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1.
Cancers (Basel) ; 16(3)2024 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-38339346

RESUMO

R-loops (RNA-DNA hybrids with displaced single-stranded DNA) have emerged as a potent source of DNA damage and genomic instability. The termination of defective RNA polymerase II (RNAPII) is one of the major sources of R-loop formation. 5'-3'-exoribonuclease 2 (XRN2) promotes genome-wide efficient RNAPII termination, and XRN2-deficient cells exhibit increased DNA damage emanating from elevated R-loops. Recently, we showed that DNA damage instigated by XRN2 depletion in human fibroblast cells resulted in enhanced poly(ADP-ribose) polymerase 1 (PARP1) activity. Additionally, we established a synthetic lethal relationship between XRN2 and PARP1. However, the underlying cellular stress response promoting this synthetic lethality remains elusive. Here, we delineate the molecular consequences leading to the synthetic lethality of XRN2-deficient cancer cells induced by PARP inhibition. We found that XRN2-deficient lung and breast cancer cells display sensitivity to two clinically relevant PARP inhibitors, Rucaparib and Olaparib. At a mechanistic level, PARP inhibition combined with XRN2 deficiency exacerbates R-loop and DNA double-strand break formation in cancer cells. Consistent with our previous findings using several different siRNAs, we also show that XRN2 deficiency in cancer cells hyperactivates PARP1. Furthermore, we observed enhanced replication stress in XRN2-deficient cancer cells treated with PARP inhibitors. Finally, the enhanced stress response instigated by compromised PARP1 catalytic function in XRN2-deficient cells activates caspase-3 to initiate cell death. Collectively, these findings provide mechanistic insights into the sensitivity of XRN2-deficient cancer cells to PARP inhibition and strengthen the underlying translational implications for targeted therapy.

2.
Ecotoxicol Environ Saf ; 246: 114138, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36201921

RESUMO

Diclofenac is an emerging surface water contaminant, yet the environmental impact of its degradation products remains elusive. The current study focuses on mineralogy-controlled diclofenac photo-degradation and its potential health impacts. Under irradiated conditions, we studied the effects of kaolinite, hematite, and anatase on diclofenac degradation. Our results showed that kaolinite doubled the diclofenac degradation rate, which can be attributed to the high catalytic effect, mediated via increased surface area and pore size of mineral surface in the low pH. Conversely, anatase, a crystal phase of titanium dioxide (TiO2), diminished the diclofenac degradation compared to treatments without TiO2. Hematite, on the other hand, showed no effect on diclofenac degradation. Photo-degradation products also varied with the mineral surface. We further assessed in vitro toxicological effects of photo-degraded products on two human cell lines, HEK293T and HepG2. Biological assays confirmed that photo-degraded compound 6 (1-(2,6-dichlorophenyl)indolin-2-one) decreased HEK293T cell survival significantly (p < 0.05) when compared to diclofenac in all concentrations. At lower concentrations, inhibition of HEK293T cells caused by compounds 4 (2-(8-chloro-9H-carbazol-1-yl)acetic acid), and 5 (2-(9H-carbazol-1-yl)acetic acid) was greater than diclofenac. Compound 7 (1-phenylindolin-2-one) was toxic only at 250 µM. Additionally, compound 6 decreased HepG2 cell viability significantly when compared to diclofenac. Overall, our data highlighted that mineralogy plays a vital role in environmental diclofenac transformation and its photo-degraded products. Some photo-degraded compounds can be more cytotoxic than the parent compound, diclofenac.


Assuntos
Diclofenaco , Poluentes Químicos da Água , Humanos , Diclofenaco/toxicidade , Diclofenaco/química , Caulim , Células HEK293 , Titânio/toxicidade , Titânio/química , Poluentes Químicos da Água/toxicidade , Poluentes Químicos da Água/química
3.
R Soc Open Sci ; 9(9): 220358, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36177203

RESUMO

A series of cystargolide-based ß-lactone analogues containing nitrogen atoms at the Pz portion of the scaffold were prepared and evaluated as proteasome inhibitors, and for their cytotoxicity profile toward several cancer cell lines. Inclusion of one, two or even three nitrogen atoms at the Pz portion of the cystargolide scaffold is well tolerated, producing analogues with low nanomolar proteasome inhibition activity, in many cases superior to carfilzomib. Additionally, analogue 8g, containing an ester and pyrazine group at Pz, was shown to possess significant activity toward RPMI 8226 cells (IC50 = 21 nM) and to be less cytotoxic toward the normal tissue model MCF10A cells than carfilzomib.

4.
Sci Rep ; 10(1): 20210, 2020 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-33214574

RESUMO

The overall prognosis for pancreatic cancer remains dismal and potent chemotherapeutic agents that selectively target this cancer are critically needed. Elevated expression of NAD(P)H:quinone oxidoreductase 1 (NQO1) is frequent in pancreatic cancer, and it offers promising tumor-selective targeting. Recently, KP372-1 was identified as a novel NQO1 redox cycling agent that induces cytotoxicity in cancer cells by creating redox imbalance; however, the mechanistic basis of KP372-1-induced cytotoxicity remains elusive. Here, we show that KP372-1 sensitizes NQO1-expressing pancreatic cancer cells and spares immortalized normal pancreatic duct cells, hTERT-HPNE. Notably, we found that KP372-1 is ~ 10- to 20-fold more potent than ß-lapachone, another NQO1 substrate, against pancreatic cancer cells. Mechanistically, our data strongly suggest that reactive oxygen species produced by NQO1-dependent redox cycling of KP372-1 cause robust DNA damage, including DNA breaks. Furthermore, we found that KP372-1-induced DNA damage hyperactivates the central DNA damage sensor protein poly(ADP-ribose) polymerase 1 (PARP1) and activates caspase-3 to initiate cell death. Our data also show that the combination of KP372-1 with PARP inhibition creates enhanced cytotoxicity in pancreatic cancer cells. Collectively, our study provides mechanistic insights into the cytotoxicity instigated by KP372-1 and lays an essential foundation to establish it as a promising chemotherapeutic agent against cancer.


Assuntos
Antineoplásicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Neoplasias Pancreáticas/tratamento farmacológico , Poli(ADP-Ribose) Polimerase-1/metabolismo , Tetrazóis/farmacologia , Antineoplásicos/uso terapêutico , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Compostos Heterocíclicos de 4 ou mais Anéis/uso terapêutico , Humanos , NAD(P)H Desidrogenase (Quinona)/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Espécies Reativas de Oxigênio/metabolismo , Tetrazóis/uso terapêutico
5.
Sci Rep ; 10(1): 14253, 2020 08 28.
Artigo em Inglês | MEDLINE | ID: mdl-32859985

RESUMO

Persistent R-loops (RNA-DNA hybrids with a displaced single-stranded DNA) create DNA damage and lead to genomic instability. The 5'-3'-exoribonuclease 2 (XRN2) degrades RNA to resolve R-loops and promotes transcription termination. Previously, XRN2 was implicated in DNA double strand break (DSB) repair and in resolving replication stress. Here, using tandem affinity purification-mass spectrometry, bioinformatics, and biochemical approaches, we found that XRN2 associates with proteins involved in DNA repair/replication (Ku70-Ku80, DNA-PKcs, PARP1, MCM2-7, PCNA, RPA1) and RNA metabolism (RNA helicases, PRP19, p54(nrb), splicing factors). Novel major pathways linked to XRN2 include cell cycle control of chromosomal replication and DSB repair by non-homologous end joining. Investigating the biological implications of these interactions led us to discover that XRN2 depletion compromised cell survival after additional knockdown of specific DNA repair proteins, including PARP1. XRN2-deficient cells also showed enhanced PARP1 activity. Consistent with concurrent depletion of XRN2 and PARP1 promoting cell death, XRN2-deficient fibroblast and lung cancer cells also demonstrated sensitivity to PARP1 inhibition. XRN2 alterations (mutations, copy number/expression changes) are frequent in cancers. Thus, PARP1 inhibition could target cancers exhibiting XRN2 functional loss. Collectively, our data suggest XRN2's association with novel protein partners and unravel synthetic lethality between XRN2 depletion and PARP1 inhibition.


Assuntos
Exorribonucleases/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Estruturas R-Loop/fisiologia , Células A549 , Quebras de DNA de Cadeia Dupla , Dano ao DNA/fisiologia , Reparo do DNA por Junção de Extremidades/fisiologia , Reparo do DNA/fisiologia , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/genética , Exorribonucleases/fisiologia , Instabilidade Genômica/fisiologia , Células HEK293 , Células HeLa , Humanos , Poli(ADP-Ribose) Polimerase-1/fisiologia , Poli(ADP-Ribose) Polimerases/metabolismo , Estruturas R-Loop/genética , RNA Helicases/metabolismo , Mutações Sintéticas Letais/genética
6.
Ecotoxicol Environ Saf ; 188: 109892, 2020 Jan 30.
Artigo em Inglês | MEDLINE | ID: mdl-31732272

RESUMO

Increasing quantities of pharmaceutical waste in the environment have disrupted the balance of ecosystems, and may have subsequent effects on human health. Although a handful of previous studies have shown the impacts of pharmaceutically active compounds on the environment, the toxicological effects of their degradation products remain largely unknown. In the current study, the photo-degradation products of environmental ibuprofen were assessed for both ecotoxicological and human health effects using a series of in vitro assays. Here, six of the major degradation products are synthesized with high purity (>98%) and characterized with 1HNMR, 13CNMR, FT-IR and HRMS. To evaluate human health effects, three gut microbiota species, Lactobacillus acidophilus, Enterococcus faecalis and Escherichia coli, and two human cell lines, HEK293T and HepG2, are exposed to various concentrations of ibuprofen and its degradation products. On L. acidophilus, the ibuprofen degradation product (±)-(2R,3R)-2-(4-isobutylphenyl)-5-methylhexan-3-ol shows a greater toxic effect while ibuprofen enhances its growth at lower concentrations. At higher concentrations, ibuprofen shows at least a 2-fold higher toxicity compared to that of its degradation products. However, E. faecalis shows little or no effect upon exposure to these compounds. An induction of the SOS response in E. coli is observed but limited to only ibuprofen and 4-acetylbenzoic acid. In human cell line studies, survival of both HEK293T and HepG2 cell lines is profoundly impaired by the photo-degradation products of (±)- (2R,3R)-2-(4-isobutylphenyl)-5-methylhexan-3-ol, (±)-(2R,3S)-2-(4-isobutylphenyl)-5-methylhexan-3-ol, and (±)-1-(4-(1-hydroxy-2methylpropyl)phenyl)ethan-1-one. In this work, the bioluminescence bacterium, Aliivibrio fischeri, is used as a model to assess environmental impact. Both ibuprofen and its degradation products inhibit the growth of this gram-negative bacteria with the primary compound showing the most significant impact. Overall, our results highlight that some of the degradation products of ibuprofen can be more toxic to human kidney cell line and liver cell line than the parent compound while ibuprofen can be more toxic to human gut microbiota and A. fischeri than ibuprofen degradation products.


Assuntos
Aliivibrio fischeri/efeitos dos fármacos , Microbioma Gastrointestinal/efeitos dos fármacos , Ibuprofeno/toxicidade , Fotólise , Poluentes Químicos da Água/toxicidade , Sobrevivência Celular/efeitos dos fármacos , Ecossistema , Ecotoxicologia , Microbioma Gastrointestinal/genética , Células HEK293 , Células Hep G2 , Humanos , Ibuprofeno/química , Resposta SOS em Genética/efeitos dos fármacos , Poluentes Químicos da Água/química
7.
Clin Cancer Res ; 24(24): 6459-6470, 2018 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-30108102

RESUMO

PURPOSE: Identification of novel strategies to expand the use of PARP inhibitors beyond BRCA deficiency is of great interest in personalized medicine. Here, we investigated the unannotated role of Kub5-HeraRPRD1B (K-H) in homologous recombination (HR) repair and its potential clinical significance in targeted cancer therapy. EXPERIMENTAL DESIGN: Functional characterization of K-H alterations on HR repair of double-strand breaks (DSB) were assessed by targeted gene silencing, plasmid reporter assays, immunofluorescence, and Western blots. Cell survival with PARP inhibitors was evaluated through colony-forming assays and statistically analyzed for correlation with K-H expression in various BRCA1/2 nonmutated breast cancers. Gene expression microarray/qPCR analyses, chromatin immunoprecipitation, and rescue experiments were used to investigate molecular mechanisms of action. RESULTS: K-H expression loss correlates with rucaparib LD50 values in a panel of BRCA1/2 nonmutated breast cancers. Mechanistically, K-H depletion promotes BRCAness, where extensive upregulation of PARP1 activity was required for the survival of breast cancer cells. PARP inhibition in these cells led to synthetic lethality that was rescued by wild-type K-H reexpression, but not by a mutant K-H (p.R106A) that weakly binds RNAPII. K-H mediates HR by facilitating recruitment of RNAPII to the promoter region of a critical DNA damage response and repair effector, cyclin-dependent kinase 1 (CDK1). CONCLUSIONS: Cancer cells with low K-H expression may have exploitable BRCAness properties that greatly expand the use of PARP inhibitors beyond BRCA mutations. Our results suggest that aberrant K-H alterations may have vital translational implications in cellular responses/survival to DNA damage, carcinogenesis, and personalized medicine.


Assuntos
Neoplasias da Mama/etiologia , Neoplasias da Mama/metabolismo , Proteínas de Ciclo Celular/deficiência , Genes BRCA1 , Genes BRCA2 , Proteínas de Neoplasias/deficiência , Poli(ADP-Ribose) Polimerases/metabolismo , Animais , Neoplasias da Mama/patologia , Proteína Quinase CDC2/química , Proteína Quinase CDC2/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Dano ao DNA , Modelos Animais de Doenças , Feminino , Regulação Neoplásica da Expressão Gênica , Genes Reporter , Humanos , Camundongos , Mutação , Proteínas de Neoplasias/química , Proteínas de Neoplasias/metabolismo , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Regiões Promotoras Genéticas , Mutações Sintéticas Letais , Ensaios Antitumorais Modelo de Xenoenxerto
8.
Nat Commun ; 8: 15282, 2017 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-28513583

RESUMO

RecN is a cohesin-like protein involved in DNA double-strand break repair in bacteria. The RecA recombinase functions to mediate repair via homologous DNA strand invasion to form D-loops. Here we provide evidence that the RecN protein stimulates the DNA strand invasion step of RecA-mediated recombinational DNA repair. The intermolecular DNA tethering activity of RecN protein described previously cannot fully explain this novel activity since stimulation of RecA function is species-specific and requires RecN ATP hydrolysis. Further, DNA-bound RecA protein increases the rate of ATP hydrolysis catalysed by RecN during the DNA pairing reaction. DNA-dependent RecN ATPase kinetics are affected by RecA protein in a manner suggesting a specific order of protein-DNA assembly, with RecN acting after RecA binds DNA. We present a model for RecN function that includes presynaptic stimulation of the bacterial repair pathway perhaps by contributing to the RecA homology search before ternary complex formation.


Assuntos
Proteínas de Bactérias/metabolismo , Quebras de DNA de Cadeia Dupla , Enzimas de Restrição do DNA/metabolismo , Deinococcus/genética , Recombinases Rec A/metabolismo , Reparo de DNA por Recombinação , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/isolamento & purificação , Enzimas de Restrição do DNA/isolamento & purificação , DNA de Cadeia Simples/metabolismo , Deinococcus/metabolismo , Hidrólise , Ligação Proteica , Recombinases Rec A/isolamento & purificação
9.
Cancer Cell ; 30(6): 940-952, 2016 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-27960087

RESUMO

Therapeutic drugs that block DNA repair, including poly(ADP-ribose) polymerase (PARP) inhibitors, fail due to lack of tumor-selectivity. When PARP inhibitors and ß-lapachone are combined, synergistic antitumor activity results from sustained NAD(P)H levels that refuel NQO1-dependent futile redox drug recycling. Significant oxygen-consumption-rate/reactive oxygen species cause dramatic DNA lesion increases that are not repaired due to PARP inhibition. In NQO1+ cancers, such as non-small-cell lung, pancreatic, and breast cancers, cell death mechanism switches from PARP1 hyperactivation-mediated programmed necrosis with ß-lapachone monotherapy to synergistic tumor-selective, caspase-dependent apoptosis with PARP inhibitors and ß-lapachone. Synergistic antitumor efficacy and prolonged survival were noted in human orthotopic pancreatic and non-small-cell lung xenograft models, expanding use and efficacy of PARP inhibitors for human cancer therapy.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Neoplasias Pulmonares/tratamento farmacológico , NAD(P)H Desidrogenase (Quinona)/genética , Naftoquinonas/administração & dosagem , Neoplasias Pancreáticas/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases/administração & dosagem , Animais , Carcinoma Pulmonar de Células não Pequenas/genética , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , Sinergismo Farmacológico , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Pulmonares/genética , Camundongos , Naftoquinonas/farmacologia , Neoplasias Pancreáticas/genética , Espécies Reativas de Oxigênio/metabolismo , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto
10.
PLoS Genet ; 12(7): e1006107, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27437695

RESUMO

XRN2 is a 5'-3' exoribonuclease implicated in transcription termination. Here we demonstrate an unexpected role for XRN2 in the DNA damage response involving resolution of R-loop structures and prevention of DNA double-strand breaks (DSBs). We show that XRN2 undergoes DNA damage-inducible nuclear re-localization, co-localizing with 53BP1 and R loops, in a transcription and R-loop-dependent process. XRN2 loss leads to increased R loops, genomic instability, replication stress, DSBs and hypersensitivity of cells to various DNA damaging agents. We demonstrate that the DSBs that arise with XRN2 loss occur at transcriptional pause sites. XRN2-deficient cells also exhibited an R-loop- and transcription-dependent delay in DSB repair after ionizing radiation, suggesting a novel role for XRN2 in R-loop resolution, suppression of replication stress, and maintenance of genomic stability. Our study highlights the importance of regulating transcription-related activities as a critical component in maintaining genetic stability.


Assuntos
Dano ao DNA , Replicação do DNA , Exorribonucleases/genética , Exorribonucleases/fisiologia , Transcrição Gênica , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Antineoplásicos/química , Núcleo Celular/metabolismo , DNA Helicases , Reparo do DNA , Regulação Neoplásica da Expressão Gênica , Instabilidade Genômica , Genômica , Células HeLa , Humanos , Microscopia de Fluorescência , Enzimas Multifuncionais , Neoplasias/tratamento farmacológico , Neoplasias/genética , Plasmídeos/metabolismo , RNA Helicases/metabolismo , RNA Interferente Pequeno/metabolismo
11.
Nucleic Acids Res ; 44(4): 1718-31, 2016 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-26819409

RESUMO

Ku70-binding protein 5 (Kub5)-Hera (K-H)/RPRD1B maintains genetic integrity by concomitantly minimizing persistent R-loops and promoting repair of DNA double strand breaks (DSBs). We used tandem affinity purification-mass spectrometry, co-immunoprecipitation and gel-filtration chromatography to define higher-order protein complexes containing K-H scaffolding protein to gain insight into its cellular functions. We confirmed known protein partners (Ku70, RNA Pol II, p15RS) and discovered several novel associated proteins that function in RNA metabolism (Topoisomerase 1 and RNA helicases), DNA repair/replication processes (PARP1, MSH2, Ku, DNA-PKcs, MCM proteins, PCNA and DNA Pol δ) and in protein metabolic processes, including translation. Notably, this approach directed us to investigate an unpredicted involvement of K-H in DNA mismatch repair (MMR) where K-H depletion led to concomitant MMR deficiency and compromised global microsatellite stability. Mechanistically, MMR deficiency in K-H-depleted cells was a consequence of reduced stability of the core MMR proteins (MLH1 and PMS2) caused by elevated basal caspase-dependent proteolysis. Pan-caspase inhibitor treatment restored MMR protein loss. These findings represent a novel mechanism to acquire MMR deficiency/microsatellite alterations. A significant proportion of colon, endometrial and ovarian cancers exhibit k-h expression/copy number loss and may have severe mutator phenotypes with enhanced malignancies that are currently overlooked based on sporadic MSI+ screening.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Reparo de Erro de Pareamento de DNA/genética , Instabilidade Genômica , Proteínas de Neoplasias/metabolismo , Neoplasias/genética , Antígenos Nucleares/genética , Proteínas de Ciclo Celular/genética , Quebras de DNA de Cadeia Dupla , DNA Topoisomerases Tipo I/genética , Proteínas de Ligação a DNA/genética , Células HeLa , Humanos , Autoantígeno Ku , Complexos Multiproteicos/genética , Proteínas de Neoplasias/genética , Neoplasias/metabolismo , RNA Helicases/genética , RNA Polimerase II/genética , Proteínas Repressoras/genética
12.
Nucleic Acids Res ; 42(8): 4996-5006, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24589584

RESUMO

Functions of Kub5-Hera (In Greek Mythology Hera controlled Artemis) (K-H), the human homolog of the yeast transcription termination factor Rtt103, remain undefined. Here, we show that K-H has functions in both transcription termination and DNA double-strand break (DSB) repair. K-H forms distinct protein complexes with factors that repair DSBs (e.g. Ku70, Ku86, Artemis) and terminate transcription (e.g. RNA polymerase II). K-H loss resulted in increased basal R-loop levels, DSBs, activated DNA-damage responses and enhanced genomic instability. Significantly lowered Artemis protein levels were detected in K-H knockdown cells, which were restored with specific K-H cDNA re-expression. K-H deficient cells were hypersensitive to cytotoxic agents that induce DSBs, unable to reseal complex DSB ends, and showed significantly delayed γ-H2AX and 53BP1 repair-related foci regression. Artemis re-expression in K-H-deficient cells restored DNA-repair function and resistance to DSB-inducing agents. However, R loops persisted consistent with dual roles of K-H in transcription termination and DSB repair.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Reparo do DNA , Proteínas de Neoplasias/metabolismo , Terminação da Transcrição Genética , Animais , Antineoplásicos/toxicidade , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/fisiologia , Células Cultivadas , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Proteínas de Ligação a DNA , Endonucleases , Instabilidade Genômica , Humanos , Camundongos , Proteínas de Neoplasias/química , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiologia , Proteínas Nucleares/metabolismo , Estrutura Terciária de Proteína , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética
13.
J Biol Chem ; 285(22): 16521-9, 2010 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-20360008

RESUMO

The bacterial RecN protein is involved in the recombinational repair of DNA double-stranded breaks, and recN mutants are sensitive to DNA-damaging agents. Little is known about the biochemical function of RecN. Protein sequence analysis suggests that RecN is related to the SMC (structural maintenance of chromosomes) family of proteins, predicting globular N- and C-terminal domains connected by an extensive coil-coiled domain. The N- and C-domains contain the nucleotide-binding sequences Walker A and Walker B, respectively. We have purified the RecN protein from Deinococcus radiodurans and characterized its DNA-dependent and DNA-independent ATPase activity. The RecN protein hydrolyzes ATP with a k(cat) of 24 min(-1), and this rate is stimulated 4-fold by duplex DNA but not by single-stranded DNA. This DNA-dependent ATP turnover rate exhibits a dependence on the concentration of RecN protein, suggesting that RecN-RecN interactions are required for efficient ATP hydrolysis, and those interactions are stabilized only by duplex DNA. Finally, we show that RecN stimulates the intermolecular ligation of linear DNA molecules in the presence of DNA ligase. This DNA bridging activity is strikingly similar to that of the cohesin complex, an SMC family member, to which RecN is related.


Assuntos
Proteínas de Bactérias/metabolismo , Enzimas de Restrição do DNA/metabolismo , DNA/genética , Deinococcus/metabolismo , Adenosina Trifosfatases/metabolismo , Clonagem Molecular , Simulação por Computador , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/metabolismo , Dimerização , Genoma Bacteriano , Hidrólise , Técnicas In Vitro , Cinética , Modelos Biológicos , Modelos Genéticos , Ligação Proteica
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