The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Richard D Wood - One of the best experts on this subject based on the ideXlab platform.
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strong functional interactions of tfiih with xpc and xpg in human dna Nucleotide Excision Repair without a preassembled Repairosome
Molecular and Cellular Biology, 2001Co-Authors: Erich A Nigg, Sofia J Araujo, Richard D WoodAbstract:In mammalian cells, the core factors involved in the damage recognition and incision steps of DNA Nucleotide Excision Repair are XPA, TFIIH complex, XPC-HR23B, replication protein A (RPA), XPG, and ERCC1-XPF. Many interactions between these components have been detected, using different physical methods, in human cells and for the homologous factors in Saccharomyces cerevisiae. Several human Nucleotide Excision Repair (NER) complexes, including a high-molecular-mass Repairosome complex, have been proposed. However, there have been no measurements of activity of any mammalian NER protein complex isolated under native conditions. In order to assess relative strengths of interactions between NER factors, we captured TFIIH from cell extracts with an anti-cdk7 antibody, retaining TFIIH in active form attached to magnetic beads. Coimmunoprecipitation of other NER proteins was then monitored functionally in a reconstituted Repair system with purified proteins. We found that all detectable TFIIH in gently prepared human cell extracts was present in the intact nine-subunit form. There was no evidence for a Repair complex that contained all of the NER components. At low ionic strength TFIIH could associate with functional amounts of each NER factor except RPA. At physiological ionic strength, TFIIH associated with significant amounts of XPC-HR23B and XPG but not other Repair factors. The strongest interaction was between TFIIH and XPC-HR23B, indicating a coupled role of these proteins in early steps of Repair. A panel of antibodies was used to estimate that there are on the order of 10(5) molecules of each core NER factor per HeLa cell.
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removal of oxygen free radical induced 5 8 purine cyclodeoxynucleosides from dna by the Nucleotide Excision Repair pathway in human cells
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Isao Kuraoka, Richard D Wood, Christina Bender, Anthony Romieu, Jean Cadet, Tomas LindahlAbstract:Exposure of cellular DNA to reactive oxygen species generates several classes of base lesions, many of which are removed by the base Excision-Repair pathway. However, the lesions include purine cyclodeoxynucleoside formation by intramolecular crosslinking between the C-8 position of adenine or guanine and the 5′ position of 2-deoxyribose. This distorting form of DNA damage, in which the purine is attached by two covalent bonds to the sugar-phosphate backbone, occurs as distinct diastereoisomers. It was observed here that both diastereoisomers block primer extension by mammalian and microbial replicative DNA polymerases, using DNA with a site-specific purine cyclodeoxynucleoside residue as template, and consequently appear to be cytotoxic lesions. Plasmid DNA containing either the 5′R or 5′S form of 5′,8-cyclo-2-deoxyadenosine was a substrate for the human Nucleotide Excision-Repair enzyme complex. The R diastereoisomer was more efficiently Repaired than the S isomer. No correction of the lesion by direct damage reversal or base Excision Repair was detected. Dual incision around the lesion depended on the core Nucleotide Excision-Repair protein XPA. In contrast to several other types of oxidative DNA damage, purine cyclodeoxynucleosides are chemically stable and would be expected to accumulate at a slow rate over many years in the DNA of nonregenerating cells from xeroderma pigmentosum patients. High levels of this form of DNA damage might explain the progressive neurodegeneration seen in XPA individuals.
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damage recognition in Nucleotide Excision Repair of dna
Gene, 2000Co-Authors: Dawn P Batty, Richard D WoodAbstract:Abstract Nucleotide Excision Repair (NER) is found throughout nature, in eubacteria, eukaryotes and archaea. In human cells it is the main pathway for the removal of damage caused by UV light, but it also acts on a wide variety of other bulky helix-distorting lesions caused by chemical mutagens. An ongoing challenge is to understand how a site of DNA damage is located during NER and distinguished from non-damaged sites. This article reviews information on damage recognition in mammalian cells and the bacterium Escherichia coli. In mammalian cells the XPC–hHR23B, XPA, RPA and TFIIH factors may all have a role in damage recognition. XPC–hHR23B has the strongest affinity for damaged DNA in some assays, as does the similar budding yeast complex Rad4–Rad23. There is current discussion as to whether XPC or XPA acts first in the Repair process to recognise damage or distortions. TFIIH may play a role in distinguishing the damaged strand from the non-damaged one, if translocation along a DNA strand by the TFIIH DNA helicases is interrupted by encountering a lesion. The recognition and incision steps of human NER use 15 to 18 polypeptides, whereas E. coli requires only three proteins to obtain a similar result. Despite this, many remarkable similarities in the NER mechanism have emerged between eukaryotes and bacteria. These include use of a distortion-recognition factor, a strand separating helicase to create an open preincision complex, participation of structure-specific endonucleases and the lack of a need for certain factors when a region containing damage is already sufficiently distorted.
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the evolutionarily conserved zinc finger motif in the largest subunit of human replication protein a is required for dna replication and mismatch Repair but not for Nucleotide Excision Repair
Journal of Biological Chemistry, 1998Co-Authors: Mahmud K K Shivji, Clark C Chen, Richard D Kolodner, Richard D Wood, Anindya DuttaAbstract:Abstract The largest subunit of the replication protein A (RPA) contains an evolutionarily conserved zinc finger motif that lies outside of the domains required for binding to single-stranded DNA or forming the RPA holocomplex. In previous studies, we showed that a point mutation in this motif (RPAm) cannot support SV40 DNA replication. We have now investigated the role of this motif in several steps of DNA replication and in two DNA Repair pathways. RPAm associates with T antigen, assists the unwinding of double-stranded DNA at an origin of replication, stimulates DNA polymerases α and δ, and supports the formation of the initial short Okazaki fragments. However, the synthesis of a leading strand and later Okazaki fragments is impaired. In contrast, RPAm can function well during the incision step of Nucleotide Excision Repair and in a full Repair synthesis reaction, with either UV-damaged or cisplatin-adducted DNA. Two deletion mutants of the Rpa1 subunit (eliminating amino acids 1–278 or 222–411) were not functional in Nucleotide Excision Repair. We report for the first time that wild type RPA is required for a mismatch Repair reaction in vitro. Neither the deletion mutants nor RPAm can support this reaction. Therefore, the zinc finger of the largest subunit of RPA is required for a function that is essential for DNA replication and mismatch Repair but not for Nucleotide Excision Repair.
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mechanism of open complex and dual incision formation by human Nucleotide Excision Repair factors
The EMBO Journal, 1997Co-Authors: Elizabeth Evans, Jean Marc Egly, Jonathan G Moggs, Jae R Hwang, Richard D WoodAbstract:During Nucleotide Excision Repair in human cells, a damaged DNA strand is cleaved by two endonucleases, XPG on the 3' side of the lesion and ERCC1-XPF on the 5' side. These structure-specific enzymes act at junctions between duplex and single-stranded DNA. ATP-dependent formation of an open DNA structure of approximately 25 nt around the adduct precedes this dual incision. We investigated the mechanism of open complex formation and find that mutations in XPB or XPD, the DNA helicase subunits of the transcription and Repair factor TFIIH, can completely prevent opening and dual incision in cell-free extracts. A deficiency in XPC protein also prevents opening. The absence of RPA, XPA or XPG activities leads to an intermediate level of strand separation. In contrast, XPF or ERCC1-defective extracts open normally and generate a 3' incision, but fail to form the 5' incision. This same Repair defect was observed in extracts from human xeroderma pigmentosum cells with an alteration in the C-terminal domain of XPB, suggesting that XPB has an additional role in facilitating 5' incision by ERCC1-XPF nuclease. These data support a mechanism in which TFIIH-associated helicase activity and XPC protein catalyze initial formation of the key open intermediate, with full extension to the cleavage sites promoted by the other core Nucleotide Excision Repair factors. Opening is followed by dual incision, with the 3' cleavage made first.
Jan H J Hoeijmakers - One of the best experts on this subject based on the ideXlab platform.
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understanding Nucleotide Excision Repair and its roles in cancer and ageing
Nature Reviews Molecular Cell Biology, 2014Co-Authors: Jurgen A Marteijn, Wim Vermeulen, Hannes Lans, Jan H J HoeijmakersAbstract:Nucleotide Excision Repair (NER) eliminates various structurally unrelated DNA lesions by a multiwise 'cut and patch'-type reaction. The global genome NER (GG-NER) subpathway prevents mutagenesis by probing the genome for helix-distorting lesions, whereas transcription-coupled NER (TC-NER) removes transcription-blocking lesions to permit unperturbed gene expression, thereby preventing cell death. Consequently, defects in GG-NER result in cancer predisposition, whereas defects in TC-NER cause a variety of diseases ranging from ultraviolet radiation-sensitive syndrome to severe premature ageing conditions such as Cockayne syndrome. Recent studies have uncovered new aspects of DNA-damage detection by NER, how NER is regulated by extensive post-translational modifications, and the dynamic chromatin interactions that control its efficiency. Based on these findings, a mechanistic model is proposed that explains the complex genotype-phenotype correlations of transcription-coupled Repair disorders.
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dna damage triggers Nucleotide Excision Repair dependent monoubiquitylation of histone h2a
Genes & Development, 2006Co-Authors: Steven Bergink, Florian A Salomons, Deborah Hoogstraten, Tom A M Groothuis, Harm De Waard, Li Yuan, Elisabetta Citterio, Adriaan B Houtsmuller, Jacques Neefjes, Jan H J HoeijmakersAbstract:Chromatin changes within the context of DNA Repair remain largely obscure. Here we show that DNA damage induces monoubiquitylation of histone H2A in the vicinity of DNA lesions. Ultraviolet (UV)-induced monoubiquitylation of H2A is dependent on functional Nucleotide Excision Repair and occurs after incision of the damaged strand. The ubiquitin ligase Ring2 is required for the DNA damage-induced H2A ubiquitylation. UV-induced ubiquitylation of H2A is dependent on the DNA damage signaling kinase ATR (ATM- and Rad3-related) but not the related kinase ATM (ataxia telangiectasia-mutated). Although the response coincides with phosphorylation of variant histone H2AX, H2AX was not required for H2A ubiquitylation. Together our data show that monoubiquitylation of H2A forms part of the cellular response to UV damage and suggest a role of this modification in DNA Repair-induced chromatin remodeling.
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Sequential assembly of the Nucleotide Excision Repair factors in vivo
Molecular Cell, 2001Co-Authors: Marcel Volker, Martijn J. Moné, Wouter Schul, Parimal Karmakar, Wim Vermeulen, Anneke Van Hoffen, Albert A Van Zeeland, Roel Van Driel, Jan H J Hoeijmakers, Leon H F MullendersAbstract:Abstract Here, we describe the assembly of the Nucleotide Excision Repair (NER) complex in normal and Repair-deficient (xeroderma pigmentosum) human cells, employing a novel technique of local UV irradiation combined with fluorescent antibody labeling. The damage recognition complex XPC-hHR23B appears to be essential for the recruitment of all subsequent NER factors in the preincision complex, including transcription Repair factor TFIIH. XPA associates relatively late, is required for anchoring of ERCC1-XPF, and may be essential for activation of the endonuclease activity of XPG. These findings identify XPC as the earliest known NER factor in the reaction mechanism, give insight into the order of subsequent NER components, provide evidence for a dual role of XPA, and support a concept of sequential assembly of Repair proteins at the site of the damage rather than a preassembled Repairosome.
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Nucleotide Excision Repair and human syndromes
Carcinogenesis, 2000Co-Authors: Jan De Boer, Jan H J HoeijmakersAbstract:DNA damage is implicated in cancer and aging, and several DNA Repair mechanisms exist that safeguard the genome from these deleterious consequences. Nucleotide Excision Repair (NER) removes a wide diversity of lesions, the main of which include UV-induced lesions, bulky chemical adducts and some forms of oxidative damage. The NER process involves the action of at least 30 proteins in a 'cut-and-paste'-like mechanism. The consequences of a defect in one of the NER proteins are apparent from three rare recessive syndromes: xeroderma pigmentosum (XP), Cockayne syndrome (CS) and the photosensitive form of the brittle hair disorder trichothiodystrophy (TTD). Sun-sensitive skin is associated with skin cancer predisposition in the case of XP, but remarkably not in CS and TTD. Moreover, the spectrum of clinical symptoms differs considerably between the three syndromes. CS and TTD patients exhibit a spectrum of neurodevelopmental abnormalities and, in addition, TTD is associated with ichthyosis and brittle hair. These typical CS and TTD abnormalities are difficult to comprehend as a consequence of defective NER. This review briefly describes the biochemistry of the NER process, summarizes the clinical features of the NER disorders and speculates on the molecular basis underlying these pleitropic syndromes.
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xeroderma pigmentosum group c protein complex is the initiator of global genome Nucleotide Excision Repair
Molecular Cell, 1998Co-Authors: Kaoru Sugasawa, Shigenori Iwai, Chikahide Masutani, Fumio Hanaoka, Peter J Van Der Spek, Andre P M Eker, D Bootsma, Jan H J HoeijmakersAbstract:The XPC-HR23B complex is specifically involved in global genome but not transcription-coupled Nucleotide Excision Repair (NER). Its function is unknown. Using a novel DNA damage recognition-competition assay, we identified XPC-HR23B as the earliest damage detector to initiate NER: it acts before the known damage-binding protein XPA. Coimmunoprecipitation and DNase I footprinting show that XPC-HR23B binds to a variety of NER lesions. These results resolve the function of XPC-HR23B, define the first NER stages, and suggest a two-step mechanism of damage recognition involving damage detection by XPC-HR23B followed by damage verification by XPA. This provides a plausible explanation for the extreme damage specificity exhibited by global genome Repair. In analogy, in the transcription-coupled NER subpathway, RNA polymerase II may take the role of XPC. After this subpathway-specific initial lesion detection, XPA may function as a common damage verifier and adaptor to the core of the NER apparatus.
Aziz Sancar - One of the best experts on this subject based on the ideXlab platform.
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Nucleotide Excision Repair in human cells fate of the excised oligoNucleotide carrying dna damage in vivo
Journal of Biological Chemistry, 2013Co-Authors: Joyce T. Reardon, Michael G Kemp, Jun Hyuk Choi, Shobhan Gaddameedhi, Aziz SancarAbstract:Nucleotide Excision Repair is the sole mechanism for removing the major UV photoproducts from genomic DNA in human cells. In vitro with human cell-free extract or purified Excision Repair factors, the damage is removed from naked DNA or nucleosomes in the form of 24- to 32-Nucleotide-long oligomers (nominal 30-mer) by dual incisions. Whether the DNA damage is removed from chromatin in vivo in a similar manner and what the fate of the excised oligomer was has not been known previously. Here, we demonstrate that dual incisions occur in vivo identical to the in vitro reaction. Further, we show that transcription-coupled Repair, which operates in the absence of the XPC protein, also generates the nominal 30-mer in UV-irradiated XP-C mutant cells. Finally, we report that the excised 30-mer is released from the chromatin in complex with the Repair factors TFIIH and XPG. Taken together, our results show the congruence of in vivo and in vitro data on Nucleotide Excision Repair in humans.
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circadian oscillation of Nucleotide Excision Repair in mammalian brain
Proceedings of the National Academy of Sciences of the United States of America, 2009Co-Authors: Taehong Kang, Joyce T. Reardon, Michael G Kemp, Aziz SancarAbstract:The circadian clock regulates the daily rhythms in the physiology and behavior of many organisms, including mice and humans. These cyclical changes at molecular and macroscopic levels affect the organism's response to environmental stimuli such as light and food intake and the toxicity and efficacy of chemo- and radiotherapeutic agents. In this work, we investigated the circadian behavior of the Nucleotide Excision Repair capacity in the mouse cerebrum to gain some insight into the optimal circadian time for favorable therapeutic response with minimal side effects in cancer treatment with chemotherapeutic drugs that produce bulky adducts in DNA. We find that Nucleotide Excision Repair activity in the mouse cortex is highest in the afternoon/evening hours and is at its lowest in the night/early morning hours. The circadian oscillation of the Repair capacity is caused at least in part by the circadian oscillation of the xeroderma pigmentosum A DNA damage recognition protein.
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purification and characterization of escherichia coli and human Nucleotide Excision Repair enzyme systems
Methods in Enzymology, 2006Co-Authors: Joyce T. Reardon, Aziz SancarAbstract:Nucleotide Excision Repair is a multicomponent, multistep enzymatic system that removes a wide spectrum of DNA damage by dual incisions in the damaged strand on both sides of the lesion. The basic steps are damage recognition, dual incisions, resynthesis to replace the excised DNA, and ligation. Each step has been studied in vitro using cell extracts or highly purified Repair factors and radiolabeled DNA of known sequence with DNA damage at a defined site. This chapter describes procedures for preparation of DNA substrates designed for analysis of damage recognition, either the 5′ or the 3′ incision event, Excision (resulting from concerted dual incisions), and Repair synthesis. Excision in Escherichia coli is accomplished by the three‐subunit Uvr(A)BC Excision nuclease and in humans by six Repair factors: XPA, RPA, XPC⋅hR23B, TFIIH, XPF⋅ERCC1, and XPG. This chapter outlines methods for expression and purification of these essential Repair factors and provides protocols for performing each of the in vitro Repair assays with either the E. coli or the human Excision nuclease.
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Nucleotide Excision Repair.
Progress in Nucleic Acid Research and Molecular Biology, 2005Co-Authors: Joyce T. Reardon, Aziz SancarAbstract:Publisher Summary DNA damage is a common occurrence that compromises the functional integrity of DNA. It is not surprising then that cells have multiple mechanisms for coping with DNA damage, including those introduced by sunlight and other environmental agents. Nucleotide Excision Repair is a pluripotent pathway for the recognition and removal of a broad spectrum of DNA lesions. Nucleotide Excision Repair involves the removal of damaged DNA bases by dual incisions bracketing the damaged base, release of the damaged base in the form of 12–13 Nucleotide-long oligomers in prokaryotes and 24– 32 Nucleotide-long oligomers in eukaryotes followed by polymerase-mediated replacement of the excised Nucleotides and sealing the Repair patch with ligase. Structural work currently being carried out by numerous groups is expected to provide an understanding at the atomic level for both prokaryotic and eukaryotic Excision nucleases, which will aid in designing further biochemical experiments to refine current models.
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efficient Nucleotide Excision Repair of cisplatin oxaliplatin and bis aceto ammine dichloro cyclohexylamine platinum iv jm216 platinum intrastrand dna diadducts
Cancer Research, 1999Co-Authors: Joyce T. Reardon, Alexandra Vaisman, Stephen G Chaney, Aziz SancarAbstract:Tumors exhibit a spectrum of cellular responses to chemotherapy ranging from extreme sensitivity to resistance, either intrinsic or acquired. These variable responses are both patient and tumor specific. For platinum DNA-damaging agents, drug resistance depends on the carrier ligand of the platinum complex and is due to a combination of mechanisms including DNA Repair. Nucleotide Excision Repair is the only known mechanism by which bulky adducts, including those generated by platinum chemotherapeutic agents, are removed from DNA in human cells. In this report, we show that the types of DNA lesions generated by three platinum drugs, cisplatin, oxaliplatin, and (Bis-aceto-ammine-dichloro-cyclohexylamine-platinum(IV) (JM216), are Repaired in vitro with similar kinetics by the mammalian Nucleotide Excision Repair pathway.
Jean Marc Egly - One of the best experts on this subject based on the ideXlab platform.
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a ubiquitin binding domain in cockayne syndrome b required for transcription coupled Nucleotide Excision Repair
Molecular Cell, 2010Co-Authors: Roy Anindya, Leon H F Mullenders, Wim Vermeulen, Jean Marc Egly, Ulrik Kristensen, Pierreolivier Mari, Hanneke Kool, Giuseppina Gigliamari, Maria Fousteri, Jesper Q SvejstrupAbstract:Transcription-coupled Nucleotide Excision Repair (TC-NER) allows RNA polymerase II (RNAPII)-blocking lesions to be rapidly removed from the transcribed strand of active genes. Defective TCR in humans is associated with Cockayne syndrome (CS), typically caused by defects in either CSA or CSB. Here, we show that CSB contains a ubiquitin-binding domain (UBD). Cells expressing UBD-less CSB (CSB(del)) have phenotypes similar to those of cells lacking CSB, but these can be suppressed by appending a heterologous UBD, so ubiquitin binding is essential for CSB function. Surprisingly, CSB(del) remains capable of assembling Nucleotide Excision Repair factors and Repair synthesis proteins around damage-stalled RNAPII, but such Repair complexes fail to excise the lesion. Together, our results indicate an essential role for protein ubiquitylation and CSB's UBD in triggering damage incision during TC-NER and allow us to integrate the function of CSA and CSB in a model for the process.
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Nucleotide Excision Repair driven by the dissociation of CAK from TFIIH.
Molecular Cell, 2008Co-Authors: Frédéric Coin, Valentyn Oksenych, Vincent Mocquet, Stefanie Groh, Christine Blattner, Jean Marc EglyAbstract:The transcription/DNA Repair factor TFIIH is organized into a core that associates with the CDK-activating kinase (CAK) complex. Using chromatin immunoprecipitation, we have followed the composition of TFIIH over time after UV irradiation of Repair-proficient or -deficient human cells. We show that TFIIH changes subunit composition in response to DNA damage. The CAK is released from the core during Nucleotide Excision Repair (NER). Using reconstituted in vitro NER assay, we show that XPA catalyzes the detachment of the CAK from the core, together with the arrival of the other NER-specific factors. The release of the CAK from the core TFIIH promotes the incision/Excision of the damaged oligoNucleotide and thereby the Repair of the DNA. Following Repair, the CAK reappears with the core TFIIH on the chromatin, together with the resumption of transcription. Our findings demonstrate that the composition of TFIIH is dynamic to adapt its engagement in distinct cellular processes.
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the comings and goings of Nucleotide Excision Repair factors on damaged dna
The EMBO Journal, 2003Co-Authors: Thilo Riedl, Fumio Hanaoka, Jean Marc EglyAbstract:To understand the mechanism of Nucleotide Excision Repair (NER), one of the major human DNA Repair pathways, we have set up a DNA Repair system in which a linear damaged DNA substrate is immobilized by its terminus. By isolating functionally active intermediate complexes, our data dissect the ordered arrival and displacement of NER factors in the progress of the dual incision step. We describe (i) the role of ATP in remodelling the NER-initiating complex of XPC/TFIIH/damaged DNA as a prerequisite for the recruitment of the next NER factors; (ii) the coordination between damage removal and DNA resynthesis and the release of XPC-HR23B, TFIIH and XPA upon arrival of XPG and XPF-ERCC1, respectively; (iii) how RPA remains associated with the excised DNA initiating the assembly of resynthesis factors such as PCNA; (iv) the recycling of XPC-HR23B, TFIIH and XPA in the NER; and the shuttling of TFIIH between NER and transcription. Thus, our findings define multiple functions of NER factors to explain the molecular basis of human NER disorders.
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mechanism of open complex and dual incision formation by human Nucleotide Excision Repair factors
The EMBO Journal, 1997Co-Authors: Elizabeth Evans, Jean Marc Egly, Jonathan G Moggs, Jae R Hwang, Richard D WoodAbstract:During Nucleotide Excision Repair in human cells, a damaged DNA strand is cleaved by two endonucleases, XPG on the 3' side of the lesion and ERCC1-XPF on the 5' side. These structure-specific enzymes act at junctions between duplex and single-stranded DNA. ATP-dependent formation of an open DNA structure of approximately 25 nt around the adduct precedes this dual incision. We investigated the mechanism of open complex formation and find that mutations in XPB or XPD, the DNA helicase subunits of the transcription and Repair factor TFIIH, can completely prevent opening and dual incision in cell-free extracts. A deficiency in XPC protein also prevents opening. The absence of RPA, XPA or XPG activities leads to an intermediate level of strand separation. In contrast, XPF or ERCC1-defective extracts open normally and generate a 3' incision, but fail to form the 5' incision. This same Repair defect was observed in extracts from human xeroderma pigmentosum cells with an alteration in the C-terminal domain of XPB, suggesting that XPB has an additional role in facilitating 5' incision by ERCC1-XPF nuclease. These data support a mechanism in which TFIIH-associated helicase activity and XPC protein catalyze initial formation of the key open intermediate, with full extension to the cleavage sites promoted by the other core Nucleotide Excision Repair factors. Opening is followed by dual incision, with the 3' cleavage made first.
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mammalian dna Nucleotide Excision Repair reconstituted with purified protein components
Cell, 1995Co-Authors: Abdelilah Aboussekhra, Jean Marc Egly, Mahmud K K Shivji, Maureen Biggerstaff, Juhani Vilpo, Vincent Moncollin, Vladimir N Podust, Miroslava Protic, Ulrich Hubscher, Richard D WoodAbstract:Abstract Nucleotide Excision Repair is the principal way by which human cells remove UV damage from DNA. Human cell extracts were fractionated to locate active components, including xeroderma pigmentosum (XP) and ERCC factors. The incision reaction was then reconstituted with the purified proteins RPA, XPA, TFIIH (containing XPB and XPD), XPC, UV-DDB, XPG, partially purified ERCC1/XPF complex, and a factor designated IF7. UV-DDB (related to XPE protein) stimulated Repair but was not essential. ERCC1- and XPF-correcting activity copurified with an ERCC1-binding polypeptide of 110 kDa that was absent in XP-F cell extract. Complete Repair synthesis was achieved by combining these factors with DNA polymerase e, RFC, PCNA, and DNA ligase I. The reconstituted core reaction requires about 30 polypeptides.
Kaoru Sugasawa - One of the best experts on this subject based on the ideXlab platform.
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Mechanism and regulation of DNA damage recognition in Nucleotide Excision Repair
Genes and Environment, 2019Co-Authors: Masayuki Kusakabe, Shigenori Iwai, Yuki Onishi, Haruto Tada, Fumika Kurihara, Kanako Kusao, Mari Furukawa, Masayuki Yokoi, Wataru Sakai, Kaoru SugasawaAbstract:Nucleotide Excision Repair (NER) is a versatile DNA Repair pathway, which can remove an extremely broad range of base lesions from the genome. In mammalian global genomic NER, the XPC protein complex initiates the Repair reaction by recognizing sites of DNA damage, and this depends on detection of disrupted/destabilized base pairs within the DNA duplex. A model has been proposed that XPC first interacts with unpaired bases and then the XPD ATPase/helicase in concert with XPA verifies the presence of a relevant lesion by scanning a DNA strand in 5′-3′ direction. Such multi-step strategy for damage recognition would contribute to achieve both versatility and accuracy of the NER system at substantially high levels. In addition, recognition of ultraviolet light (UV)-induced DNA photolesions is facilitated by the UV-damaged DNA-binding protein complex (UV-DDB), which not only promotes recruitment of XPC to the damage sites, but also may contribute to remodeling of chromatin structures such that the DNA lesions gain access to XPC and the following Repair proteins. Even in the absence of UV-DDB, however, certain types of histone modifications and/or chromatin remodeling could occur, which eventually enable XPC to find sites with DNA lesions. Exploration of novel factors involved in regulation of the DNA damage recognition process is now ongoing.
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tripartite dna lesion recognition and verification by xpc tfiih and xpa in Nucleotide Excision Repair
Molecular Cell, 2015Co-Authors: Filip M Golebiowski, Kaoru Sugasawa, Yuki Onishi, Nadine L Samara, Wei YangAbstract:Transcription factor IIH (TFIIH) is essential for both transcription and Nucleotide Excision Repair (NER). DNA lesions are initially detected by NER factors XPC and XPE or stalled RNA polymerases, but only bulky lesions are preferentially Repaired by NER. To elucidate substrate specificity in NER, we have prepared homogeneous human ten-subunit TFIIH and its seven-subunit core (Core7) without the CAK module and show that bulky lesions in DNA inhibit the ATPase and helicase activities of both XPB and XPD in Core7 to promote NER, whereas non-genuine NER substrates have no such effect. Moreover, the NER factor XPA activates unwinding of normal DNA by Core7, but inhibits the Core7 helicase activity in the presence of bulky lesions. Finally, the CAK module inhibits DNA binding by TFIIH and thereby enhances XPC-dependent specific recruitment of TFIIH. Our results support a tripartite lesion verification mechanism involving XPC, TFIIH, and XPA for efficient NER.
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centrin 2 stimulates Nucleotide Excision Repair by interacting with xeroderma pigmentosum group c protein
Molecular and Cellular Biology, 2005Co-Authors: Ryotaro Nishi, Kaoru Sugasawa, Yuki Okuda, Eriko Watanabe, Toshio Mori, Shigenori Iwai, Chikahide Masutani, Fumio HanaokaAbstract:Xeroderma pigmentosum group C (XPC) protein plays a key role in DNA damage recognition in global genome Nucleotide Excision Repair (NER). The protein forms in vivo a heterotrimeric complex involving one of the two human homologs of Saccharomyces cerevisiae Rad23p and centrin 2, a centrosomal protein. Because centrin 2 is dispensable for the cell-free NER reaction, its role in NER has been unclear. Binding experiments with a series of truncated XPC proteins allowed the centrin 2 binding domain to be mapped to a presumed α-helical region near the C terminus, and three amino acid substitutions in this domain abrogated interaction with centrin 2. Human cell lines stably expressing the mutant XPC protein exhibited a significant reduction in global genome NER activity. Furthermore, centrin 2 enhanced the cell-free NER dual incision and damaged DNA binding activities of XPC, which likely require physical interaction between XPC and centrin 2. These results reveal a novel vital function for centrin 2 in NER, the potentiation of damage recognition by XPC.
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a multistep damage recognition mechanism for global genomic Nucleotide Excision Repair
Genes & Development, 2001Co-Authors: Kaoru Sugasawa, Shigenori Iwai, Chikahide Masutani, Tomoko Okamoto, Yuichiro Shimizu, Fumio HanaokaAbstract:A mammalian Nucleotide Excision Repair (NER) factor, the XPC–HR23B complex, can specifically bind to certain DNA lesions and initiate the cell-free Repair reaction. Here we describe a detailed analysis of its binding specificity using various DNA substrates, each containing a single defined lesion. A highly sensitive gel mobility shift assay revealed that XPC–HR23B specifically binds a small bubble structure with or without damaged bases, whereas dual incision takes place only when damage is present in the bubble. This is evidence that damage recognition for NER is accomplished through at least two steps; XPC–HR23B first binds to a site that has a DNA helix distortion, and then the presence of injured bases is verified prior to dual incision. Cyclobutane pyrimidine dimers (CPDs) were hardly recognized by XPC–HR23B, suggesting that additional factors may be required for CPD recognition. Although the presence of mismatched bases opposite a CPD potentiated XPC–HR23B binding, probably due to enhancement of the helix distortion, cell-free Excision of such compound lesions was much more efficient than expected from the observed affinity for XPC–HR23B. This also suggests that additional factors and steps are required for the recognition of some types of lesions. A multistep mechanism of this sort may provide a molecular basis for ensuring the high level of damage discrimination that is required for global genomic NER.
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xeroderma pigmentosum group c protein complex is the initiator of global genome Nucleotide Excision Repair
Molecular Cell, 1998Co-Authors: Kaoru Sugasawa, Shigenori Iwai, Chikahide Masutani, Fumio Hanaoka, Peter J Van Der Spek, Andre P M Eker, D Bootsma, Jan H J HoeijmakersAbstract:The XPC-HR23B complex is specifically involved in global genome but not transcription-coupled Nucleotide Excision Repair (NER). Its function is unknown. Using a novel DNA damage recognition-competition assay, we identified XPC-HR23B as the earliest damage detector to initiate NER: it acts before the known damage-binding protein XPA. Coimmunoprecipitation and DNase I footprinting show that XPC-HR23B binds to a variety of NER lesions. These results resolve the function of XPC-HR23B, define the first NER stages, and suggest a two-step mechanism of damage recognition involving damage detection by XPC-HR23B followed by damage verification by XPA. This provides a plausible explanation for the extreme damage specificity exhibited by global genome Repair. In analogy, in the transcription-coupled NER subpathway, RNA polymerase II may take the role of XPC. After this subpathway-specific initial lesion detection, XPA may function as a common damage verifier and adaptor to the core of the NER apparatus.
