Publications by authors named "Fumio Hanaoka"

176 Publications

The intrinsically disordered N-terminal region of mouse DNA polymerase alpha mediates its interaction with POT1a/b at telomeres.

Genes Cells 2021 Mar 12. Epub 2021 Mar 12.

Department of Applied Chemistry, Faculty of Science, Tokyo University of Science, Shinjuku-ku, Tokyo, 162-8601, Japan.

Mouse telomerase and the DNA polymerase alpha-primase complex elongate the leading and lagging strands of telomeres, respectively. To elucidate the molecular mechanism of lagging strand synthesis, we investigated the interaction between DNA polymerase alpha and two paralogs of the mouse POT1 telomere-binding protein (POT1a and POT1b). Yeast two-hybrid analysis and a glutathione-S-transferase pull-down assay indicated that the C-terminal region of POT1a binds to the intrinsically disordered N-terminal region of p180, the catalytic subunit of mouse DNA polymerase alpha. Subcellular distribution analyses showed that although POT1a, POT1b, and TPP1 were localized to the cytoplasm, POT1a-TPP1 and POT1b-TPP1 coexpressed with TIN2 localized to the nucleus in a TIN2 dose-dependent manner. Coimmunoprecipitation and cell cycle synchronization experiments indicated that POT1b-TPP1-TIN2 was more strongly associated with p180 than POT1a-TPP1-TIN2, and this complex accumulated during the S phase. Fluorescence in situ hybridization and proximity ligation assays showed that POT1a and POT1b interacted with p180 and TIN2 on telomeric chromatin. Based on the present study and a previous study, we propose a model in which POT1a/b-TPP1-TIN2 translocates into the nucleus in a TIN2-dose dependent manner to target the telomere, where POT1a/b interacts with DNA polymerase alpha for recruitment at the telomere for lagging strand synthesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/gtc.12845DOI Listing
March 2021

Functional impacts of the ubiquitin-proteasome system on DNA damage recognition in global genome nucleotide excision repair.

Sci Rep 2020 11 12;10(1):19704. Epub 2020 Nov 12.

Biosignal Research Center, Kobe University, Kobe, 657-8501, Japan.

The ubiquitin-proteasome system (UPS) plays crucial roles in regulation of various biological processes, including DNA repair. In mammalian global genome nucleotide excision repair (GG-NER), activation of the DDB2-associated ubiquitin ligase upon UV-induced DNA damage is necessary for efficient recognition of lesions. To date, however, the precise roles of UPS in GG-NER remain incompletely understood. Here, we show that the proteasome subunit PSMD14 and the UPS shuttle factor RAD23B can be recruited to sites with UV-induced photolesions even in the absence of XPC, suggesting that proteolysis occurs at DNA damage sites. Unexpectedly, sustained inhibition of proteasome activity results in aggregation of PSMD14 (presumably with other proteasome components) at the periphery of nucleoli, by which DDB2 is immobilized and sequestered from its lesion recognition functions. Although depletion of PSMD14 alleviates such DDB2 immobilization induced by proteasome inhibitors, recruitment of DDB2 to DNA damage sites is then severely compromised in the absence of PSMD14. Because all of these proteasome dysfunctions selectively impair removal of cyclobutane pyrimidine dimers, but not (6-4) photoproducts, our results indicate that the functional integrity of the proteasome is essential for the DDB2-mediated lesion recognition sub-pathway, but not for GG-NER initiated through direct lesion recognition by XPC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-020-76898-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7665181PMC
November 2020

Effect of sequence context on Polζ-dependent error-prone extension past (6-4) photoproducts.

DNA Repair (Amst) 2020 03 14;87:102771. Epub 2019 Dec 14.

Department of Life Science, Faculty of Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo, 171-8588, Japan; Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamada-oka, Suita, Osaka, 565-0871, Japan; National Institute of Genetics, 1111 Yata, Mishima, Shizuoka, 411-8540, Japan. Electronic address:

The (6-4) pyrimidine-pyrimidone photoproduct [(6-4)PP] is a major DNA lesion induced by ultraviolet radiation. (6-4)PP induces complex mutations opposite its downstream bases, in addition to opposite 3' or 5' base, as has been observed through a site-specific translesion DNA synthesis (TLS) assay. The mechanism by which these mutations occur is not well understood. To elucidate the mechanisms underlying mutagenesis induced by (6-4)PP, we performed an intracellular TLS assay using a replicative vector with site-specific T(thymidine)-T (6-4)PP. Rev3p53 mouse embryonic fibroblast (MEF) cells (defective in Polζ) were almost completely defective in bypassing T-T (6-4)PP, whereas both Rev1 and PolhPoliPolk MEF cells (defective in Polη, Polι, and Polκ) presented bypassing activity comparable to that of wild-type cells, indicating that Y-family TLS polymerases are dispensable for bypassing activity, whereas Polζ plays an essential role, probably at the extension step. Among all cells tested, misincorporation occurred most frequently just beyond the lesion (position +1), indicating that the Polζ-dependent extension step is crucial for (6-4)PP-induced mutagenesis. We then examined the effects of sequence context on T-T (6-4)PP bypass using a series of T-T (6-4)PP templates with different sequences at position +1 or -1 to the lesion, and found that the dependency of T-T (6-4)PP bypass on Polζ is not sequence specific. However, the misincorporation frequency at position +1 differed significantly among these templates. The misincorporation of A at position +1 occurred frequently when a purine base was located at position -1. These results indicate that Polζ-dependent extension plays a major role in inducing base substitutions in (6-4)PP-induced mutagenesis, and its fidelity is affected by sequence context surrounding a lesion.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.dnarep.2019.102771DOI Listing
March 2020

Replication stress triggers microsatellite destabilization and hypermutation leading to clonal expansion in vitro.

Nat Commun 2019 09 2;10(1):3925. Epub 2019 Sep 2.

Division of Carcinogenesis and Cancer Prevention, National Cancer Center Research Institute, Tsukiji, Chuo-ku, Tokyo, 104-0045, Japan.

Mismatch repair (MMR)-deficient cancers are characterized by microsatellite instability (MSI) and hypermutation. However, it remains unclear how MSI and hypermutation arise and contribute to cancer development. Here, we show that MSI and hypermutation are triggered by replication stress in an MMR-deficient background, enabling clonal expansion of cells harboring ARF/p53-module mutations and cells that are resistant to the anti-cancer drug camptothecin. While replication stress-associated DNA double-strand breaks (DSBs) caused chromosomal instability (CIN) in an MMR-proficient background, they induced MSI with concomitant suppression of CIN via a PARP-mediated repair pathway in an MMR-deficient background. This was associated with the induction of mutations, including cancer-driver mutations in the ARF/p53 module, via chromosomal deletions and base substitutions. Immortalization of MMR-deficient mouse embryonic fibroblasts (MEFs) in association with ARF/p53-module mutations was ~60-fold more efficient than that of wild-type MEFs. Thus, replication stress-triggered MSI and hypermutation efficiently lead to clonal expansion of cells with abrogated defense systems.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41467-019-11760-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6718401PMC
September 2019

Polη, a Y-family translesion synthesis polymerase, promotes cellular tolerance of Myc-induced replication stress.

J Cell Sci 2018 06 25;131(12). Epub 2018 Jun 25.

Laboratory of Molecular Genetics, Institute for Molecular and Cellular Regulation, Gunma University, 371-8512 Gunma, Japan

Growth of precancerous and cancer cells relies on their tolerance of oncogene-induced replication stress (RS). Translesion synthesis (TLS) plays an essential role in the cellular tolerance of various types of RS and bypasses replication barriers by employing specialized polymerases. However, limited information is available about the role of TLS polymerases in oncogene-induced RS. Here, we report that Polη, a Y-family TLS polymerase, promotes cellular tolerance of Myc-induced RS. Polη was recruited to Myc-induced RS sites, and Polη depletion enhanced the Myc-induced slowing and stalling of replication forks and the subsequent generation of double-strand breaks (DSBs). Overexpression of a catalytically dead Polη also promoted Myc-induced DSB formation. In the absence of Polη, Myc-induced DSB formation depended on MUS81-EME2 (the S-phase-specific endonuclease complex), and concomitant depletion of MUS81-EME2 and Polη enhanced RS and cell death in a synergistic manner. Collectively, these results indicate that Polη facilitates fork progression during Myc-induced RS, thereby helping cells tolerate the resultant deleterious effects. Additionally, the present study highlights the possibility of a synthetic sickness or lethality between Polη and MUS81-EME2 in cells experiencing Myc-induced RS.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/jcs.212183DOI Listing
June 2018

Novel function of HATs and HDACs in homologous recombination through acetylation of human RAD52 at double-strand break sites.

PLoS Genet 2018 03 28;14(3):e1007277. Epub 2018 Mar 28.

Research Center for Radiation Emergency Medicine, National Institute of Radiological Sciences (NIRS), Anagawa, Inage-ku, Chiba, Japan.

The p300 and CBP histone acetyltransferases are recruited to DNA double-strand break (DSB) sites where they induce histone acetylation, thereby influencing the chromatin structure and DNA repair process. Whether p300/CBP at DSB sites also acetylate non-histone proteins, and how their acetylation affects DSB repair, remain unknown. Here we show that p300/CBP acetylate RAD52, a human homologous recombination (HR) DNA repair protein, at DSB sites. Using in vitro acetylated RAD52, we identified 13 potential acetylation sites in RAD52 by a mass spectrometry analysis. An immunofluorescence microscopy analysis revealed that RAD52 acetylation at DSBs sites is counteracted by SIRT2- and SIRT3-mediated deacetylation, and that non-acetylated RAD52 initially accumulates at DSB sites, but dissociates prematurely from them. In the absence of RAD52 acetylation, RAD51, which plays a central role in HR, also dissociates prematurely from DSB sites, and hence HR is impaired. Furthermore, inhibition of ataxia telangiectasia mutated (ATM) protein by siRNA or inhibitor treatment demonstrated that the acetylation of RAD52 at DSB sites is dependent on the ATM protein kinase activity, through the formation of RAD52, p300/CBP, SIRT2, and SIRT3 foci at DSB sites. Our findings clarify the importance of RAD52 acetylation in HR and its underlying mechanism.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1371/journal.pgen.1007277DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5891081PMC
March 2018

Hypersensitivity of mouse embryonic fibroblast cells defective for DNA polymerases η, ι and κ to various genotoxic compounds: Its potential for application in chemical genotoxic screening.

DNA Repair (Amst) 2018 01 26;61:76-85. Epub 2017 Nov 26.

Division of Pathology, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan.

Genotoxic agents cause modifications of genomic DNA, such as alkylation, oxidation, bulky adduct formation, and strand breaks, which potentially induce mutations and changes to the structure or number of genes. Majority of point mutations are generated during error-prone bypass of modified nucleotides (translesion DNA synthesis, TLS); however, when TLS fails, replication forks stalled at lesions eventually result in more lethal effects, formation of double-stranded breaks (DSBs). Here we compared sensitivities to various compounds among mouse embryonic fibroblasts derived from wild-type and knock-out mice lacking one of the three Y-family TLS DNA polymerases (Polη, Polι, and Polκ) or all of them (TKO). The compounds tested in this study include genotoxins such as methyl methanesulfonate (MMS) and nongenotoxins such as ammonium chloride. We found that TKO cells exhibited the highest sensitivities to most of the tested genotoxins, but not to the non-genotoxins. In order to quantitatively evaluate the hypersensitivity of TKO cells to different chemicals, we calculated ratios of half-maximal inhibitory concentration for WT and TKO cells. The ratios for 9 out of 10 genotoxins ranged from 2.29 to 5.73, while those for 5 nongenotoxins ranged from 0.81 to 1.63. Additionally, the two markers for DNA damage, ubiquitylated proliferating cell nuclear antigen and γ-H2AX after MMS treatment, were accumulated in TKO cells more greatly than in WT cells. Furthermore, following MMS treatment, TKO cells exhibited increased frequency of sister chromatid exchange compared with WT cells. These results indicated that the hypersensitivity of TKO cells to genotoxins resulted from replication fork stalling and subsequent DNA double-strand breaks, thus demonstrating that TKO cells should be useful for evaluating chemical genotoxicity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.dnarep.2017.11.006DOI Listing
January 2018

The Mcm2-7-interacting domain of human mini-chromosome maintenance 10 (Mcm10) protein is important for stable chromatin association and origin firing.

J Biol Chem 2017 08 22;292(31):13008-13021. Epub 2017 Jun 22.

Department of Life Science, Faculty of Science, Gakushuin University, Tokyo 171-8588, Japan; Life Science Center of Tsukuba Advanced Research Alliance, University of Tsukuba, Ibaraki 305-8577, Japan.

The protein mini-chromosome maintenance 10 (Mcm10) was originally identified as an essential yeast protein in the maintenance of mini-chromosome plasmids. Subsequently, Mcm10 has been shown to be required for both initiation and elongation during chromosomal DNA replication. However, it is not fully understood how the multiple functions of Mcm10 are coordinated or how Mcm10 interacts with other factors at replication forks. Here, we identified and characterized the Mcm2-7-interacting domain in human Mcm10. The interaction with Mcm2-7 required the Mcm10 domain that contained amino acids 530-655, which overlapped with the domain required for the stable retention of Mcm10 on chromatin. Expression of truncated Mcm10 in HeLa cells depleted of endogenous Mcm10 via siRNA revealed that the Mcm10 conserved domain (amino acids 200-482) is essential for DNA replication, whereas both the conserved and the Mcm2-7-binding domains were required for its full activity. Mcm10 depletion reduced the initiation frequency of DNA replication and interfered with chromatin loading of replication protein A, DNA polymerase (Pol) α, and proliferating cell nuclear antigen, whereas the chromatin loading of Cdc45 and Pol ϵ was unaffected. These results suggest that human Mcm10 is bound to chromatin through the interaction with Mcm2-7 and is primarily involved in the initiation of DNA replication after loading of Cdc45 and Pol ϵ.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M117.779371DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5546039PMC
August 2017

Direct single-molecule observations of DNA unwinding by SV40 large tumor antigen under a negative DNA supercoil state.

J Biomol Struct Dyn 2018 01 5;36(1):32-44. Epub 2017 Jan 5.

a Department of Environmental Engineering Science, Graduate School of Science and Technology , Gunma University , Kiryu , Japan.

Superhelices, which are induced by the twisting and coiling of double-helical DNA in chromosomes, are thought to affect transcription, replication, and other DNA metabolic processes. In this study, we report the effects of negative supercoiling on the unwinding activity of simian virus 40 large tumor antigen (SV40 TAg) at a single-molecular level. The supercoiling density of linear DNA templates was controlled using magnetic tweezers and monitored using a fluorescent microscope in a flow cell. SV40 TAg-mediated DNA unwinding under relaxed and negative supercoil states was analyzed by the direct observation of both single- and double-stranded regions of single DNA molecules. Increased negative superhelicity stimulated SV40 TAg-mediated DNA unwinding more strongly than a relaxed state; furthermore, negative superhelicity was associated with an increased probability of SV40 TAg-mediated DNA unwinding. These results suggest that negative superhelicity helps to regulate the initiation of DNA replication.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1080/07391102.2016.1269689DOI Listing
January 2018

Two mammalian homologs of yeast Rad23, HR23A and HR23B, as multifunctional proteins.

Gene 2017 Jan 20;597:1-9. Epub 2016 Oct 20.

Faculty of Science, Gakushuin University, Toshima-ku, Tokyo 171-8588, Japan; TARA Center, University of Tsukuba, Tsukuba, Ibaraki 305-8577, Japan. Electronic address:

Mammalian cells express two homologs of yeast Rad23, the so-called homolog of Rad23 (HR23) proteins. The HR23 proteins were identified more than two decades ago as factors involved in initiation of global genome nucleotide excision repair (GG-NER) along with their interacting partner, xeroderma pigmentosum group C (XPC) protein. Because the HR23 genes encode proteins harboring ubiquitin-like (UBL) domains at their N-termini and two ubiquitin-associated (UBA) domains in their central- and C-terminal regions, the link between HR23 proteins and proteolytic degradation has been widely explored by several methods, including yeast two-hybrid screening and co-affinity purification. To date, various HR23 protein partners have been identified, and these proteins are involved not only in DNA repair, but also in ubiquitin-dependent protein degradation, transcriptional regulation, and cell cycle control. In addition, establishment of mouse strains lacking the HR23 genes and RNA silencing of these genes in human cells demonstrated their significance in animal development and cell growth. Through these studies, the functional differences between the two HR23 proteins have been gradually revealed. Furthermore, recent comprehensive proteomic analyses will help to elucidate the functional protein-protein networks involving the HR23 proteins.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.gene.2016.10.027DOI Listing
January 2017

USP7 Is a Suppressor of PCNA Ubiquitination and Oxidative-Stress-Induced Mutagenesis in Human Cells.

Cell Rep 2015 Dec 8;13(10):2072-80. Epub 2015 Dec 8.

Department of Genome Dynamics, Research Institute of Environmental Medicine, Nagoya University, Nagoya 464-8601, Japan. Electronic address:

Mono-ubiquitinated PCNA activates error-prone DNA polymerases; therefore, strict regulation of PCNA mono-ubiquitination is crucial in avoiding undesired mutagenesis. In this study, we used an in vitro assay system to identify USP7 as a deubiquitinating enzyme of mono-ubiquitinated PCNA. Suppression of USP1, a previously identified PCNA deubiquitinase, or USP7 increased UV- and H2O2-induced PCNA mono-ubiquitination in a distinct and additive manner, suggesting that USP1 and USP7 make different contributions to PCNA deubiquitination in human cells. Cell-cycle-synchronization analyses revealed that USP7 suppression increased H2O2-induced PCNA ubiquitination throughout interphase, whereas USP1 suppression specifically increased ubiquitination in S-phase cells. UV-induced mutagenesis was elevated in USP1-suppressed cells, whereas H2O2-induced mutagenesis was elevated in USP7-suppressed cells. These results suggest that USP1 suppresses UV-induced mutations produced in a manner involving DNA replication, whereas USP7 suppresses H2O2-induced mutagenesis involving cell-cycle-independent processes such as DNA repair.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2015.11.014DOI Listing
December 2015

Structural basis of pyrimidine-pyrimidone (6-4) photoproduct recognition by UV-DDB in the nucleosome.

Sci Rep 2015 Nov 17;5:16330. Epub 2015 Nov 17.

Laboratory of Structural Biology, Graduate School of Advanced Science and Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japan.

UV-DDB, an initiation factor for the nucleotide excision repair pathway, recognizes 6-4PP lesions through a base flipping mechanism. As genomic DNA is almost entirely accommodated within nucleosomes, the flipping of the 6-4PP bases is supposed to be extremely difficult if the lesion occurs in a nucleosome, especially on the strand directly contacting the histone surface. Here we report that UV-DDB binds efficiently to nucleosomal 6-4PPs that are rotationally positioned on the solvent accessible or occluded surface. We determined the crystal structures of nucleosomes containing 6-4PPs in these rotational positions, and found that the 6-4PP DNA regions were flexibly disordered, especially in the strand exposed to the solvent. This characteristic of 6-4PP may facilitate UV-DDB binding to the damaged nucleosome. We present the first atomic-resolution pictures of the detrimental DNA cross-links of neighboring pyrimidine bases within the nucleosome, and provide the mechanistic framework for lesion recognition by UV-DDB in chromatin.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep16330DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4648065PMC
November 2015

Different types of interaction between PCNA and PIP boxes contribute to distinct cellular functions of Y-family DNA polymerases.

Nucleic Acids Res 2015 Sep 13;43(16):7898-910. Epub 2015 Jul 13.

Department of Genome Dynamics, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan

Translesion DNA synthesis (TLS) by the Y-family DNA polymerases Polη, Polι and Polκ, mediated via interaction with proliferating cell nuclear antigen (PCNA), is a crucial pathway that protects human cells against DNA damage. We report that Polη has three PCNA-interacting protein (PIP) boxes (PIP1, 2, 3) that contribute differentially to two distinct functions, stimulation of DNA synthesis and promotion of PCNA ubiquitination. The latter function is strongly associated with formation of nuclear Polη foci, which co-localize with PCNA. We also show that Polκ has two functionally distinct PIP boxes, like Polη, whereas Polι has a single PIP box involved in stimulation of DNA synthesis. All three polymerases were additionally stimulated by mono-ubiquitinated PCNA in vitro. The three PIP boxes and a ubiquitin-binding zinc-finger of Polη exert redundant and additive effects in vivo via distinct molecular mechanisms. These findings provide an integrated picture of the orchestration of TLS polymerases.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkv712DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4652755PMC
September 2015

The Protein Level of Rev1, a TLS Polymerase in Fission Yeast, Is Strictly Regulated during the Cell Cycle and after DNA Damage.

PLoS One 2015 6;10(7):e0130000. Epub 2015 Jul 6.

Institute for Biomolecular Science, Faculty of Science, Gakushuin University, Toshima-ku, Tokyo, Japan.

Translesion DNA synthesis provides an alternative DNA replication mechanism when template DNA is damaged. In fission yeast, Eso1 (polη), Kpa1/DinB (polκ), Rev1, and Polζ (a complex of Rev3 and Rev7) have been identified as translesion synthesis polymerases. The enzymatic characteristics and protein-protein interactions of these polymerases have been intensively characterized; however, how these proteins are regulated during the cell cycle remains unclear. Therefore, we examined the cell cycle oscillation of translesion polymerases. Interestingly, the protein levels of Rev1 peaked during G1 phase and then decreased dramatically at the entry of S phase; this regulation was dependent on the proteasome. Temperature-sensitive proteasome mutants, such as mts2-U31 and mts3-U32, stabilized Rev1 protein when the temperature was shifted to the restrictive condition. In addition, deletion of pop1 or pop2, subunits of SCF ubiquitin ligase complexes, upregulated Rev1 protein levels. Besides these effects during the cell cycle, we also observed upregulation of Rev1 protein upon DNA damage. This upregulation was abolished when rad3, a checkpoint protein, was deleted or when the Rev1 promoter was replaced with a constitutive promoter. From these results, we hypothesize that translesion DNA synthesis is strictly controlled through Rev1 protein levels in order to avoid unwanted mutagenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0130000PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4493104PMC
March 2016

SUMOylation of xeroderma pigmentosum group C protein regulates DNA damage recognition during nucleotide excision repair.

Sci Rep 2015 Jun 4;5:10984. Epub 2015 Jun 4.

1] Biosignal Research Center, Organization of Advanced Science and Technology, Kobe University, Kobe 657-8501, Japan [2] Graduate School of Science, Kobe University, Kobe 657-8501, Japan [3] Cellular Physiology Laboratory, RIKEN, Wako 351-0198, Japan.

The xeroderma pigmentosum group C (XPC) protein complex is a key factor that detects DNA damage and initiates nucleotide excision repair (NER) in mammalian cells. Although biochemical and structural studies have elucidated the interaction of XPC with damaged DNA, the mechanism of its regulation in vivo remains to be understood in more details. Here, we show that the XPC protein undergoes modification by small ubiquitin-related modifier (SUMO) proteins and the lack of this modification compromises the repair of UV-induced DNA photolesions. In the absence of SUMOylation, XPC is normally recruited to the sites with photolesions, but then immobilized profoundly by the UV-damaged DNA-binding protein (UV-DDB) complex. Since the absence of UV-DDB alleviates the NER defect caused by impaired SUMOylation of XPC, we propose that this modification is critical for functional interactions of XPC with UV-DDB, which facilitate the efficient damage handover between the two damage recognition factors and subsequent initiation of NER.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep10984DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4455304PMC
June 2015

Excision of translesion synthesis errors orchestrates responses to helix-distorting DNA lesions.

J Cell Biol 2015 Apr;209(1):33-46

Department of Human Genetics, Leiden University Medical Center, 2300 RC Leiden, Netherlands

In addition to correcting mispaired nucleotides, DNA mismatch repair (MMR) proteins have been implicated in mutagenic, cell cycle, and apoptotic responses to agents that induce structurally aberrant nucleotide lesions. Here, we investigated the mechanistic basis for these responses by exposing cell lines with single or combined genetic defects in nucleotide excision repair (NER), postreplicative translesion synthesis (TLS), and MMR to low-dose ultraviolet light during S phase. Our data reveal that the MMR heterodimer Msh2/Msh6 mediates the excision of incorrect nucleotides that are incorporated by TLS opposite helix-distorting, noninstructive DNA photolesions. The resulting single-stranded DNA patches induce canonical Rpa-Atr-Chk1-mediated checkpoints and, in the next cell cycle, collapse to double-stranded DNA breaks that trigger apoptosis. In conclusion, a novel MMR-related DNA excision repair pathway controls TLS a posteriori, while initiating cellular responses to environmentally relevant densities of genotoxic lesions. These results may provide a rationale for the colorectal cancer tropism in Lynch syndrome, which is caused by inherited MMR gene defects.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.201408017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4395481PMC
April 2015

UV-induced mutations in epidermal cells of mice defective in DNA polymerase η and/or ι.

DNA Repair (Amst) 2015 May 16;29:139-46. Epub 2015 Feb 16.

Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamada-oka, Suita, Osaka 565-0871, Japan; Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamada-oka, Suita, Osaka 565-0871, Japan; Faculty of Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588, Japan; Solution Oriented Research for Science and Technology, Japan Science and Technology Agency, Tokyo, Japan. Electronic address:

Xeroderma pigmentosum variant (XP-V) is a human rare inherited recessive disease, predisposed to sunlight-induced skin cancer, which is caused by deficiency in DNA polymerase η (Polη). Polη catalyzes accurate translesion synthesis (TLS) past pyrimidine dimers, the most prominent UV-induced lesions. DNA polymerase ι (Polι) is a paralog of Polη that has been suggested to participate in TLS past UV-induced lesions, but its function in vivo remains uncertain. We have previously reported that Polη-deficient and Polη/Polι double-deficient mice showed increased susceptibility to UV-induced carcinogenesis. Here, we investigated UV-induced mutation frequencies and spectra in the epidermal cells of Polη- and/or Polι-deficient mice. While Polη-deficient mice showed significantly higher UV-induced mutation frequencies than wild-type mice, Polι deficiency did not influence the frequencies in the presence of Polη. Interestingly, the frequencies in Polη/Polι double-deficient mice were statistically lower than those in Polη-deficient mice, although they were still higher than those of wild-type mice. Sequence analysis revealed that most of the UV-induced mutations in Polη-deficient and Polη/Polι double-deficient mice were base substitutions at dipyrimidine sites. An increase in UV-induced mutations at both G:C and A:T pairs associated with Polη deficiency suggests that Polη contributes to accurate TLS past both thymine- and cytosine-containing dimers in vivo. A significant decrease in G:C to A:T transition in Polη/Polι double-deficient mice when compared with Polη-deficient mice suggests that Polι is involved in error-prone TLS past cytosine-containing dimers when Polη is inactivated.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.dnarep.2015.02.006DOI Listing
May 2015

Relevance of simultaneous mono-ubiquitinations of multiple units of PCNA homo-trimers in DNA damage tolerance.

PLoS One 2015 18;10(2):e0118775. Epub 2015 Feb 18.

Department of Genome Dynamics, Research Institute of Environmental Medicine, Nagoya University, Nagoya, Japan; Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan.

DNA damage tolerance (DDT) pathways, including translesion synthesis (TLS) and additional unknown mechanisms, enable recovery from replication arrest at DNA lesions. DDT pathways are regulated by post-translational modifications of proliferating cell nuclear antigen (PCNA) at its K164 residue. In particular, mono-ubiquitination by the ubiquitin ligase RAD18 is crucial for Polη-mediated TLS. Although the importance of modifications of PCNA to DDT pathways is well known, the relevance of its homo-trimer form, in which three K164 residues are present in a single ring, remains to be elucidated. Here, we show that multiple units of a PCNA homo-trimer are simultaneously mono-ubiquitinated in vitro and in vivo. RAD18 catalyzed sequential mono-ubiquitinations of multiple units of a PCNA homo-trimer in a reconstituted system. Exogenous PCNA formed hetero-trimers with endogenous PCNA in WI38VA13 cell transformants. When K164R-mutated PCNA was expressed in these cells at levels that depleted endogenous PCNA homo-trimers, multiple modifications of PCNA complexes were reduced and the cells showed defects in DDT after UV irradiation. Notably, ectopic expression of mutant PCNA increased the UV sensitivities of Polη-proficient, Polη-deficient, and REV1-depleted cells, suggesting the disruption of a DDT pathway distinct from the Polη- and REV1-mediated pathways. These results suggest that simultaneous modifications of multiple units of a PCNA homo-trimer are required for a certain DDT pathway in human cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0118775PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4332867PMC
December 2015

Functional regulation of the DNA damage-recognition factor DDB2 by ubiquitination and interaction with xeroderma pigmentosum group C protein.

Nucleic Acids Res 2015 Feb 27;43(3):1700-13. Epub 2015 Jan 27.

Biosignal Research Center, Organization of Advanced Science and Technology, Kobe University, Kobe 657-8501, Japan Graduate School of Science, Kobe University, Kobe 657-8501, Japan

In mammalian nucleotide excision repair, the DDB1-DDB2 complex recognizes UV-induced DNA photolesions and facilitates recruitment of the XPC complex. Upon binding to damaged DNA, the Cullin 4 ubiquitin ligase associated with DDB1-DDB2 is activated and ubiquitinates DDB2 and XPC. The structurally disordered N-terminal tail of DDB2 contains seven lysines identified as major sites for ubiquitination that target the protein for proteasomal degradation; however, the precise biological functions of these modifications remained unknown. By exogenous expression of mutant DDB2 proteins in normal human fibroblasts, here we show that the N-terminal tail of DDB2 is involved in regulation of cellular responses to UV. By striking contrast with behaviors of exogenous DDB2, the endogenous DDB2 protein was stabilized even after UV irradiation as a function of the XPC expression level. Furthermore, XPC competitively suppressed ubiquitination of DDB2 in vitro, and this effect was significantly promoted by centrin-2, which augments the DNA damage-recognition activity of XPC. Based on these findings, we propose that in cells exposed to UV, DDB2 is protected by XPC from ubiquitination and degradation in a stochastic manner; thus XPC allows DDB2 to initiate multiple rounds of repair events, thereby contributing to the persistence of cellular DNA repair capacity.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkv038DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4330392PMC
February 2015

Association of the winged helix motif of the TFIIEα subunit of TFIIE with either the TFIIEβ subunit or TFIIB distinguishes its functions in transcription.

Genes Cells 2015 Mar 10;20(3):203-16. Epub 2014 Dec 10.

Laboratory of Gene Regulation, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan.

In eukaryotes, the general transcription factor TFIIE consists of two subunits, α and β, and plays essential roles in transcription. Structure-function studies indicate that TFIIE has three-winged helix (WH) motifs, with one in TFIIEα and two in TFIIEβ. Recent studies suggested that, by binding to the clamp region of RNA polymerase II, TFIIEα-WH promotes the conformational change that transforms the promoter-bound inactive preinitiation complex to the active complex. Here, to elucidate its roles in transcription, functional analyses of point-mutated human TFIIEα-WH proteins were carried out. In vitro transcription analyses identified two classes of mutants. One class was defective in transcription initiation, and the other was defective in the transition from initiation to elongation. Analyses of the binding of this motif to other general transcription factors showed that the former class was defective in binding to the basic helix-loop-helix motif of TFIIEβ and the latter class was defective in binding to the N-terminal cyclin homology region of TFIIB. Furthermore, TFIIEα-WH bound to the TFIIH XPB subunit at a third distinct region. Therefore, these results provide further insights into the mechanisms underlying RNA polymerase II activation at the initial stages of transcription.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/gtc.12212DOI Listing
March 2015

Both high-fidelity replicative and low-fidelity Y-family polymerases are involved in DNA rereplication.

Mol Cell Biol 2015 Feb 8;35(4):699-715. Epub 2014 Dec 8.

Laboratory of Molecular Genetics, The Institute for Molecular and Cellular Regulation, Gunma University, Maebashi, Gunma, Japan

DNA rereplication is a major form of aberrant replication that causes genomic instabilities, such as gene amplification. However, little is known about which DNA polymerases are involved in the process. Here, we report that low-fidelity Y-family polymerases (Y-Pols), Pol η, Pol ι, Pol κ, and REV1, significantly contribute to DNA synthesis during rereplication, while the replicative polymerases, Pol δ and Pol ε, play an important role in rereplication, as expected. When rereplication was induced by depletion of geminin, these polymerases were recruited to rereplication sites in human cell lines. This finding was supported by RNA interference (RNAi)-mediated knockdown of the polymerases, which suppressed rereplication induced by geminin depletion. Interestingly, epistatic analysis indicated that Y-Pols collaborate in a common pathway, independently of replicative polymerases. We also provide evidence for a catalytic role for Pol η and the involvement of Pol η and Pol κ in cyclin E-induced rereplication. Collectively, our findings indicate that, unlike normal S-phase replication, rereplication induced by geminin depletion and oncogene activation requires significant contributions of both Y-Pols and replicative polymerases. These findings offer important mechanistic insights into cancer genomic instability.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1128/MCB.01153-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4301717PMC
February 2015

Transcription cofactor PC4 plays essential roles in collaboration with the small subunit of general transcription factor TFIIE.

Genes Cells 2014 Dec 13;19(12):879-90. Epub 2014 Oct 13.

Laboratory of Gene Regulation, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama, 930-0194, Japan.

In eukaryotes, positive cofactor 4 (PC4) stimulates activator-dependent transcription by facilitating transcription initiation and the transition from initiation to elongation. It also forms homodimers and binds to single-stranded DNA and various transcriptional activators, including the general transcription factor TFIIH. In this study, we further investigated PC4 from Homo sapiens and the nematode Caenorhabditis elegans (hPC4 and cePC4, respectively). hPC4 strongly stimulated transcription on a linearized template, whereas it alleviated transcription on a supercoiled template. Transcriptional stimulation by PC4 was also alleviated by increasing the amount of TFIID. GST pull-down studies with general transcription factors indicated that both hPC4 and cePC4 bind strongly to TFIIB, TFIIEβ, TFIIFα, TFIIFβ and TFIIH XPB subunits and weakly to TBP and TFIIH p62. However, only hPC4 bound to CDK7. The effect of each PC4 on transcription was studied in combination with TFIIEβ. hPC4 stimulated both basal and activated transcription, whereas cePC4 primarily stimulated activated transcription, especially in the presence of TFIIEβ from C. elegans. Finally, hPC4 bound to the C-terminal region of hTFIIEβ adjacent to the basic region. These results indicate that PC4 plays essential roles in the transition step from transcription initiation to elongation by binding to melted DNA in collaboration with TFIIEβ.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1111/gtc.12187DOI Listing
December 2014

Remarkable induction of UV-signature mutations at the 3'-cytosine of dipyrimidine sites except at 5'-TCG-3' in the UVB-exposed skin epidermis of xeroderma pigmentosum variant model mice.

DNA Repair (Amst) 2014 Oct 14;22:112-22. Epub 2014 Aug 14.

Department of Life Science, Faculty of Science, Gakushuin University, Tokyo 171-8588, Japan.

The human POLH gene is responsible for the variant form of xeroderma pigmentosum (XP-V), a genetic disease highly susceptible to cancer on sun-exposed skin areas, and encodes DNA polymerase η (polη), which is specialized for translesion DNA synthesis (TLS) of UV-induced DNA photolesions. We constructed polη-deficient mice transgenic with lacZ mutational reporter genes to study the effect of Polh null mutation (Polh(-/-)) on mutagenesis in the skin after UVB irradiation. UVB induced lacZ mutations with remarkably higher frequency in the Polh(-/-) epidermis and dermis than in the wild-type (Polh(+/+)) and heterozygote. DNA sequences of a hundred lacZ mutants isolated from the epidermis of four UVB-exposed Polh(-/-) mice were determined and compared with mutant sequences from irradiated Polh(+)(/)(+) mice. The spectra of the mutations in the two genotypes were both highly UV-specific and dominated by C→T transitions at dipyrimidines, namely UV-signature mutations. However, sequence preferences of the occurrence of UV-signature mutations were quite different between the two genotypes: the mutations occurred at a higher frequency preferentially at the 5'-TCG-3' sequence context than at the other dipyrimidine contexts in the Polh(+/+) epidermis, whereas the mutations were induced remarkably and exclusively at the 3'-cytosine of almost all dipyrimidine contexts with no preference for 5'-TCG-3' in the Polh(-/-) epidermis. In addition, in Polh(-/-) mice, a small but remarkable fraction of G→T transversions was also observed exclusively at the 3'-cytosine of dipyrimidine sites, strongly suggesting that these transversions resulted not from oxidative damage but from UV photolesions. These results would reflect the characteristics of the error-prone TLS functioning in the bypass of UV photolesions in the absence of polη, which would be mediated by mechanisms based on the two-step model of TLS. On the other hand, the deamination model would explain well the mutation spectrum in the Polh(+/+) genotype.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.dnarep.2014.07.012DOI Listing
October 2014

Sphingosine, a modulator of human translesion DNA polymerase activity.

J Biol Chem 2014 Aug 13;289(31):21663-72. Epub 2014 Jun 13.

From the Departments of Pathology and Biochemistry, The Gottstein Memorial Cancer Research Laboratory, University of Washington, Seattle, Washington 98195,

Translesion (TLS) DNA polymerases are specialized, error-prone enzymes that synthesize DNA across bulky, replication-stalling DNA adducts. In so doing, they facilitate the progression of DNA synthesis and promote cell proliferation. To potentiate the effect of cancer chemotherapeutic regimens, we sought to identify inhibitors of TLS DNA polymerases. We screened five libraries of ∼ 3000 small molecules, including one comprising ∼ 600 nucleoside analogs, for their effect on primer extension activity of DNA polymerase η (Pol η). We serendipitously identified sphingosine, a lipid-signaling molecule that robustly stimulates the activity of Pol η by ∼ 100-fold at low micromolar concentrations but inhibits it at higher concentrations. This effect is specific to the Y-family DNA polymerases, Pols η, κ, and ι. The addition of a single phosphate group on sphingosine completely abrogates this effect. Likewise, the inclusion of other sphingolipids, including ceramide and sphingomyelin to extension reactions does not elicit this response. Sphingosine increases the rate of correct and incorrect nucleotide incorporation while having no effect on polymerase processivity. Endogenous Pol η activity is modulated similarly as the recombinant enzyme. Importantly, sphingosine-treated cells exhibit increased lesion bypass activity, and sphingosine tethered to membrane lipids mimics the effects of free sphingosine. Our studies have uncovered sphingosine as a modulator of TLS DNA polymerase activity; this property of sphingosine may be associated with its known role as a signaling molecule in regulating cell proliferation in response to cellular stress.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1074/jbc.M114.570242DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4118125PMC
August 2014

Guanine- 5-carboxylcytosine base pairs mimic mismatches during DNA replication.

Sci Rep 2014 Jun 9;4:5220. Epub 2014 Jun 9.

Graduate School of Engineering Science, Osaka University Graduate School of Engineering Science, 1-3 Machikaneyama, Toyonaka, Osaka 560-8531 Japan.

The genetic information encoded in genomes must be faithfully replicated and transmitted to daughter cells. The recent discovery of consecutive DNA conversions by TET family proteins of 5-methylcytosine into 5-hydroxymethylcytosine, 5-formylcytosine, and 5-carboxylcytosine (5caC) suggests these modified cytosines act as DNA lesions, which could threaten genome integrity. Here, we have shown that although 5caC pairs with guanine during DNA replication in vitro, G·5caC pairs stimulated DNA polymerase exonuclease activity and were recognized by the mismatch repair (MMR) proteins. Knockdown of thymine DNA glycosylase increased 5caC in genome, affected cell proliferation via MMR, indicating MMR is a novel reader for 5caC. These results suggest the epigenetic modification products of 5caC behave as DNA lesions.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/srep05220DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4048885PMC
June 2014

A cyclobutane thymine-N4-methylcytosine dimer is resistant to hydrolysis but strongly blocks DNA synthesis.

Nucleic Acids Res 2014 Feb 31;42(3):2075-84. Epub 2013 Oct 31.

Division of Chemistry, Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama, Toyonaka, Osaka 560-8531, Japan, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan and Faculty of Science, Gakushuin University, 1-5-1 Mejiro, Toshima-ku, Tokyo 171-8588, Japan.

Exposure of DNA to ultraviolet light produces harmful crosslinks between adjacent pyrimidine bases, to form cyclobutane pyrimidine dimers (CPDs) and pyrimidine(6-4)pyrimidone photoproducts. The CPD is frequently formed, and its repair mechanisms have been exclusively studied by using a CPD formed at a TT site. On the other hand, biochemical analyses using CPDs formed within cytosine-containing sequence contexts are practically difficult, because saturated cytosine easily undergoes hydrolytic deamination. Here, we found that N-alkylation of the exocyclic amino group of 2'-deoxycytidine prevents hydrolysis in CPD formation, and an N-methylated cytosine-containing CPD was stable enough to be derivatized into its phosphoramidite building block and incorporated into oligonucleotides. Kinetic studies of the CPD-containing oligonucleotide indicated that its lifetime under physiological conditions is relatively long (∼ 7 days). In biochemical analyses using human DNA polymerase η, incorporation of TMP opposite the N-methylcytosine moiety of the CPD was clearly detected, in addition to dGMP incorporation, and the incorrect TMP incorporation blocked DNA synthesis. The thermodynamic parameters confirmed the formation of this unusual base pair.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkt1039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3919605PMC
February 2014

Structure-function analysis of the EF-hand protein centrin-2 for its intracellular localization and nucleotide excision repair.

Nucleic Acids Res 2013 Aug 28;41(14):6917-29. Epub 2013 May 28.

Biosignal Research Center, Organization of Advanced Science and Technology, Kobe University, Kobe, Hyogo 657-8501, Japan.

Centrin-2 is an evolutionarily conserved, calmodulin-related protein, which is involved in multiple cellular functions including centrosome regulation and nucleotide excision repair (NER) of DNA. Particularly to exert the latter function, complex formation with the XPC protein, the pivotal NER damage recognition factor, is crucial. Here, we show that the C-terminal half of centrin-2, containing two calcium-binding EF-hand motifs, is necessary and sufficient for both its localization to the centrosome and interaction with XPC. In XPC-deficient cells, nuclear localization of overexpressed centrin-2 largely depends on co-overexpression of XPC, and mutational analyses of the C-terminal domain suggest that XPC and the major binding partner in the centrosome share a common binding surface on the centrin-2 molecule. On the other hand, the N-terminal domain of centrin-2 also contains two EF-hand motifs but shows only low-binding affinity for calcium ions. Although the N-terminal domain is dispensable for enhancement of the DNA damage recognition activity of XPC, it contributes to augmenting rather weak physical interaction between XPC and XPA, another key factor involved in NER. These results suggest that centrin-2 may have evolved to bridge two protein factors, one with high affinity and the other with low affinity, thereby allowing delicate regulation of various biological processes.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1093/nar/gkt434DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3737541PMC
August 2013

Mechanism of somatic hypermutation at the WA motif by human DNA polymerase η.

Proc Natl Acad Sci U S A 2013 May 29;110(20):8146-51. Epub 2013 Apr 29.

Institute of Nuclear-Agricultural Sciences, Zhejiang University, Hangzhou 310029, China.

Somatic hypermutation is programmed base substitutions in the variable regions of Ig genes for high-affinity antibody generation. Two motifs, RGYW and WA (R, purine; Y, pyrimidine; W, A or T), have been found to be somatic hypermutation hotspots. Overwhelming evidence suggests that DNA polymerase η (Pol η) is responsible for converting the WA motif to WG by misincorporating dGTP opposite the templating T. To elucidate the molecular mechanism, crystal structures and kinetics of human Pol η substituting dGTP for dATP in four sequence contexts, TA, AA, GA, and CA, have been determined and compared. The T:dGTP wobble base pair is stabilized by Gln-38 and Arg-61, two uniquely conserved residues among Pol η. Weak base paring of the W (T:A or A:T) at the primer end and their distinct interactions with Pol η lead to misincorporation of G in the WA motif. Between two WA motifs, our kinetic and structural data indicate that A-to-G mutation occurs more readily in the TA context than AA. Finally, Pol η can extend the T:G mispair efficiently to complete the mutagenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1073/pnas.1303126110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3657764PMC
May 2013

Trapping DNA replication origins from the human genome.

Genes (Basel) 2013 Apr 17;4(2):198-225. Epub 2013 Apr 17.

Cellular Physiology Laboratory, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.

Synthesis of chromosomal DNA is initiated from multiple origins of replication in higher eukaryotes; however, little is known about these origins' structures. We isolated the origin-derived nascent DNAs from a human repair-deficient cell line by blocking the replication forks near the origins using two different origin-trapping methods (i.e., UV- or chemical crosslinker-treatment and cell synchronization in early S phase using DNA replication inhibitors). Single-stranded DNAs (of 0.5-3 kb) that accumulated after such treatments were labeled with bromodeoxyuridine (BrdU). BrdU-labeled DNA was immunopurified after fractionation by alkaline sucrose density gradient centrifugation and cloned by complementary-strand synthesis and PCR amplification. Competitive PCR revealed an increased abundance of DNA derived from known replication origins (c-myc and lamin B2 genes) in the nascent DNA fractions from the UV-treated or crosslinked cells. Nucleotide sequences of 85 and 208 kb were obtained from the two libraries (I and II) prepared from the UV-treated log-phase cells and early S phase arrested cells, respectively. The libraries differed from each other in their G+C composition and replication-related motif contents, suggesting that differences existed between the origin fragments isolated by the two different origin-trapping methods. The replication activities for seven out of 12 putative origin loci from the early-S phase cells were shown by competitive PCR. We mapped 117 (library I) and 172 (library II) putative origin loci to the human genome; approximately 60% and 50% of these loci were assigned to the G-band and intragenic regions, respectively. Analyses of the flanking sequences of the mapped loci suggested that the putative origin loci tended to associate with genes (including conserved sites) and DNase I hypersensitive sites; however, poor correlations were found between such loci and the CpG islands, transcription start sites, and K27-acetylated histone H3 peaks.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.3390/genes4020198DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3899975PMC
April 2013

Identification of new scavengers for hydroxyl radicals and superoxide dismutase by utilising ultraviolet A photoreaction of 8-methoxypsoralen and a variety of mutants of Escherichia coli: implications on certain diseases of DNA repair deficiency.

J Photochem Photobiol B 2012 Nov 5;116:30-6. Epub 2012 Aug 5.

School of Science and Technology, Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, England, United Kingdom.

8-Methoxypsoralen+UVA (ultraviolet light of 320-400 nm) known as PUVA has been in use for a number of years for the treatment of psoriasis and vitiligo. The treatment possibly works on the basis of UVA photoactivated 8-methoxypsoralen binding to DNA forming both single strand and double strand type damage. We have used Escherichia coli as model system in studying PUVA induced DNA damage and repair. It has been known for some time that the photoactivated 8-methoxypsoralen, besides intercalating with DNA, generates at least two reactive oxygen species (ROS): hydroxyl radicals and superoxide anions, and also singlet oxygen. In this study it has been found that, in E. coli, malate dehydrogenase, succinate dehydrogenase and NADH:ubiquinone oxidoreductase can protect cells from PUVA killing presumably by scavenging these ROS. Possible mechanisms have been proposed for these enzymes as cell protectors. Studies also suggest the potential for the use of PUVA in the treatment of a large number of human diseases. This study also finds that, unlike 8-methoxypsoralen, trioxsalen (4,5',8-trimethylpsoralen, another derivative of psoralens) does not generate ROS by UVA photoactivation; and hence the mode of action of trioxsalen and PUVA overlaps only in the binding of these molecules to DNA in the presence of UVA.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.jphotobiol.2012.07.004DOI Listing
November 2012