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1.
Biol Pharm Bull ; 45(2): 200-206, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35110507

RESUMO

Werner helicase-interacting protein 1 (WRNIP1) belongs to the AAA+ ATPase family and is conserved from Escherichia coli to human. In addition to an ATPase domain in the middle region of WRNIP1, WRNIP1 contains a ubiquitin-binding zinc-finger (UBZ) domain and two leucine zipper motifs in the N-terminal and C-terminal regions, respectively. Here, we report that the UBZ domain of WRNIP1 is responsible for the reduced levels of UV-induced proliferating cell nuclear antigen (PCNA) monoubiquitylation in POLH-disrupted (polymerase η (Polη)-deficient) cells, and that the ATPase domain of WRNIP1 is involved in regulating the level of the PrimPol protein. The suppression of UV sensitivity of Polη-deficient cells by deletion of WRNIP1 was abolished by expression of the mutant WRNIP1 lacking the UBZ domain or ATPase domain, but not by the mutant lacking the leucine zipper domain in WRNIP1/POLH double-disrupted cells. The leucine zipper domain of WRNIP1 was required for its interaction with RAD18, a key factor in TLS (DNA translesion synthesis), and DNA polymerase δ catalytic subunit, POLD1. On the basis of these findings, we discuss the possible role of WRNIP1 in TLS.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica/fisiologia , Regulação da Expressão Gênica/efeitos da radiação , ATPases Associadas a Diversas Atividades Celulares/genética , Proteínas de Ligação a DNA/genética , Deleção de Genes , Células HEK293 , Humanos , Domínios Proteicos , Raios Ultravioleta
2.
Biol Pharm Bull ; 42(5): 764-769, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31061318

RESUMO

Werner helicase-interacting protein 1 (WRNIP1) was originally identified as a protein that interacts with WRN, the product of the gene responsible for Werner syndrome. Our previous studies suggested that WRNIP1 is implicated in translesion synthesis (TLS), a process in which specialized TLS polymerases replace replicative DNA polymerase and take over DNA synthesis on damaged templates. We proposed that a novel error-free pathway involving DNA polymerase δ and primase-polymerase (PrimPol) functions to synthesize DNA on UV-damaged DNA templates in the absence of WRNIP1 and the TLS polymerase Polη. Hence, in the current study, we analyzed the relationship between WRNIP1 and PrimPol. We found that WRNIP1 and PrimPol form a complex in cells. PrimPol protein expression was reduced in cells overexpressing WRNIP1, but was increased in WRNIP1-depleted cells. The WRNIP1-mediated reduction in the amount of PrimPol was suppressed by treatment of the cells with proteasome inhibitors, suggesting that WRNIP1 is involved in the degradation of PrimPol via the proteasome.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , DNA Primase/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Enzimas Multifuncionais/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , DNA Primase/genética , DNA Complementar/genética , Proteínas de Ligação a DNA/genética , DNA Polimerase Dirigida por DNA/genética , Células HEK293 , Humanos , Enzimas Multifuncionais/genética , Plasmídeos , Inibidores de Proteassoma/farmacologia , RNA Mensageiro/metabolismo , Transfecção
3.
Biochem Biophys Res Commun ; 452(1): 48-52, 2014 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-25139235

RESUMO

WRNIP1 (WRN-interacting protein 1) was first identified as a factor that interacts with WRN, the protein that is defective in Werner syndrome (WS). WRNIP1 associates with DNA polymerase η (Polη), but the biological significance of this interaction remains unknown. In this study, we analyzed the functional interaction between WRNIP1 and Polη by generating knockouts of both genes in DT40 chicken cells. Disruption of WRNIP1 in Polη-disrupted (POLH(-/-)) cells suppressed the phenotypes associated with the loss of Polη: sensitivity to ultraviolet light (UV), delayed repair of cyclobutane pyrimidine dimers (CPD), elevated frequency of mutation, elevated levels of UV-induced sister chromatid exchange (SCE), and reduced rate of fork progression after UV irradiation. These results suggest that WRNIP1 functions upstream of Polη in the response to UV irradiation.


Assuntos
Proteínas de Transporte/fisiologia , Dano ao DNA , Proteínas de Ligação a DNA/fisiologia , DNA Polimerase Dirigida por DNA/metabolismo , DNA/efeitos da radiação , Raios Ultravioleta , ATPases Associadas a Diversas Atividades Celulares , Linhagem Celular , Ensaio de Imunoadsorção Enzimática , Humanos , Troca de Cromátide Irmã
4.
J Biol Chem ; 286(35): 30504-30512, 2011 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-21757688

RESUMO

Ordered nucleosome disassembly and reassembly are required for eukaryotic DNA replication. The facilitates chromatin transcription (FACT) complex, a histone chaperone comprising Spt16 and SSRP1, is involved in DNA replication as well as transcription. FACT associates with the MCM helicase, which is involved in DNA replication initiation and elongation. Although the FACT-MCM complex is reported to regulate DNA replication initiation, its functional role in DNA replication elongation remains elusive. To elucidate the functional role of FACT in replication fork progression during DNA elongation in the cells, we generated and analyzed conditional SSRP1 gene knock-out chicken (Gallus gallus) DT40 cells. SSRP1-depleted cells ceased to grow and exhibited a delay in S-phase cell cycle progression, although SSRP1 depletion did not affect the level of chromatin-bound DNA polymerase α or nucleosome reassembly on daughter strands. The tracking length of newly synthesized DNA, but not origin firing, was reduced in SSRP1-depleted cells, suggesting that the S-phase cell cycle delay is mainly due to the inhibition of replication fork progression rather than to defects in the initiation of DNA replication in these cells. We discuss the mechanisms of how FACT promotes replication fork progression in the cells.


Assuntos
Cromatina/química , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/metabolismo , Chaperonas de Histonas/química , Transcrição Gênica , Fatores de Elongação da Transcrição/metabolismo , Animais , Ciclo Celular , Galinhas , Epigênese Genética , Citometria de Fluxo/métodos , Histonas/química , Humanos , Chaperonas Moleculares/metabolismo , Nucleossomos/metabolismo , Fase S
5.
Biochim Biophys Acta ; 1813(3): 473-9, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21256165

RESUMO

Rothmund-Thomson syndrome (RTS) is a rare genetic disorder characterized by premature aging, developmental abnormalities, and a predisposition to cancer. RTS is caused by mutations in the RECQL4 gene, which encodes one of the five human RecQ helicases. To identify the cellular functions of RECQL4, we generated a chicken DT40 cell line in which RECQL4 expression could be turned off by doxycycline (Dox). Upon exposure to Dox, cells stopped growing and underwent apoptosis. The cells could be rescued by expression of the N-terminal region of RECQL4 (amino acids 1-496), which lacks the helicase domain and has sequence similarity to yeast Sld2, which plays an essential function in the initiation of DNA replication in Saccharomyces cerevisiae. Smaller fragments of the N-terminal region of RECQL4 did not rescue the cells from lethality. RECQL4 gene knockout cells complemented with RECQL4 (1-496) showed relatively high sensitivity to DNA damaging agents that induce double strand breaks and cross-links, suggesting that the C-terminal region including the helicase domain of RECQL4 is involved in the repair of certain types of DNA lesions.


Assuntos
Sobrevivência Celular , RecQ Helicases/genética , RecQ Helicases/metabolismo , Síndrome de Rothmund-Thomson/genética , Animais , Antibacterianos/farmacologia , Morte Celular , Linhagem Celular , Galinhas , DNA Helicases/metabolismo , Reparo do DNA , Doxiciclina/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Inativação de Genes , Humanos , Estrutura Terciária de Proteína , RecQ Helicases/química , Síndrome de Rothmund-Thomson/metabolismo
6.
Biochem Biophys Res Commun ; 417(4): 1145-50, 2012 Jan 27.
Artigo em Inglês | MEDLINE | ID: mdl-22209848

RESUMO

WRNIP1 (Werner helicase-interacting protein 1) was originally identified as a protein that interacts with the Werner syndrome responsible gene product. WRNIP1 contains a ubiquitin-binding zinc-finger (UBZ) domain in the N-terminal region and two leucine zipper motifs in the C-terminal region. In addition, it possesses an ATPase domain in the middle of the molecule and the lysine residues serving as ubiquitin acceptors in the entire of the molecule. Here, we report that WRNIP1 accumulates in laser light irradiated sites very rapidly via its ubiquitin-binding zinc finger domain, which is known to bind polyubiquitin and to be involved in ubiquitination of WRNIP1 itself. The accumulation of WRNIP1 in laser light irradiated sites also required the C-terminal region containing two leucine zippers, which is reportedly involved in the oligomerization of WRNIP1. Mutated WRNIP1 with a deleted ATPase domain or with mutations in lysine residues, which serve as ubiquitin acceptors, accumulated in laser light irradiated sites, suggesting that the ATPase domain of WRNIP1 and ubiquitination of WRNIP1 are dispensable for the accumulation.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Ubiquitina/metabolismo , Dedos de Zinco , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/genética , Proteínas de Transporte/genética , Proteínas de Ligação a DNA/genética , Células HeLa , Humanos , Lasers , Zíper de Leucina , Luz , Lisina/genética , Lisina/metabolismo , Mutação , Estrutura Terciária de Proteína , Ubiquitinação
7.
J Cell Biol ; 179(1): 53-63, 2007 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-17923529

RESUMO

Bloom's syndrome (BS), which is caused by mutations in the BLM gene, is characterized by a predisposition to a wide variety of cancers. BS cells exhibit elevated frequencies of sister chromatid exchanges (SCEs), interchanges between homologous chromosomes (mitotic chiasmata), and sensitivity to several DNA-damaging agents. To address the mechanism that confers these phenotypes in BS cells, we characterize a series of double and triple mutants with mutations in BLM and in other genes involved in repair pathways. We found that XRCC3 activity generates substrates that cause the elevated SCE in blm cells and that BLM with DNA topoisomerase IIIalpha suppresses the formation of SCE. In addition, XRCC3 activity also generates the ultraviolet (UV)- and methyl methanesulfonate (MMS)-induced mitotic chiasmata. Moreover, disruption of XRCC3 suppresses MMS and UV sensitivity and the MMS- and UV-induced chromosomal aberrations of blm cells, indicating that BLM acts downstream of XRCC3.


Assuntos
Adenosina Trifosfatases/metabolismo , Síndrome de Bloom/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/fisiologia , Animais , Síndrome de Bloom/genética , Linhagem Celular , Galinhas , Aberrações Cromossômicas , DNA Helicases/genética , DNA Helicases/fisiologia , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/fisiologia , Predisposição Genética para Doença , Humanos , Mutação , RecQ Helicases , Recombinação Genética , Troca de Cromátide Irmã , Raios Ultravioleta
8.
Biochem Biophys Res Commun ; 414(2): 298-303, 2011 Oct 22.
Artigo em Inglês | MEDLINE | ID: mdl-21945441

RESUMO

Claspin was originally identified as a Check1 (Chk1)-interacting protein. Claspin and Rad17 are reportedly involved in the DNA damage-induced phosphorylation of Chk1, a hallmark of checkpoint activation. To understand the cellular functions of Claspin and the functional relationship between Claspin and Rad17, we generated Claspin(-/-) and Claspin(-/-)/RAD17(-) cells using chicken DT40 cells, which contain an exogenously introduced Claspin that can be suppressed by the addition of doxycycline (Dox). In the presence of Dox, Claspin(-/-) cells ceased growth within 2 days, leading to cell death. In addition, a remarkable reduction in the rate of DNA elongation was observed in Claspin-depleted cells, suggesting that Claspin plays a critical role in DNA replication in the absence of exogenous stress. When cells were exposed to methyl methanesulfonate (MMS), a DNA damaging agent, RAD17(-) cells showed a greater defect in checkpoint activation than Claspin(-/-) cells as monitored by progression of cell cycle and phosphorylation of Chk1. Knocking out RAD17 gene showed almost no additive effects on cell death and DNA elongation rates in Claspin-depleted cells.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Ciclo Celular/genética , Linhagem Celular , Galinhas , Replicação do DNA/genética , Técnicas de Inativação de Genes , Humanos
9.
Biol Pharm Bull ; 34(8): 1314-8, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21804224

RESUMO

Werner interacting protein 1 (WRNIP1) that is highly conserved from Escherichia coli to human was originally identified as a protein that interacts with the Werner syndrome responsible gene product (WRN). Here, human WRNIP1 and WRN are shown to bind to template-primer DNA, and WRNIP1, but not WRN, requires ATP for DNA binding. Under conditions of a limiting amount of WRN, WRNIP1 facilitated binding of WRN to DNA in a dose-dependent manner. However, WRNIP1 did not stimulate the DNA helicase activity of WRN, and WRN displaced pre-bound WRNIP1 from DNA. Functional relationships between WRNIP1 and WRN will be discussed.


Assuntos
Proteínas de Transporte/metabolismo , Primers do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Exodesoxirribonucleases/metabolismo , RecQ Helicases/metabolismo , Síndrome de Werner/genética , ATPases Associadas a Diversas Atividades Celulares , Trifosfato de Adenosina/metabolismo , Humanos , Ligação Proteica , Síndrome de Werner/metabolismo , Helicase da Síndrome de Werner
10.
Genes Environ ; 43(1): 35, 2021 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-34353368

RESUMO

BACKGROUND: Peroxiredoxin 1 (PRDX1) is a member of a ubiquitous family of thiol peroxidases that catalyze the reduction of peroxides, including hydrogen peroxide. It functions as an antioxidant enzyme, similar to catalase and glutathione peroxidase. PRDX1 was recently shown act as a sensor of reactive oxygen species (ROS) and play a role in ROS-dependent intracellular signaling pathways. To investigate its physiological functions, PRDX1 was conditionally disrupted in chicken DT40 cells in the present study. RESULTS: The depletion of PRDX1 resulted in cell death with increased levels of intracellular ROS. PRDX1-depleted cells did not show the accumulation of chromosomal breaks or sister chromatid exchange (SCE). These results suggest that cell death in PRDX1-depleted cells was not due to DNA damage. 2-Mercaptoethanol protected against cell death in PRDX1-depleted cells and also suppressed elevations in ROS. CONCLUSIONS: PRDX1 is essential in chicken DT40 cells and plays an important role in maintaining intracellular ROS homeostasis (or in the fine-tuning of cellular ROS levels). Cells deficient in PRDX1 may be used as an endogenously deregulated ROS model to elucidate the physiological roles of ROS in maintaining proper cell growth.

11.
Biochim Biophys Acta ; 1782(2): 75-81, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18078829

RESUMO

RECQL1 and RECQL5 as well as BLM reportedly interact with TOP3alpha whose defect is lethal for the cell. Therefore in this study, we characterized recql5/recql1/blm triple mutants from DT40 cells to determine whether the triple mutants show a top3alpha disrupted cell-like phenotype. The triple mutants are viable. Moreover, both blm/recql1 and recql5/blm cells, and recql5/recql1/blm cells grew slightly slower than blm cells, that is, triple mutant cells grew almost the same rate as either of the double mutant cells. The blm cells showed sensitivity to methyl methanesulfonate (MMS) and ultraviolet light (UV), about a 10-fold increase in sister chromatid exchange (SCE), and about a 3-fold increase in damage-induced mitotic chiasma compared to wild-type cells. The triple mutants showed the same sensitivity to MMS or UV and the same frequency of damage-induced mitotic chiasma compared to those of blm cells, indicating that unlike BLM, RECQL1 and RECQL5 play a little role in the repair of or tolerance to DNA damages. However, recql5/blm cells showed higher frequency of SCE than blm cells, whereas the RECQL1 gene disruption had no effect on SCE in blm cells and even in recql5/blm cells.


Assuntos
Adenosina Trifosfatases/genética , Adenosina Trifosfatases/fisiologia , DNA Helicases/genética , DNA Helicases/fisiologia , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases Tipo I/fisiologia , RecQ Helicases/genética , RecQ Helicases/fisiologia , Adenosina Trifosfatases/metabolismo , Animais , Células Cultivadas , Galinhas , Dano ao DNA/fisiologia , DNA Helicases/metabolismo , Metanossulfonato de Metila/farmacologia , Modelos Biológicos , Proteínas Mutantes/metabolismo , Proteínas Mutantes/fisiologia , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/genética , Domínios e Motivos de Interação entre Proteínas/fisiologia , RecQ Helicases/metabolismo , Troca de Cromátide Irmã/efeitos da radiação , Raios Ultravioleta
12.
Biochem Biophys Res Commun ; 379(2): 233-8, 2009 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-19100713

RESUMO

Manganese-dependent superoxide dismutase (SOD2) serves as the primary defense against mitochondrial superoxide, and decreased SOD2 activity results in a range of pathologies. To investigate the events occurring soon after depletion of SOD2, we generated SOD2 gene knockout chicken DT40 cells complemented with a human SOD2 (hSOD2) cDNA, whose expression can be switched off by doxycycline (Dox). When SOD2 was depleted by the addition of Dox, the cells grew slightly slower and formed fewer colonies than cells expressing hSOD2. In addition, these cells showed a high sensitivity to paraquat, which produces superoxide, and died through apoptosis. In contrast to results obtained with mouse and DrosophilaSod2 mutants, we found no indication of an increase in DNA lesions due to depletion of SOD2.


Assuntos
Mitocôndrias/enzimologia , Superóxido Dismutase/genética , Animais , Apoptose/genética , Linhagem Celular , Sobrevivência Celular/genética , Galinhas , Dano ao DNA/genética , Técnicas de Inativação de Genes , Teste de Complementação Genética , Humanos , Camundongos , Mutação , Paraquat/farmacologia , Superóxidos/metabolismo
13.
Cell Signal ; 20(11): 1978-85, 2008 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-18674614

RESUMO

KU70(-/-) and DNA-PKcs(-/-/-)chicken DT40 cells are reportedly highly sensitive to the DNA topoisomerase II inhibitor etoposide. Here we report that KU70 and DNA-PKcs unexpectedly function together during the induction of apoptosis after exposure to high levels of etoposide. In the presence of 100 microM etoposide, apoptosis was induced within 1 h in wild type DT40 cells but not in KU70(-/-) and DNA-PKcs(-/-/-) cells. In addition, the DNA-PK inhibitors NU7026 and wortmannin, as well as the caspase inhibitor Z-VAD-FMK, inhibited etoposide-induced apoptosis in wild type cells. Although Artemis(-/-) cells also showed defects in the etoposide-induced apoptosis, the other mutants defective in nonhomologous end-joining (NHEJ), LIG4(-/-), XRCC4(-), and XLF(-/-) cells were capable to induce apoptosis. When cells were treated with high doses of etoposide, the chromatin binding of DNA-PKcs was impaired by deletion of KU70 but not of Artemis, suggesting that KU70 acts upstream of DNA-PKcs and Artemis acts downstream of DNA-PKcs in the apoptotic pathway like the NHEJ pathway. These results suggest that the proteins involved in the early stage of NHEJ pathway including Artemis but not the downstream factors decide the cell fate by selecting apoptosis or DNA repair according to the degree of DNA damage.


Assuntos
Antígenos Nucleares/metabolismo , Apoptose , Quebras de DNA de Cadeia Dupla , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Galinhas , Cromatina/enzimologia , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Proteína Quinase Ativada por DNA/deficiência , Proteínas de Ligação a DNA/deficiência , Etoposídeo/farmacologia , Autoantígeno Ku , Mutação/genética , Inibidores de Proteínas Quinases/farmacologia , Recombinação Genética/efeitos dos fármacos
14.
Mol Cell Biol ; 26(16): 6299-307, 2006 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-16880537

RESUMO

Bloom's syndrome (BS) is an autosomal disorder characterized by predisposition to a wide variety of cancers. The gene product whose mutation leads to BS is the RecQ family helicase BLM, which forms a complex with DNA topoisomerase IIIalpha (Top3alpha). However, the physiological relevance of the interaction between BLM and Top3alpha within the cell remains unclear. We show here that Top3alpha depletion causes accumulation of cells in G2 phase, enlargement of nuclei, and chromosome gaps and breaks that occur at the same position in sister chromatids. The transition from metaphase to anaphase is also inhibited. All of these phenomena except cell lethality are suppressed by BLM gene disruption. Taken together with the biochemical properties of BLM and Top3alpha, these data indicate that BLM and Top3alpha execute the dissolution of sister chromatids.


Assuntos
Adenosina Trifosfatases/metabolismo , Cromátides/enzimologia , Cromátides/genética , DNA Helicases/metabolismo , DNA Topoisomerases Tipo I/metabolismo , 2-Aminopurina/farmacologia , Anáfase/efeitos dos fármacos , Animais , Apoptose , Galinhas , Cromátides/efeitos dos fármacos , Aberrações Cromossômicas , DNA Topoisomerases Tipo I/deficiência , Fase G2/efeitos dos fármacos , Marcação de Genes , Humanos , Isoenzimas/metabolismo , Metáfase/efeitos dos fármacos , Camundongos , Modelos Genéticos , Mutação/genética , Fenótipo , RecQ Helicases
15.
Genes Genet Syst ; 84(2): 171-8, 2009 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19556710

RESUMO

WRN interacting protein 1 (WRNIP1) was originally identified as a protein that interacts with the Werner syndrome responsible gene product (WRN). WRNIP1 is a highly conserved protein from E. coli to humans. Genetic studies in budding yeast suggested that the yeast orthlog of WRNIP1, Mgs1, may function in a DNA damage tolerance pathway that is similar to, but distinct from, the template-switch damage avoidance pathway involving Rad6, Rad18, Rad5, Mms2, and Ubc13. Here we report that human WRNIP1 binds in an ATP dependent manner to both forked DNA that mimics stalled replication forks and to template/primer DNA. We found that WRNIP1 interacts physically with RAD18 and interferes with the binding of RAD18 to forked DNA and to template/primer DNA. In contrast, RAD18 enhances the binding of WRNIP1 to these DNAs, suggesting that WRNIP1 targets DNA bound by RAD18.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mapeamento de Interação de Proteínas/métodos , ATPases Associadas a Diversas Atividades Celulares , Trifosfato de Adenosina/metabolismo , Animais , Proteínas de Transporte/genética , Linhagem Celular , Proteínas de Ligação a DNA/genética , Humanos , Imunoprecipitação , Conformação de Ácido Nucleico , Oligonucleotídeos/química , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Ligação Proteica , Spodoptera , Transfecção , Ubiquitina-Proteína Ligases
16.
Genes Genet Syst ; 83(1): 95-100, 2008 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-18379138

RESUMO

Bloom syndrome (BS) is rare autosomal recessive disorder associated with chromosomal instability. The gene responsible for BS, BLM, encodes a protein belonging to the RecQ helicase family. Disruptions of the SGS1 gene of Saccharomyces cerevisiae, which encodes the RecQ helicase homologue in the budding yeast, causes accelerated aging, and this phenotype is enhanced by the disruption of MGS1, the budding yeast homologue for WRNIP1. To examine the functional relationship between RecQ and WRNIP1 in vertebrate cells, we generated and characterized wrnip1/blm cells derived from the chicken B-lymphocyte line DT40. wrnip1/blm cells showed an additive elevation of sister chromatid exchange (SCE), suggesting that both genes independently contribute to the suppression of excess SCE formation. The double mutants were more sensitive to DNA damage from camptothecin (CPT), but not to damage from methyl methanesulfonate, than either single mutant. This result suggests that WRNIP1 and BLM function independently to repair DNA or induce tolerance to the lesions induced by CPT.


Assuntos
Adenosina Trifosfatases/fisiologia , DNA Helicases/fisiologia , Proteínas de Ligação a DNA/fisiologia , Instabilidade Genômica , Adenosina Trifosfatases/genética , Animais , Síndrome de Bloom/genética , Células Cultivadas , Galinhas , Dano ao DNA , DNA Helicases/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Humanos , Mutação , RecQ Helicases , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Troca de Cromátide Irmã
17.
Genes Genet Syst ; 83(5): 427-31, 2008 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-19168994

RESUMO

Replication checkpoint protein Rad17 senses DNA lesions during DNA replication and halts progression of replication fork. The cells derived from Bloom syndrome individuals show some defects in DNA replication. In order to investigate the functional relationship between the replication checkpoint protein Rad17 and BLM, which is the product of the causative gene of Bloom syndrome, we generated BLM/RAD17 double knockout (blm/rad17) cells using chicken DT40 cells. The blm/rad17 cells showed exaggerated growth defects as determined by analysis of their growth curves and plating efficiency compared to those of either of the single gene mutants. These defects seem to be due to an increase in DNA lesions that cause spontaneous cell death, suggesting that Rad17 and BLM execute different functions in the progression of replication forks. We also demonstrate that targeting integration was dramatically compromised by a lack of Rad17. In addition, the elevated frequency of sister chromatid exchange (SCE) due to homologous recombination in BLM knockout (blm) cells was greatly reduced by disruption of the RAD17 gene. Thus, in addition to its role in the replication checkpoint, Rad17 appears to play a role in homologous recombination.


Assuntos
Proteínas de Ciclo Celular/fisiologia , Recombinação Genética , Animais , Síndrome de Bloom/genética , Síndrome de Bloom/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , RecQ Helicases/genética , RecQ Helicases/metabolismo , Troca de Cromátide Irmã
18.
DNA Repair (Amst) ; 5(11): 1307-16, 2006 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-16931176

RESUMO

In Saccharomyces cerevisiae, Rad18 functions in post-replication repair pathways, such as error-free damage bypass involving Rad30 (Poleta) and error-prone damage bypass involving Rev3/7 (Polzeta). Chicken DT40 RAD18(-/-) cells were found to be hypersensitive to camptothecin (CPT), while RAD30(-/-) and REV3(-/-) cells, which are defective in translesion DNA synthesis, were not. RAD18(-/-) cells also showed higher levels of H2AX phosphorylation and chromosomal aberrations, particularly chromosomal gaps and breaks, upon exposure to CPT. Detailed analysis by alkaline sucrose density gradient centrifugation revealed that RAD18(-/-) and wild type cells exhibited similar rates of elongation of newly synthesized DNA in the presence or absence of low concentrations of CPT but that DNA breaks frequently occurred on both parental and nascent strands within 1h after a brief exposure to an elevated concentration of CPT, with more breaks induced in RAD18(-/-) cells than in wild type cells. These data suggest a previously unanticipated role for Rad18 in dealing with replication forks upon encountering DNA lesions induced by CPT.


Assuntos
Camptotecina/toxicidade , Dano ao DNA , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/fisiologia , Animais , Linhagem Celular , Galinhas/genética , Galinhas/metabolismo , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/fisiologia , Genoma
19.
DNA Repair (Amst) ; 5(7): 816-28, 2006 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-16769258

RESUMO

Werner was originally identified as a protein that interacts with the product of the Werner syndrome (WS) gene, WRN. To examine the function of the WRNIP1/WRN complex in cells, we generated knock-out cell lines that were deficient in either WRN (WRN(-/-)), WRNIP1 (WRNIP10(-/-/-)), or both (WRNIP1(-/-/-)/WRN(-/-)), using a chicken B lymphocyte cell line, DT40. WRNIP1(-/-/-)/WRN(-/-) DT40 cells grew at a similar rate as wild-type cells, but the rate of spontaneous sister-chromatid exchange was augmented compared to that of either of the single mutant cell lines. Moreover, while WRNIP1(-/-/-) and WRN(-/-) cells were moderately sensitive to camptothecin (CPT), double mutant cells showed a synergistic increase in CPT sensitivity. This suggested that WRNIP1 and WRN do not always function cooperatively to repair DNA lesions. The lack of a discernable functional interaction between WRNIP1 and WRN prompted us to reevaluate the nature of the physical interaction between these proteins. We found that MBP-tagged WRNIP1 interacted directly with WRN, and that the interaction was enhanced by the addition of ATP. Mutations in the Walker A motifs of the two proteins revealed that WRNIP1, but not WRN, must bind ATP before an efficient interaction can occur.


Assuntos
Proteínas de Transporte/metabolismo , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Trifosfato de Adenosina/metabolismo , Motivos de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação/genética , Proteínas de Transporte/genética , Linhagem Celular , Proliferação de Células , Galinhas , DNA Helicases/deficiência , DNA Helicases/genética , Primers do DNA/genética , Reparo do DNA , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/genética , Exodesoxirribonucleases , Humanos , Técnicas In Vitro , Camundongos , RecQ Helicases , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Troca de Cromátide Irmã , Técnicas do Sistema de Duplo-Híbrido , Síndrome de Werner/genética , Síndrome de Werner/metabolismo , Helicase da Síndrome de Werner
20.
Mol Cell Biol ; 23(10): 3527-35, 2003 May.
Artigo em Inglês | MEDLINE | ID: mdl-12724411

RESUMO

Human RECQL1 and RECQL5 belong to the RecQ family that includes Bloom syndrome, Werner syndrome, and Rothmund-Thomson syndrome causative genes. Cells derived from individuals suffering from these syndromes show significant levels of genomic instability. However, neither RECQL1 nor RECQL5 has been related to a disease, and nothing is known about the functions of RecQL1 and RecQL5. We generated here RECQL1(-/-), RECQL5(-/-), RECQL1(-/-)/RECQL5(-/-), RECQL1(-/-)/BLM(-/-), and RECQL5(-/-)/BLM(-/-) cells from chicken B-lymphocyte line DT40 cells. Although BLM(-/-) DT40 cells showed a slow-growth phenotype, a higher sensitivity to methyl methanesulfonate than the wild type, and an approximately 10-fold increase in the frequency of sister chromatid exchange (SCE) compared to wild-type cells, RECQL1(-/-), RECQL5(-/-), and RECQL1(-/-)/RECQL5(-/-) cells showed no significant difference from the wild-type cells in growth, sensitivity to DNA-damaging agents, and the frequency of SCE. However, both RECQL1(-/-)/BLM(-/-) and RECQL5(-/-)/BLM(-/-) cells grew more slowly than BLM(-/-) cells because of the increase in the population of dead cells, indicating that RecQL1 and RecQL5 are somehow involved in cell viability under the BLM function-impaired condition. Surprisingly, RECQL5(-/-)/BLM(-/-) cells showed a higher frequency of SCE than BLM(-/-) cells, indicating that RecQL5 suppresses SCE under the BLM function-impaired condition.


Assuntos
Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , DNA Helicases/química , DNA Helicases/metabolismo , Animais , Bromodesoxiuridina/farmacologia , Técnicas de Cultura de Células , Divisão Celular , Galinhas , Aberrações Cromossômicas , DNA/metabolismo , Citometria de Fluxo , Vetores Genéticos , Humanos , Mitomicina/farmacologia , Modelos Genéticos , Dados de Sequência Molecular , Mutação , Fenótipo , RecQ Helicases , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/metabolismo , Troca de Cromátide Irmã , Fatores de Tempo , Transfecção , Raios Ultravioleta
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