Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 20 de 28
Filter
1.
Biochem Biophys Res Commun ; 587: 63-68, 2022 01 08.
Article in English | MEDLINE | ID: mdl-34864548

ABSTRACT

The concept of tRNA recycling has recently emerged from the studies of ribosome-associated quality control. Therein tRNase ZS removes the 2', 3'>p from the ANKZF1-cleaved tRNA and the subsequent TRNT1 action re-generates the intact tRNA. To know the roles of the tRNA recycling in vivo, we investigated how viral infection affects the tRNA recycling system by analyzing the mRNA levels of tRNase ZS and TRNT1. We found that both genes in HeLa cells are upregulated in response to infection of Theiler's mouse encephalitis virus but not to that of an influenza A virus. Upregulation was also observed in cells infected with encephalomyocarditis virus with reduced efficiency. The levels of the IFN-ß mRNA appeared to positively correlate with those of the tRNase ZS and TRNT1 mRNAs. The tRNase ZS gene may be regulated post-transcriptionally in the cells infected with Theiler's mouse encephalitis virus.


Subject(s)
Endoribonucleases/genetics , Host-Pathogen Interactions/genetics , Nucleotidyltransferases/genetics , RNA Processing, Post-Transcriptional , RNA, Transfer/genetics , Theilovirus/genetics , Carrier Proteins/genetics , Carrier Proteins/metabolism , Encephalomyocarditis virus/genetics , Encephalomyocarditis virus/growth & development , Encephalomyocarditis virus/metabolism , Endoribonucleases/metabolism , HeLa Cells , Humans , Influenza A virus/genetics , Influenza A virus/growth & development , Influenza A virus/metabolism , Interferon-beta/genetics , Interferon-beta/metabolism , Isoenzymes/genetics , Isoenzymes/metabolism , Nucleotidyltransferases/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Transfer/metabolism , Ribosomes/genetics , Ribosomes/metabolism , Theilovirus/growth & development , Theilovirus/metabolism , Viral Load
2.
Biochem Biophys Res Commun ; 585: 162-168, 2021 12 31.
Article in English | MEDLINE | ID: mdl-34808499

ABSTRACT

tRNase ZS (ELAC1) and TRNT1 function in tRNA recycling. Recently, we have shown that these genes are upregulated in the cells infected with Theiler's mouse encephalitis virus (TMEV), implying that tRNA recycling functions in response to viral infection. To address the molecular mechanism underlying the ELAC1 upregulation in the cells infected with TMEV, we performed luciferase assays using various plasmid constructs harboring the ELAC1 promoter region. The luciferase expression from a construct containing the full-length ELAC1 promoter was augmented by TMEV, poly IC, IFN-ß, or IFN-γ. We identified four IFN-stimulated responsible elements (ISREs) in the proximal promoter region. The luciferase expression from the constructs that lack all the ISREs was strongly reduced compared with that from the constructs with the four ISREs in the presence of IFN-ß or IFN-γ. The observation that the ISREs from the ELAC1 promoter are essential for the gene upregulation by IFN-ß or IFN-γ suggests that the ELAC1 gene is upregulated by IFNs.


Subject(s)
Interferons/pharmacology , Promoter Regions, Genetic/genetics , RNA, Transfer/genetics , Transcription, Genetic , Tumor Suppressor Proteins/genetics , Up-Regulation/drug effects , Antiviral Agents/pharmacology , Base Sequence , HeLa Cells , Humans , Interferon-beta/pharmacology , Interferon-gamma/pharmacology , RNA, Transfer/metabolism , Response Elements/genetics , Theilovirus/drug effects , Theilovirus/physiology , Up-Regulation/genetics
3.
J Nucleic Acids ; 2021: 2458470, 2021.
Article in English | MEDLINE | ID: mdl-34824869

ABSTRACT

Although antisense oligonucleotide (ASO) therapeutics can be taken up by living cells without carrier molecules, a large part of incorporated ASOs are trapped in the endosomes and do not exert therapeutic effects. To improve their therapeutic effects, it would be important to elucidate the mechanism of cellular uptake and intracellular trafficking of ASOs. In this study, we investigated how SIDT1 affects cellular uptake and intracellular trafficking of ASOs. Fluorescence microscopic analysis suggested that most of naked ASOs are trafficked to the lysosomes via the endosomes. The data obtained from flow cytometry and fluorescence microscopy together showed that although the SIDT1 level barely affects the total cellular uptake of ASOs, it appears to affect the intracellular trafficking of ASOs. We also showed that SIDT1 exists mainly in the endoplasmic reticulum and that perturbing the normal level of SIDT1 suppresses the antisense effect of the naked ASO targeting miR-16.

4.
Biochem Biophys Res Commun ; 573: 140-144, 2021 10 08.
Article in English | MEDLINE | ID: mdl-34411896

ABSTRACT

We have been developing a gene silencing technology by harnessing a tRNA 3' processing endoribonuclease, tRNase ZL, with antisense oligonucleotides. Here, to further improve this technology, we investigated how the length and the modifications of naked oligonucleotides affect the efficiency of their uptake by HeLa, HEK293, and HL60 cells by flow cytometry and fluorescence microscopy. 7-30-nt Alexa-Fluor-568-labeled DNAs with phosphorothioate linkages and 7-30-nt Alexa-Fluor-568-labeled, 2'-O-methylated RNAs without phosphorothioate linkages were examined, and, on the whole, longer oligonucleotides were shown to be intracellularly taken up more efficiently. In addition, a 2'-O-methoxyethylated RNA without phosphorothioate linkages, a 2'-fluoriated RNA without phosphorothioate linkages, a 2'-O-methylated RNA with phosphorothioate linkages, and a 2'-O-methylated RNA with phosphorothioate linkages and LNA modifications of 5'-/3'-terminal nucleotides were examined. The oligonucleotides with phosphorothioate linkages were taken up by the cells more efficiently than those without the linkages. Furthermore, we examined how the phosphorothioate linkages of oligonucleotides affect their antisense effects using 22-nt anti-miR16 oligonucleotides with and without phosphorothioate linkages. The latter oligonucleotide decreased the miR16 level much more intensively than the former, although the latter was intracellularly taken up much less efficiently. These observations may be not generalized and differ depending on features of oligonucleotides and cell types. Taken together these results suggest that the productive uptake efficiency for an antisense oligonucleotide needs to be considered to select its length and modifications.


Subject(s)
Oligonucleotides, Antisense/metabolism , Phosphates/metabolism , Cells, Cultured , Humans , Oligonucleotides, Antisense/chemistry , Phosphates/chemistry
5.
Biochem Biophys Rep ; 26: 100918, 2021 Jul.
Article in English | MEDLINE | ID: mdl-33553691

ABSTRACT

TRUE gene silencing is one of the gene suppression technologies. This technology exploits the enzymatic property of the tRNA 3' processing endoribonuclease tRNase ZL, which is that it can cleave a target RNA under the direction of a small guide RNA (sgRNA). We have been working on the development of therapeutic sgRNAs for hematological malignancies. In the course of an experiment to examine the ability of the heptamer-type sgRNA H15792 targeting the OCT4 mRNA to differentiate human amnion stem cells, we observed unexpectedly that the amnion cells exhibited a morphology resembling initialized cells. Here we investigated the effect of H15792 on human HL60 leukemia cells, and found that H15792 can upregulate the OCT4 expression and the expression of alkaline phosphatase in the cells.

6.
Blood Cells Mol Dis ; 86: 102503, 2021 02.
Article in English | MEDLINE | ID: mdl-32920464

ABSTRACT

Multiple myeloma is a refractory cancer of plasma cells. Although treatment strategies for multiple myeloma are getting improved year by year, in most cases patients relapse due to the emergence of drug-resistant mutations in the myeloma cells. The interplay between myeloma cells and tumor-associated macrophages (TAM) is important for the pathology. We thought that some heptamer-type sgRNAs for TRUE gene silencing would be able to transform TAM toward the M1 state and might become therapeutic drugs for myeloma. Here, we searched for heptamer-type sgRNAs that can shift macrophages toward the M1 state. We screened a heptamer-type sgRNA library for the ability to up-regulate IL-12b gene expression in human macrophage-like cell lines, and found three such sgRNAs. One of the sgRNAs, H12960, which also showed such ability in human fresh macrophages and mouse macrophage-like cell lines, efficiently suppressed human myeloma cell growth in SCID/NOD mice.


Subject(s)
Macrophages , Multiple Myeloma/therapy , RNA, Guide, Kinetoplastida/therapeutic use , Tumor-Associated Macrophages , Animals , Cell Line , Cell Line, Tumor , Cells, Cultured , Gene Expression Regulation, Neoplastic , Gene Silencing , Genetic Therapy , Humans , Interleukin-12/genetics , Interleukin-12/immunology , Macrophage Activation , Macrophages/immunology , Macrophages/metabolism , Mice, Inbred NOD , Mice, SCID , Multiple Myeloma/genetics , Multiple Myeloma/immunology , RNA, Guide, Kinetoplastida/genetics , Tumor-Associated Macrophages/immunology , Tumor-Associated Macrophages/metabolism
7.
PLoS One ; 15(10): e0241557, 2020.
Article in English | MEDLINE | ID: mdl-33126240

ABSTRACT

Metallo-ß-lactamases (MBLs) hydrolyze a wide range of ß-lactam antibiotics. While all MBLs share a common αß/ßα-fold, there are many other proteins with the same folding pattern that exhibit different enzymatic activities. These enzymes, together with MBLs, form the MBL superfamily. Thermotoga maritima tRNase Z, a tRNA 3' processing endoribonuclease of MBL-superfamily, and IMP-1, a clinically isolated MBL, showed a striking similarity in tertiary structure, despite low sequence homology. IMP-1 hydrolyzed both total cellular RNA and synthetic small unstructured RNAs. IMP-1 also hydrolyzed pre-tRNA, but its cleavage site was different from those of T. maritima tRNase Z and human tRNase Z long form, indicating a key difference in substrate recognition. Single-turnover kinetic assays suggested that substrate-binding affinity of T. maritima tRNase Z is much higher than that of IMP-1.


Subject(s)
RNA/metabolism , Thermotoga maritima/enzymology , beta-Lactamases/metabolism , Amino Acid Sequence , Humans , Hydrolysis , Models, Molecular , Protein Conformation , Substrate Specificity , Thermotoga maritima/chemistry , Thermotoga maritima/metabolism , beta-Lactamases/chemistry
8.
Anticancer Res ; 40(8): 4719-4727, 2020 Aug.
Article in English | MEDLINE | ID: mdl-32727798

ABSTRACT

BACKGROUND/AIM: We investigated whether mastication affects microglia, whose activity is thought to be associated with cognition and brain tumor progression. MATERIALS AND METHODS: We kept mice by feeding either a hard or soft diet for 2, 4 or 8 months. After each period, we removed the whole brains and isolated microglia. The total RNA extracted from each brain's microglia was subjected to DNA microarray analysis. RESULTS: Many genes were found to be significantly differentially expressed between hard- and soft-diet-fed mice in each group of the same feeding period. The expression of several genes involved in the regulation of actin cytoskeleton was down-regulated in the soft-diet-fed mice. CONCLUSION: Mastication may affect microglia's roles in cognition as well as their neuroimmune activity through their activity of patrolling the brain.


Subject(s)
Mastication/physiology , Microglia/physiology , Transcriptome/physiology , Animals , Brain/physiology , Male , Mice , Mice, Inbred C3H
9.
Noncoding RNA Res ; 5(1): 37-40, 2020 Mar.
Article in English | MEDLINE | ID: mdl-32206739

ABSTRACT

The 31- and 32-nt 5'-fragment of Y4-RNA (Y4RNAfr) exists abundantly in human peripheral blood plasma. Although physiological roles of the plasma Y4RNAfr are not well established, its potential utility as a diagnostic/prognostic marker for acute coronary syndrome was suggested. In this paper, to establish a normal range of the Y4RNAfr level in plasma, we measured plasma Y4RNAfr levels of 40 healthy persons using the method we have developed, and compared them with other blood test data. From the obtained data, we tentatively regarded <0.1 fmol/ng as normal for the Y4RNAfr level in peripheral blood plasma. And the white blood cell count (WBC) and the C-reactive protein (CRP) level showed moderate positive correlations with the Y4RNAfr level, suggesting that Y4RNAfr could be a potential novel inflammatory marker. We also measured the Y4RNAfr level in peripheral blood plasma from four multiple myeloma patients. The plasma Y4RNAfr level was abnormal in all four myeloma patients, and the levels for two patients were far beyond the normal level. The WBC for each patient was normal and the CRP levels for two patients were normal. These observations together suggest that a high level of Y4RNAfr in peripheral blood plasma and a normal WBC could be indicative of multiple myeloma.

10.
Emerg Microbes Infect ; 8(1): 1688-1700, 2019.
Article in English | MEDLINE | ID: mdl-31749408

ABSTRACT

Resistance to ß-lactams is one of the most serious problems associated with Gram-negative infections. ß-Lactamases are able to hydrolyze ß-lactams such as cephalosporins and/or carbapenems. Evolutionary origin of metallo-ß-lactamases (MBLs), conferring critical antibiotic resistance threats, remains unknown. We discovered PNGM-1, the novel subclass B3 MBL, in deep-sea sediments that predate the antibiotic era. Here, our phylogenetic analysis suggests that PNGM-1 yields insights into the evolutionary origin of subclass B3 MBLs. We reveal the structural similarities between tRNase Zs and PNGM-1, and demonstrate that PNGM-1 has both MBL and tRNase Z activities, suggesting that PNGM-1 is thought to have evolved from a tRNase Z. We also show kinetic and structural comparisons between PNGM-1 and other proteins including subclass B3 MBLs and tRNase Zs. These comparisons revealed that the B3 MBL activity of PNGM-1 is a promiscuous activity and subclass B3 MBLs are thought to have evolved through PNGM-1 activity.


Subject(s)
Bacteria/enzymology , Bacterial Proteins/genetics , Evolution, Molecular , Geologic Sediments/microbiology , beta-Lactamases/genetics , Amino Acid Sequence , Bacteria/chemistry , Bacteria/classification , Bacteria/genetics , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Catalytic Domain , Phylogeny , beta-Lactamases/chemistry , beta-Lactamases/metabolism
11.
Blood Cells Mol Dis ; 79: 102341, 2019 11.
Article in English | MEDLINE | ID: mdl-31226499

ABSTRACT

Emergence of drug-resistant mutations in the course of myeloma cell evolution and subsequent relapse of myeloma appears to be currently inevitable in most patients. To remedy this situation, we are trying to develop therapeutic small guide RNAs (sgRNAs) based on tRNase ZL-utilizing efficacious gene silencing (TRUE gene silencing), an RNA-mediated gene expression control technology. We designed two sets of double heptamer-type sgRNA, which target the human BCL2 mRNA. Both sets of double heptamer-type sgRNA reduced viability of human myeloma cell lines, RPMI-8226 and KMM-1. We also performed a mouse xenograft experiment to examine how the double heptamer-type sgRNA DHa1(BCL2)/DHa2(BCL2) can reduce the growth of KMM-1 cells in vivo. Median survival periods of the sgRNA cohorts were greater than that of the control cohort by 11-43 days. Furthermore, we designed two sets of double heptamer-type sgRNA, which target the human CCND1 mRNA, and both sets synergistically reduced RPMI-8226 cell viability.


Subject(s)
Drug Design , Multiple Myeloma/drug therapy , RNA, Guide, Kinetoplastida/therapeutic use , Animals , Cell Line, Tumor , Cell Survival/drug effects , Cyclin D1/antagonists & inhibitors , Cyclin D1/genetics , Gene Silencing , Heterografts/drug effects , Humans , Mice , Multiple Myeloma/pathology , Proto-Oncogene Proteins c-bcl-2/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/genetics , RNA, Guide, Kinetoplastida/chemistry , RNA, Messenger , Survival Analysis
12.
Noncoding RNA Res ; 4(4): 135-140, 2019 Dec.
Article in English | MEDLINE | ID: mdl-32072081

ABSTRACT

The 31- and 32-nt 5'-fragments of Y4-RNA (Y4RNAfr) exist abundantly in human plasma. The Y4RNAfr can function as 5'-half-tRNA-type sgRNA for tRNase ZL, although we do not know yet what its physiological roles are and what cellular RNAs are its genuine targets. In this paper, we analyzed the effects of the Y4RNAfr on cell viability and transcriptomes using HL60, RPMI-8226, and HEK293 cells, and Y4RNAfr-binding RNAs in A549 cells. Although the Y4RNAfr hardly affected the viability of HL60, RPMI-8226, and HEK293 cells, it significantly affected their transcriptome. The DAVID analysis for > 2-fold upregulated and downregulated genes suggested that the Y4RNAfr may affect various KEGG pathways. We obtained 108 Y4RNAfr-binding RNAs in A549 cells, searched potential secondary structures of complexes between theY4RNAfr and its binding RNAs for the pre-tRNA-like structure, and found many such structures. One of the five best fitted structures was for the MKI67 mRNA, suggesting that the Y4RNAfr can decrease the cellular MKI67 level through guiding the cleavage of the MKI67 mRNA by tRNase ZL. This may be one of the underlying mechanisms for the reported observation that the Y4RNAfr suppresses the proliferation of A549 cells.

13.
In Vivo ; 31(4): 579-583, 2017.
Article in English | MEDLINE | ID: mdl-28652423

ABSTRACT

BACKGROUND/AIM: Clinically used disinfectants are often irritating and cause skin problems. Ozone water is unique among disinfectants. It does not damage skin cells and readily decomposes to oxygen without generating harmful residues. On the other hand, it rapidly loses its sanitizing activity. Recently developed nano-bubble ozone water (NBOW) can keep its sanitizing activity much longer. This study aimed to examine the microbicidal effects of NBOW after long-term storage. MATERIALS AND METHODS: The concentration of ozone in NBOW was examined by measuring the NBOW redox potential. Microbicidal activity was evaluated by colony formation assays, after incubating bacteria with NBOW for set time periods. RESULTS: NBOW lost its microbicidal activity after 1 year of storage at 4°C. Stocked frozen, NBOW retained appreciable microbicidal activity after 1 year of storage. Mycobacterium smegmatis, one of the most disinfectant-resistant bacteria, was killed within 15 min. NBOW was resistant to freeze-thawing. CONCLUSION: NBOW that had been stored frozen possessed sufficient microbicidal activity to kill bacteria even after 1 year of storage. Moreover, it was shown that NBOW is freeze-thaw resistant. NBOW possesses desirable features rendering it an attractive alternative disinfectant.


Subject(s)
Disinfectants/pharmacology , Nanotechnology , Ozone/pharmacology , Staphylococcus aureus/drug effects , Disinfectants/chemistry , Humans , Microbial Sensitivity Tests , Ozone/chemistry , Staphylococcus aureus/pathogenicity , Water/chemistry
14.
Noncoding RNA Res ; 2(2): 122-128, 2017 Jun.
Article in English | MEDLINE | ID: mdl-30159430

ABSTRACT

The 94-nt full-length Y4-RNA is thought to have roles in the initiation of DNA replication and RNA quality control. Although its 31/32-nt fragment also exists abundantly in plasma, little is known about its physiological role. Since the 31/32-nt Y4-RNA fragment in sera is reported to be more abundant in patients with coronary artery disease than healthy persons, the fragment may have a potential for a diagnostic and/or prognostic biomarker for some diseases regardless of its functionality. As a step toward further investigation of its potential utility, we examined if the 31/32-nt Y4-RNA fragment also exists in saliva that can be obtained noninvasively, and showed that, in addition to the 31/32-nt fragment, 14- and 11-nt Y4-RNA fragments are present in all saliva RNA samples from four healthy persons. We established a PCR method to accurately quantitate the amount of the 31/32-nt Y4-RNA fragment, and estimated its amount in saliva of healthy persons to be 0.06 ± 0.04 fmol per nanogram of saliva RNA. We also tried to develop an easier quantitation method using a DNA molecular beacon.

15.
J Vis Exp ; (112)2016 06 02.
Article in English | MEDLINE | ID: mdl-27285342

ABSTRACT

TRUE gene silencing (termed after tRNase Z(L)-utilizing efficacious gene silencing) is one of the RNA-directed gene silencing technologies, which utilizes an artificial small guide RNA (sgRNA) to guide tRNA 3' processing endoribonuclease, tRNase Z(L), to recognize a target RNA. sgRNAs can be taken up by cells without any transfection reagents and can downregulate their target RNA levels and/or induce apoptosis in human cancer cells. We have screened an sgRNA library containing 156 heptamer-type sgRNAs for the effect on viability of human myeloma and leukemia cells, and found that 20 of them can efficiently induce apoptosis in at least one of the cancer cell lines. Here we present a protocol for screening of a heptamer-type sgRNA library for potential therapeutic drugs against blood cancers. The protocol includes how to construct the sgRNA library, how to assess the effect of each sgRNA on cell viability, and how to further evaluate the effective sgRNAs by flow cytometry. Around 2,000 hits would be expected to be obtained by screening the full-scale sgRNA library composed of 16,384 heptamers.


Subject(s)
Gene Silencing , Apoptosis , Gene Library , Humans , Neoplasms , RNA, Guide, Kinetoplastida , Transfection
16.
J Biol Chem ; 288(23): 16680-16689, 2013 Jun 07.
Article in English | MEDLINE | ID: mdl-23585563

ABSTRACT

The emerging link between iron metabolism and genome integrity is increasingly clear. Recent studies have revealed that MMS19 and cytosolic iron-sulfur cluster assembly (CIA) factors form a complex and have central roles in CIA pathway. However, the composition of the CIA complex, particularly the involvement of the Fe-S protein IOP1, is still unclear. The roles of each component are also largely unknown. Here, we show that MMS19, MIP18, and CIAO1 form a tight "core" complex and that IOP1 is an "external" component of this complex. Although IOP1 and the core complex form a complex both in vivo and in vitro, IOP1 behaves differently in vivo. A deficiency in any core component leads to down-regulation of all of the components. In contrast, IOP1 knockdown does not affect the level of any core component. In MMS19-overproducing cells, other core components are also up-regulated, but the protein level of IOP1 remains unchanged. IOP1 behaves like a target protein in the CIA reaction, like other Fe-S helicases, and the core complex may participate in the maturation process of IOP1. Alternatively, the core complex may catch and hold IOP1 when it becomes mature to prevent its degradation. In any case, IOP1 functions in the MMS19-dependent CIA pathway. We also reveal that MMS19 interacts with target proteins. MIP18 has a role to bridge MMS19 and CIAO1. CIAO1 also binds IOP1. Based on our in vivo and in vitro data, new models of the CIA machinery are proposed.


Subject(s)
Hydrogenase/metabolism , Iron-Sulfur Proteins/metabolism , Models, Biological , Multiprotein Complexes/metabolism , Transcription Factors/metabolism , Carrier Proteins/genetics , Carrier Proteins/metabolism , HEK293 Cells , HeLa Cells , Humans , Hydrogenase/genetics , Iron-Sulfur Proteins/genetics , Metallochaperones/genetics , Metallochaperones/metabolism , Metalloproteins , Multiprotein Complexes/genetics , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Transcription Factors/genetics
17.
J Mol Biol ; 405(3): 642-52, 2011 Jan 21.
Article in English | MEDLINE | ID: mdl-21050863

ABSTRACT

DNA polymerase θ (POLQ, polθ) is a large, multidomain DNA polymerase encoded in higher eukaryotic genomes. It is important for maintaining genetic stability in cells and helping protect cells from DNA damage caused by ionizing radiation. POLQ contains an N-terminal helicase-like domain, a large central domain of indeterminate function, and a C-terminal polymerase domain with sequence similarity to the A-family of DNA polymerases. The enzyme has several unique properties, including low fidelity and the ability to insert and extend past abasic sites and thymine glycol lesions. It is not known whether the abasic site bypass activity is an intrinsic property of the polymerase domain or whether helicase activity is also required. Three "insertion" sequence elements present in POLQ are not found in any other A-family DNA polymerase, and it has been proposed that they may lend some unique properties to POLQ. Here, we analyzed the activity of the DNA polymerase in the absence of each sequence insertion. We found that the pol domain is capable of highly efficient bypass of abasic sites in the absence of the helicase-like or central domains. Insertion 1 increases the processivity of the polymerase but has little, if any, bearing on the translesion synthesis properties of the enzyme. However, removal of insertions 2 and 3 reduces activity on undamaged DNA and completely abrogates the ability of the enzyme to bypass abasic sites or thymine glycol lesions.


Subject(s)
DNA-Directed DNA Polymerase/chemistry , Amino Acid Sequence , Animals , Base Sequence , DNA Damage , DNA Repair , DNA Replication , DNA-Directed DNA Polymerase/genetics , Genes, pol , Humans , Mice , Molecular Sequence Data , Protein Structure, Tertiary , Thymine/analogs & derivatives , Thymine/chemistry , DNA Polymerase theta
18.
Mol Cell ; 39(4): 632-40, 2010 Aug 27.
Article in English | MEDLINE | ID: mdl-20797633

ABSTRACT

Xeroderma pigmentosum group D (XPD) protein is one of the subunits of TFIIH that is required for nucleotide excision repair and transcription. We found a XPD protein complex containing MMS19 that was assumed to be a regulator of TFIIH. However, the MMS19-XPD complex did not contain any other subunits of TFIIH. Instead, it included FAM96B (now designated MIP18), Ciao1, and ANT2. MMS19, MIP18, and XPD localized to the mitotic spindle during mitosis. The siRNA-mediated knockdown of MMS19, MIP18, or XPD led to improper chromosome segregation and the accumulation of nuclei with abnormal shapes. In addition, the frequency of abnormal mitosis and nuclei was increased in XP-D and XP-D/CS patients' cells. These results indicate that the MMS19-XPD protein complex, now designated MMXD (MMS19-MIP18-XPD), is required for proper chromosome segregation, an abnormality of which could contribute to the pathogenesis in some cases of XP-D and XP-D/CS.


Subject(s)
Carrier Proteins/metabolism , Chromosome Segregation , Nuclear Proteins/metabolism , Transcription Factor TFIIH/metabolism , Transcription Factors/metabolism , Xeroderma Pigmentosum Group D Protein/metabolism , Xeroderma Pigmentosum/genetics , Adenine Nucleotide Translocator 2/metabolism , Binding Sites , Carrier Proteins/genetics , Cell Nucleus Shape , Gene Knockdown Techniques , HCT116 Cells , HeLa Cells , Humans , Metallochaperones/metabolism , Metalloproteins , Microscopy, Fluorescence , Mitosis , Multiprotein Complexes , Nuclear Proteins/genetics , Protein Interaction Domains and Motifs , Protein Interaction Mapping , RNA Interference , Spindle Apparatus/metabolism , Transcription Factors/genetics , Transfection , Xeroderma Pigmentosum/metabolism , Xeroderma Pigmentosum/pathology , Xeroderma Pigmentosum Group D Protein/genetics
19.
Proc Natl Acad Sci U S A ; 107(30): 13390-5, 2010 Jul 27.
Article in English | MEDLINE | ID: mdl-20624954

ABSTRACT

"Replicative stress" is one of the main factors underlying neoplasia from its early stages. Genes involved in DNA synthesis may therefore represent an underexplored source of potential prognostic markers for cancer. To this aim, we generated gene expression profiles from two independent cohorts (France, n=206; United Kingdom, n=117) of patients with previously untreated primary breast cancers. We report here that among the 13 human nuclear DNA polymerase genes, DNA Polymerase (POLQ) is the only one significantly up-regulated in breast cancer compared with normal breast tissues. Importantly, POLQ up-regulation significantly correlates with poor clinical outcome (4.3-fold increased risk of death in patients with high POLQ expression), and this correlation is independent of Cyclin E expression or the number of positive nodes, which are currently considered as markers for poor outcome. POLQ expression provides thus an additional indicator for the survival outcome of patients with high Cyclin E tumor expression or high number of positive lymph nodes. Furthermore, to decipher the molecular consequences of POLQ up-regulation in breast cancer, we generated human MRC5-SV cell lines that stably overexpress POLQ. Strong POLQ expression was directly associated with defective DNA replication fork progression and chromosomal damage. Therefore, POLQ overexpression may be a promising genetic instability and prognostic marker for breast cancer.


Subject(s)
Breast Neoplasms/genetics , DNA Replication/drug effects , DNA-Directed DNA Polymerase/genetics , Genomic Instability , Breast Neoplasms/pathology , Cell Line , Cell Line, Tumor , Cohort Studies , Cyclin E/genetics , DNA Damage , Female , France , Gene Expression Profiling , Gene Expression Regulation, Neoplastic , Humans , Kaplan-Meier Estimate , Middle Aged , Prognosis , RNA Interference , Reverse Transcriptase Polymerase Chain Reaction , United Kingdom , Up-Regulation , DNA Polymerase theta
20.
Nucleic Acids Res ; 38(10): 3233-44, 2010 Jun.
Article in English | MEDLINE | ID: mdl-20144948

ABSTRACT

POLN is a nuclear A-family DNA polymerase encoded in vertebrate genomes. POLN has unusual fidelity and DNA lesion bypass properties, including strong strand displacement activity, low fidelity favoring incorporation of T for template G and accurate translesion synthesis past a 5S-thymine glycol (5S-Tg). We searched for conserved features of the polymerase domain that distinguish it from prokaryotic pol I-type DNA polymerases. A Lys residue (679 in human POLN) of particular interest was identified in the conserved 'O-helix' of motif 4 in the fingers sub-domain. The corresponding residue is one of the most important for controlling fidelity of prokaryotic pol I and is a nonpolar Ala or Thr in those enzymes. Kinetic measurements show that K679A or K679T POLN mutant DNA polymerases have full activity on nondamaged templates, but poorly incorporate T opposite template G and do not bypass 5S-Tg efficiently. We also found that a conserved Tyr residue in the same motif not only affects sensitivity to dideoxynucleotides, but also greatly influences enzyme activity, fidelity and bypass. Protein sequence alignment reveals that POLN has three specific insertions in the DNA polymerase domain. The results demonstrate that residues have been strictly retained during evolution that confer unique bypass and fidelity properties on POLN.


Subject(s)
DNA-Directed DNA Polymerase/chemistry , DNA-Directed DNA Polymerase/metabolism , Amino Acid Motifs , DNA-Directed DNA Polymerase/genetics , Evolution, Molecular , Humans , Lysine/analysis , Mutation , Protein Structure, Tertiary , Tyrosine/analysis
SELECTION OF CITATIONS
SEARCH DETAIL
...