhibitory effects of nucleosomes on NER have been observed in vitro and in intact cells (5)(6)(7)(8)(9), presumably reflecting the limited access of NER proteins to these lesions (10). In a similar way, certain DNAbinding proteins, such as transcription factor IIIA and HMGB1 proteins, are known to repress NER at their cognate sequences (11)(12)(13).…”

mentioning

confidence: 99%

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Inhibition of Nucleotide Excision Repair by High Mobility Group Protein HMGA1

Adair¹,

Kwon²,

Dement

et al. 2005

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The mammalian non-histone "high mobility group" A (HMGA) proteins are the primary nuclear proteins that bind to the minor groove of AT-rich DNA. They may, therefore, influence the formation and/or repair of DNA lesions that occur in AT-rich DNA, such as cyclobutane pyrimidine dimers (CPDs) induced by UV radiation. Employing both stably transfected lines of human MCF7 cells containing tetracycline-regulated HMGA1 transgenes and primary Hs578T tumor cells, which naturally overexpress HMGA1 proteins, we have shown that cells overexpressing HMGA1a protein exhibit increased UV sensitivity. Moreover, we demonstrated that knockdown of intracellular HMGA1 concentrations via two independent methods abrogated this sensitivity. Most significantly, we observed that HMGA1a overexpression inhibited global genomic nucleotide excision repair of UV-induced CPD lesions in MCF-7 cells. Consistent with these findings in intact cells, DNA repair experiments employing Xenopus oocyte nuclear extracts and lesion-containing DNA substrates demonstrated that binding of HMGA1a markedly inhibits removal of CPDs in vitro. Furthermore, UV "photo-footprinting" demonstrated that CPD formation within a long run of Ts (T 18 -tract) in a DNA substrate changes significantly when HMGA1 is bound prior to UV irradiation. Together, these results suggest that HMGA1 directly influences both the formation and repair of UV-induced DNA lesions in intact cells. These findings have important implications for the role that HMGA protein overexpression might play in the accumulation of mutations and genomic instabilities associated with many types of human cancers.In most organisms, DNA helix-distorting bulky lesions are repaired by nucleotide excision repair (NER) 5 involving the excision and replacement of 24 -32 nt of the damaged DNA strand (1). Many factors, such as (a) the type of DNA damage, (b) the DNA sequence surrounding the lesion, (c) the position in chromatin, and (d) the interactions with DNAbinding proteins, are known to affect the efficiency of NER (2-4). For instance, inhibitory effects of nucleosomes on NER have been observed in vitro and in intact cells (5-9), presumably reflecting the limited access of NER proteins to these lesions (10). In a similar way, certain DNAbinding proteins, such as transcription factor IIIA and HMGB1 proteins, are known to repress NER at their cognate sequences (11-13). Conversely, transcriptional activators and the RNA polymerase II elongation complex are associated with enhanced repair of transcribing genes (14, 15). Furthermore, in contrast to HMGB1, another member of the HMG protein superfamily that specifically binds to nucleosomes, HMGN1, has been shown to likewise enhance NER of UV-damaged DNA in vivo (16).Stable DNA photoproducts produced by UV-induced covalent linkage between adjacent bases are prototypes of helical distorting, bulky lesions (17). Among a variety of possible UV-induced photoproducts, cyclobutane pyrimidine dimers (CPDs) are the most abundant, stable forms (17) and, if unrepaired, are known...