Order of Assembly of Human DNA Repair Excision Nuclease

Human excision nuclease removes DNA damage by concerted dual incisions bracketing the lesion. The dual incisions are accomplished by sequential and partly overlapping actions of six repair factors, RPA, XPA, XPC, TFIIH, XPG, and XPF.ERCC1. Of these, RPA, XPA, and XPC have specific binding affinity for damaged DNA. To learn about the role of these three proteins in damage recognition and the order of assembly of the excision nuclease, we measured the binding affinities of XPA, RPA, and XPC to a DNA fragment containing a single (6-4) photoproduct and determined the rate of damage excision under a variety of reaction conditions. We found that XPC has the highest affinity to DNA and that RPA has the highest selectivity for damaged DNA. Under experimental conditions conducive to binding of either XPA + RPA or XPC to damaged DNA, the rate of damage removal was about 5-fold faster for reactions in which XPA + RPA was the first damage recognition factor presented to DNA compared with reactions in which XPC was the first protein that had the opportunity to bind to DNA. We conclude that RPA and XPA are the initial damage sensing factors of human excision nuclease

ヌクレオチド除去修復におけるクロマチン制御とDDBの機能

金沢大学理工研究域ヌクレオチド除去修復におけるクロマチン構造変換機構とDDBの機能を明らかにするために、主に以下の点について解析を行った。まず、DDBとクロマチンリモデリングに関わるタンパク質との関連性を調べるために、免疫沈降法によりDDBと共沈降するタンパク質をウェスタンブロッティングにより解析した。その結果、今までDDBと相互作用することが知られていなかったタンパク質が含まれることがわかったので、細胞内で共発現することによりDDBとの相互作用を確認した。また興味深いことには、細胞に紫外線を照射した時に、そのタンパク質がDNA損傷部位に集積することがわかり、紫外線によるDNA損傷応答において何らかの役割を担っていることが示唆された。現在、ヌクレオチド除去修復反応に及ぼす影響についてさらに詳細な解析を進めている。一方、ニワトリDT40細胞を用いて作成したDDB1のコンディショナルノックアウト細胞で、紫外線以外のDNA傷害剤に対する応答についても解析を行った。その結果、DNA複製を介して二本鎖切断を生じるcamptothecin(CPT)を処理すると、野生型DT40細胞はG_2/M期に停止するのに対し、DDB1を欠損させた場合にはG_2/M期まで進まず、S期の初期で停止することがわかった。さらに、ミトコンドリアの還元酵素活性を指標にCPTに対する感受性について検討したところ、DDB1を欠損させると感受性が有意に増加することがわかった。したがって、DDB1がCPTによるDNA損傷応答において何らかの役割を担っていることが推察され、このDDB1の新しい機能に注目して解析を行っている。研究課題/領域番号:18710044, 研究期間(年度):2006 – 2007出典：「ヌクレオチド除去修復におけるクロマチン制御とDDBの機能」研究成果報告書　課題番号18710044（KAKEN：科学研究費助成事業データベース（国立情報学研究所））（https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-18710044/)を加工して作

Human XPC-hHR23B interacts with XPA-RPA in the recognition of triplex-directed psoralen DNA interstrand crosslinks

DNA interstrand crosslinks (ICLs) represent a severe form of damage that blocks DNA metabolic processes and can lead to cell death or carcinogenesis. The repair of DNA ICLs in mammals is not well characterized. We have reported previously that a key protein complex of nucleotide excision repair (NER), XPA-RPA, recognizes DNA ICLs. We now report the use of triplex technology to direct a site-specific psoralen ICL to a target DNA substrate to determine whether the human global genome NER damage recognition complex, XPC-hHR23B, recognizes this lesion. Our results demonstrate that XPC-hHR23B recognizes psoralen ICLs, which have a structure fundamentally different from other lesions that XPC-hHR23B is known to bind, with high affinity and specificity. XPC-hHR23B and XPA-RPA protein complexes were also observed to bind psoralen ICLs simultaneously, demonstrating not only that psoralen ICLs are recognized by XPC-hHR23B alone, but also that XPA-RPA may interact cooperatively with XPC-hHR23B on damaged DNA, forming a multimeric complex. Since XPC-hHR23B and XPA-RPA participate in the recognition and verification of DNA damage, these results support the hypothesis that interplay between components of the global genome repair sub-pathway of NER is critical for the recognition of psoralen DNA ICLs in the mammalian genome

Characterization of Reaction Intermediates of Human Excision Repair Nuclease

Nucleotide excision repair in humans is a complex reaction involving 14 polypeptides in six repair factors for dual incisions on either sides of a DNA lesion. To identify the reaction intermediates that form by the human excision repair nuclease, we adopted three approaches: purification of functional DNA.protein complexes, permanganate footprinting, and the employment as substrate of presumptive DNA reaction intermediates containing unwound sequences 5' to, 3' to, or encompassing the DNA lesion. The first detectable reaction intermediate was formed by substrate binding of XPA, RPA, XPC.HHR23B plus TFIIH (preincision complex 1, PIC1). In this complex the DNA was unwound on either side of the lesion by no more than 10 bases. Independent of the XPG nuclease function, the XPG protein stabilized this complex, forming a long lived preincision complex 2 (PIC2). The XPF.ERCC1 complex bound to PIC2, forming PIC3, which led to dual incisions and the release of the excised oligomer. With partially unwound DNAs, thymine cyclobutane dimer was excised at a fast rate independent of XPC.HHR23B, indicating that a major function of this protein is to stabilize the unwound DNA or to aid lesion unwinding in preincision complexes

DDB Accumulates at DNA Damage Sites Immediately after UV Irradiation and Directly Stimulates Nucleotide Excision Repair

Damaged DNA-binding protein, DDB, is a heterodimer of p127 and p48 with a high specificity for binding to several types of DNA damage. Mutations in the p48 gene that cause the loss of DDB activity were found in a subset of xeroderma pigmentosum complementation group E (XP-E) patients and have linked to the deficiency in global genomic repair of cyclobutane pyrimidine dimers (CPDs) in these cells. Here we show that with a highly defined system of purified repair factors, DDB can greatly stimulate the excision reaction reconstituted with XPA, RPA, XPC.HR23B, TFIIH, XPF.ERCC1 and XPG, up to 17-fold for CPDs and approximately 2-fold for (6-4) photoproducts (6-4PPs), indicating that no additional factor is required for the stimulation by DDB. Transfection of the p48 cDNA into an SV40-transformed human cell line, WI38VA13, was found to enhance DDB activity and the in vivo removal of CPDs and 6-4PPs. Furthermore, the combined technique of recently developed micropore UV irradiation and immunostaining revealed that p48 (probably in the form of DDB heterodimer) accumulates at locally damaged DNA sites immediately after UV irradiation, and this accumulation is also observed in XP-A and XP-C cells expressing exogenous p48. These results suggest that DDB can rapidly translocate to the damaged DNA sites independent of functional XPA and XPC proteins and directly enhance the excision reaction by core repair factors

The scaffold protein JLP plays a key role in regulating ultraviolet B-induced apoptosis in mice

The ultraviolet B (UVB) component of sunlight can cause severe damage to skin cells and even induce skin cancer. Growing evidence indicates that the UVB-induced signaling network is complex and involves diverse cellular processes. In this study, we investigated the role of c-Jun NH2-terminal kinase-associated leucine zipper protein (JLP), a scaffold protein for mitogen-activated protein kinase (MAPK) signaling cascades, in UVB-induced apoptosis. We found that UVB-induced skin epidermal apoptosis was prevented in Jlp knockout (KO) as well as in keratinocyte-specific Jlp KO mice. Analysis of the repair of UVB-induced DNA damage over time showed no evidence for the involvement of JLP in this process. In contrast, UVB-stimulated p38 MAPK activation in the skin was impaired in both Jlp KO and keratinocyte-specific Jlp KO mice. Moreover, topical treatment of UVB-irradiated mouse skin with a p38 inhibitor significantly suppressed the epidermal apoptosis in wild-type mice, but not in Jlp KO mice. Our findings suggest that JLP in skin basal keratinocytes plays an important role in UVB-induced apoptosis by modulating p38 MAPK signaling pathways. This is the first study to show a critical role for JLP in an in vivo response to environmental stimulation. © 2014 by the Molecular Biology Society of Japan and Wiley Publishing Asia Pty Ltd

Physical and functional interaction between DDB and XPA in nucleotide excision repair

Damaged DNA-binding protein (DDB), consisting of DDB1 and DDB2 subunits recognizes a wide spectrum of DNA lesions. DDB is dispensable for in vitro nucleotide excision repair (NER) reaction, but stimulates this reaction especially for cyclobutane pyrimidine dimer (CPD). Here we show that DDB directly interacts with XPA, one of core NER factors, mainly through DDB2 subunit and the amino-acid residues between 185 and 226 in XPA are important for the interaction. Interestingly, the point mutation causing the substitution from Arg-207 to Gly, which was previously identified in a XP-A revertant cell-line XP129, diminished the interaction with DDB in vitro and in vivo. In a defined system containing R207G mutant XPA and other core NER factors, DDB failed to stimulate the excision of CPD, although the mutant XPA was competent for the basal NER reaction. Moreover, in vivo experiments revealed that the mutant XPA is recruited to damaged DNA sites with much less efficiency compared with wild-type XPA and fails to support the enhancement of CPD repair by ectopic expression of DDB2 in SV40-transformed human cells. These results suggest that the physical interaction between DDB and XPA plays an important role in the DDB-mediated NER reaction

Assembly, subunit composition, and footprint of human DNA repair excision nuclease

The assembly and composition of human excision nuclease were investigated by electrophoretic mobility shift assay and DNase I footprinting. Individual repair factors or any combination of up to four repair factors failed to form DNA–protein complexes of high specificity and stability. A stable complex of high specificity can be detected only when XPA/RPA, transcription factor IIH, XPC⋅HHR23B, and XPG and ATP are present in the reaction mixture. The XPF⋅ERCC1 heterodimer changes the electrophoretic mobility of the DNA–protein complex formed with the other five repair factors, but it does not confer additional specificity. By using proteins with peptide tags or antibodies to the repair factors in electrophoretic mobility shift assays, it was found that XPA, replication protein A, transcription factor IIH, XPG, and XPF⋅excision repair cross-complementing 1 but not XPC⋅HHR23B were present in the penultimate and ultimate dual incision complexes. Thus, it appears that XPC⋅HHR23B is a molecular matchmaker that participates in the assembly of the excision nuclease but is not present in the ultimate dual incision complex. The excision nuclease makes an assymmetric DNase I footprint of ≈30 bp around the damage and increases the DNase I sensitivity of the DNA on both sides of the footprint

NER中間体がもたらす新たなDNA損傷生成とその防御応答の解析

金沢大学医薬保健研究域薬学系In quiescent cells, nucleotide excision repair (NER) process generates multiple types of secondary DNA damage. However, the mechanism of secondary DNA damage formation and their biological meanings are unclear. In this study, we have examined the possible involvement of non-homologus end joining (NHEJ) in the repair of DNA double-strand break (DSB), one of the secondary DNA damages. We found that NHEJ indeed plays an important role in NER-dependent DSB repair and protects UV-induced cell death in quiescent cells. In addition, we also showed that the secondary DNA damage causes the mutations and chromosome aberrations.　 Furthermore, we revealed that at least two endonucleases are required for the activation of ATM as well as DNA-PKcs in the response to NER-dependent DSB. Several endonucleases may act on ssDNA gap intermediates for producing DSBs.ゲノム安定性の維持に重要なヌクレオチド除去修復（nucleotide excision repair; NER）は、休止期ではその後期過程が完了しない場合がある。それが原因で二次的なDNA損傷が生じるが、その生成機構や修復系、そして生物学的意義は不明である。本研究では、二次的DNA損傷のうち二本鎖切断（DNA double-strand break; DSB）には非相同末端結合が機能し、細胞死を抑制することを明らかにした。また、二次的DNA損傷がゲノム不安定性を誘発する結果を得た。そして、NER反応中間体にヌクレアーゼが作用することでDSBが生成し、複数のヌクレアーゼが関与することを示した。研究課題/領域番号:19K12319, 研究期間(年度):2019-04-01 - 2022-03-31出典：研究課題「NER中間体がもたらす新たなDNA損傷生成とその防御応答の解析」課題番号19K12319（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/report/KAKENHI-PROJECT-19K12319/19K12319seika/）を加工して作