Nucleosomal structure of undamaged DNA regions suppresses the non-specific DNA binding of the XPC complex

doi:10.1016/j.dnarep.2004.10.008

DNA Repair

Volume 4, Issue 3, 2 March 2005, Pages 389-395

https://doi.org/10.1016/j.dnarep.2004.10.008 Get rights and content

Abstract

The XPC protein complex is a DNA damage detector of human nucleotide excision repair (NER). Although the XPC complex specifically binds to certain damaged sites, it also binds to undamaged DNA in a non-specific manner. The addition of a large excess of undamaged naked DNA competitively inhibited the specific binding of the XPC complex to (6-4) photoproducts and the NER dual incision step in cell-free extracts. In contrast, the addition of undamaged nucleosomal DNA as a competitor suppressed both of these inhibitory effects. Although nucleosomes positioned on the damaged site inhibited the binding of the XPC complex, the presence of nucleosomes in undamaged DNA regions may help specific binding of the XPC complex to damaged sites by excluding its non-specific binding to undamaged DNA regions.

Introduction

Living organisms have multiple repair pathways that cope with a variety of lesions in DNA. Among these, nucleotide excision repair (NER) is responsible for the removal of helix-distorting base lesions induced by UV light and a number of chemical agents. Two subpathways, global genome repair (GGR) and transcription-coupled repair (TCR), contribute to NER. TCR removes transcription-blocking lesions on the transcribed strand, whereas GGR removes lesions from the entire genome. Human GGR has been found to employ a heterotrimeric complex that consists of XPC, HR23B and centrin 2 proteins [1]. This complex specifically binds to a variety of DNA lesions including UV-induced (6-4) photoproducts (6-4PPs) and initiates repair reactions. Although centrin 2 is known to be required for centriole duplication [2], its role in NER remains unclear because the purified recombinant XPC-HR23B heterodimer appears to be sufficient for binding damaged DNA and promoting cell-free NER. After the XPC complex binds to the damaged site, several NER factors including TFIIH, XPG, RPA and XPA are recruited via specific protein–protein interactions, leading to the local unwinding of duplex DNA. Two structure-specific endonucleases, the XPF-ERCC1 complex and XPG, subsequently excise an oligonucleotide containing the damaged site. The resulting DNA gap is filled by DNA polymerase δ or ɛ, and the final nick is sealed by DNA ligase I (reviewed in [3]).

The XPC complex is a structure-specific DNA-binding factor that has significant affinity for single- and double-stranded DNA junctions, and thereby recognizes helix distortions induced by lesions rather than recognizing lesions themselves [4], [5]. Thus the XPC complex can detect a wide variety of lesions that do not share any common features in terms of their chemical structures. In general, the damage detector that initiates DNA repair in vivo must identify damaged sites in a vast excess of undamaged DNA. Although the XPC complex has a specific binding affinity for certain damaged sites, it also binds to undamaged DNA in a non-specific manner [4]. The moderate level of damage specificity of the XPC complex seems to be insufficient for the extremely high damage discrimination required in vivo. However, eukaryotic DNA is folded into nucleosomes wrapped around histone octamers, and the influence of nucleosomal structure on the binding specificity of the XPC complex has not been extensively explored. In this report, we reconstitute nucleosomal DNA in vitro, and examine the effects of nucleosomal structure on the specific damage binding capacity of the XPC complex to 6-4PP and on the NER dual-incision step in cell-free extracts.

Section snippets

DNA substrates

Plasmid DNA containing an artificial nucleosome positioning sequence was constructed as described previously [6]. The unique symmetric AvaI site of pGEM-3zf(+) (Promega) was changed to an asymmetric AvaI site, and a single XhoI site was introduced into the resulting plasmid (Fig. 2A). Two copies of the TG motif were then introduced into the AvaI site, and the synthetic oligonucleotide sequence (5′-GTCGACTCGTCAGCGAATTCATCATACAGTCAGTG-3′) was introduced between the XbaI and PstI sites.

Results

The recognition of damaged DNA by XPC-HR23B was assayed using the electrophoretic mobility shift technique (Fig. 1). To examine the influence of nucleosomal structure on the DNA binding specificity of XPC-HR23B, we added naked or nucleosomal undamaged DNA as a competitor to reaction mixtures. The nucleosomal DNA competitor was reconstituted by the salt-dialysis method with unlabeled negatively supercoiled circular plasmid DNA and purified histone octamers. The formation of nucleosome complexes

Discussion

In this work, we showed that a large excess of undamaged naked DNA competitively inhibits the specific binding of XPC-HR23B to damaged sites and the following dual-incision step in vitro. We also found that this inhibitory effect is suppressed by the presence of nucleosomal structures in regions of undamaged DNA. Thus far, there have been several investigations of in vitro DNA repair processes in the context of nucleosomal structure. Similar inhibitory effects on NER mediated by nucleosomal

Acknowledgements

We are grateful to H. Kurumizaka for providing us with histone-overproducing plasmids. This work was supported by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, by the Core Research for Evolutional Science and Technology (CREST) from the Japan Science and Technology Agency, and by the Bioarchitect Research Project of RIKEN. T.Y. was supported by the Special Postdoctoral Researchers Program of RIKEN and by the Research

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The generation of DNA damage induces the recruitment or assembly of functional HDAC complexes and the deacetylation of local histones, which attracts XPC through an interaction with the XPC-M region. The presence of chromatin remodelling complexes, such as NuRD, may be relevant for subsequent GG-NER steps, since biochemical studies have revealed that DNA lesion sites within nucleosome cores are prevented from recognition by XPC (Hara et al., 2000; Ura et al., 2001; Yasuda et al., 2005), but not by UV-DDB (Matsumoto et al., 2019; Osakabe et al., 2015). UV-DDB in vivo appears to exist on internucleosomal DNA preferentially (Fei et al., 2011), suggesting that it induces alteration of chromatin structures, such that the lesion sites become accessible to XPC.
The XPC protein complex plays a central role in DNA lesion recognition for global genome nucleotide excision repair (GG-NER). Lesion recognition can be accomplished in either a UV-DDB-dependent or -independent manner; however, it is unclear how these sub-pathways are regulated in chromatin. Here, we show that histone deacetylases 1 and 2 facilitate UV-DDB-independent recruitment of XPC to DNA damage by inducing histone deacetylation. XPC localizes to hypoacetylated chromatin domains in a DNA damage-independent manner, mediated by its structurally disordered middle (M) region. The M region interacts directly with the N-terminal tail of histone H3, an interaction compromised by H3 acetylation. Although the M region is dispensable for in vitro NER, it promotes DNA damage removal by GG-NER in vivo, particularly in the absence of UV-DDB. We propose that histone deacetylation around DNA damage facilitates the recruitment of XPC through the M region, contributing to efficient lesion recognition and initiation of GG-NER.
Mechanism and regulation of DNA damage recognition in mammalian nucleotide excision repair
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Citation Excerpt :
These observations provide novel insights into the mechanism of subsequent lesion recognition (see below), although it should also be noted that the use of the potent nucleosome positioning sequence could mask a potential conformational shift induced by DNA damage. Collectively, the evidence obtained from biochemical assays to date indicates that assembly of lesion sites into the nucleosome hampers interaction with XPC and the subsequent NER process [204–206]. In striking contrast, UV-DDB is capable of interacting with lesion sites even within the nucleosome core.
Nucleotide excision repair (NER) is a versatile DNA repair pathway that eliminates various helix-distorting base lesions such as ultraviolet (UV)-induced photolesions. Several recessive human disorders, such as xeroderma pigmentosum (XP), are caused by hereditary defects in NER, implying that the pathway plays critical roles in suppressing diverse pathogenic processes, including carcinogenesis. In general, discrimination of lesion sites from intact DNA, which is present in vast excess, is a key determinant of the overall efficiency of DNA repair. In mammalian cells, global genomic NER lesion recognition is initiated by the XPC protein complex, which achieves broad DNA-binding specificity by sensing destabilized base pairs rather than lesions per se. To avert unnecessary incisions at lesion-free sites, and thereby ensure the fidelity of the repair system, transcription factor IIH and the XPA protein then verify the presence of relevant lesions at suspicious sites bound by XPC. In the case of UV-induced photolesions, a specialized lesion sensor called UV-damaged DNA-binding protein (UV-DDB) contributes to efficient lesion recognition and the recruitment of XPC to lesion sites. The ubiquitin–proteasome system plays a crucial role in the handoff of lesions from UV-DDB to XPC and the subsequent NER process. In addition, recognition of lesions targeted by global genomic NER is intricately regulated by higher-order chromatin structures, which play distinct roles depending on the type of lesion.
Molecular mechanisms of DNA damage recognition for mammalian nucleotide excision repair
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For faithful DNA repair, it is crucial for cells to locate lesions precisely within the vast genome. In the mammalian global genomic nucleotide excision repair (NER) pathway, this difficult task is accomplished through multiple steps, in which the xeroderma pigmentosum group C (XPC) protein complex plays a central role. XPC senses the presence of oscillating 'normal' bases in the DNA duplex, and its binding properties contribute to the extremely broad substrate specificity of NER. Unlike XPC, which acts as a versatile sensor of DNA helical distortion, the UV-damaged DNA-binding protein (UV-DDB) is more specialized, recognizing UV-induced photolesions and facilitating recruitment of XPC. Recent single-molecule analyses and structural studies have advanced our understanding of how UV-DDB finds its targets, particularly in the context of chromatin. After XPC binds DNA, it is necessary to verify the presence of damage in order to avoid potentially deleterious incisions at damage-free sites. Accumulating evidence suggests that XPA and the helicase activity of transcription factor IIH (TFIIH) cooperate to verify abnormalities in DNA chemistry. This chapter reviews recent findings about the mechanisms underlying the efficiency, versatility, and accuracy of NER.
Using deformation energy to analyze nucleosome positioning in genomes
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By modulating the accessibility of genomic regions to regulatory proteins, nucleosome positioning plays important roles in cellular processes. Although intensive efforts have been made, the rules for determining nucleosome positioning are far from satisfaction yet. In this study, we developed a biophysical model to predict nucleosomal sequences based on the deformation energy of DNA sequences, and validated it against the experimentally determined nucleosome positions in the Saccharomyces cerevisiae genome, achieving very high success rates. Furthermore, using the deformation energy model, we analyzed the distribution of nucleosomes around the following three types of DNA functional sites: (1) double strand break (DSB), (2) single nucleotide polymorphism (SNP), and (3) origin of replication (ORI). We have found from the analyzed energy spectra that a remarkable “trough” or “valley” occurs around each of these functional sites, implying a depletion of nucleosome density, fully in accordance with experimental observations. These findings indicate that the deformation energy may play a key role for accurately predicting nucleosome positions, and that it can also provide a quantitative physical approach for in-depth understanding the mechanism of nucleosome positioning.
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The cyclobutane pyrimidine dimer (CPD) is induced in genomic DNA by ultraviolet (UV) light. In mammals, this photolesion is primarily induced within nucleosomal DNA, and repaired exclusively by the nucleotide excision repair (NER) pathway. However, the mechanism by which the CPD is accommodated within the nucleosome has remained unknown. We now report the crystal structure of a nucleosome containing CPDs. In the nucleosome, the CPD induces only limited local backbone distortion, and the affected bases are accommodated within the duplex. Interestingly, one of the affected thymine bases is located within 3.0 Å from the undamaged complementary adenine base, suggesting the formation of complementary hydrogen bonds in the nucleosome. We also found that UV-DDB, which binds the CPD at the initial stage of the NER pathway, also efficiently binds to the nucleosomal CPD. These results provide important structural and biochemical information for understanding how the CPD is accommodated and recognized in chromatin.
In vitro chromatin templates to study nucleotide excision repair
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In eukaryotic cells, DNA associates with histones and exists in the form of a chromatin hierarchy. Thus, it is generally believed that many eukaryotic cellular DNA processing events such as replication, transcription, recombination and DNA repair are influenced by the packaging of DNA into chromatin. This mini-review covers the current knowledge of DNA damage and repair in chromatin based on in vitro studies. Specifically, nucleosome assembly affects DNA damage formation in both random sequences and sequences with strong nucleosome-positioning signals such as 5S rDNA. At least three systems have been used to analyze the effect of nucleosome folding on nucleotide excision repair (NER) in vitro: (a) human cell extracts that have to rely on labeling of repair synthesis to monitor DNA repair, due to very low repair efficacy; (b) Xenopus oocyte nuclear extracts, that have very robust DNA repair efficacy, have been utilized to follow direct removal of DNA damage; (c) six purified human DNA repair factors (RPA, XPA, XPC, TFIIH, XPG, and XPF-ERCC1) that have been used to reconstitute excision repair in vitro. In general, the results have shown that nucleosome folding inhibits NER and, therefore, its activity must be enhanced by chromatin remodeling factors like SWI/SNF. In addition, binding of transcription factors such as TFIIIA to the 5S rDNA promoter also modulates NER efficacy.

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Brief reportNucleosomal structure of undamaged DNA regions suppresses the non-specific DNA binding of the XPC complex

Abstract

Introduction

Section snippets

DNA substrates

Results

Discussion

Acknowledgements

J. Biol. Chem.

Curr. Biol.

DNA Repair

Methods

Meth. Enzymol.

J. Biol. Chem.

Mutat. Res.

J. Biol. Chem.

J. Biol. Chem.

Mutat. Res.

Molecular mechanism of nucleotide excision repair

Genes Dev.

A multistep recognition mechanism for global genomic nucleotide excision repair

Genes Dev.

Brief report
Nucleosomal structure of undamaged DNA regions suppresses the non-specific DNA binding of the XPC complex