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1.
Nat Genet ; 51(8): 1283-1294, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31367016

RESUMO

Germline de novo mutations are the basis of evolutionary diversity but also of genetic disease. However, the molecular origin, mechanisms and timing of germline mutagenesis are not fully understood. Here, we define a fundamental role for DNA interstrand cross-link repair in the germline. This repair process is essential for primordial germ cell (PGC) maturation during embryonic development. Inactivation of cross-link repair leads to genetic instability that is restricted to PGCs within the genital ridge during a narrow temporal window. Having successfully activated the PGC transcriptional program, a potent quality control mechanism detects and drives damaged PGCs into apoptosis. Therefore, these findings define a source of DNA damage and the nature of the subsequent DNA repair response in germ cells, which ensures faithful transmission of the genome between generations.


Assuntos
Diferenciação Celular , Reparo do DNA , DNA/química , Instabilidade Genômica , Células Germinativas/citologia , Meiose/fisiologia , Aldeídos/metabolismo , Aldeídos/toxicidade , Animais , Apoptose/efeitos dos fármacos , Reagentes para Ligações Cruzadas , DNA/genética , Dano ao DNA , Proteínas de Ligação a DNA/fisiologia , Endonucleases/fisiologia , Feminino , Fertilidade , Genoma , Células Germinativas/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout
2.
Nucleic Acids Res ; 47(13): 6796-6810, 2019 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-31114910

RESUMO

Stabilization of stalled replication forks prevents excessive fork reversal or degradation, which can undermine genome integrity. The WRN protein is unique among the other human RecQ family members to possess exonuclease activity. However, the biological role of the WRN exonuclease is poorly defined. Recently, the WRN exonuclease has been linked to protection of stalled forks from degradation. Alternative processing of perturbed forks has been associated to chemoresistance of BRCA-deficient cancer cells. Thus, we used WRN exonuclease-deficiency as a model to investigate the fate of perturbed forks undergoing degradation, but in a BRCA wild-type condition. We find that, upon treatment with clinically-relevant nanomolar doses of the Topoisomerase I inhibitor camptothecin, loss of WRN exonuclease stimulates fork inactivation and accumulation of parental gaps, which engages RAD51. Such mechanism affects reinforcement of CHK1 phosphorylation and causes persistence of RAD51 during recovery from treatment. Notably, in WRN exonuclease-deficient cells, persistence of RAD51 correlates with elevated mitotic phosphorylation of MUS81 at Ser87, which is essential to prevent excessive mitotic abnormalities. Altogether, these findings indicate that aberrant fork degradation, in the presence of a wild-type RAD51 axis, stimulates RAD51-mediated post-replicative repair and engagement of the MUS81 complex to limit genome instability and cell death.


Assuntos
Camptotecina/farmacologia , Replicação do DNA/efeitos dos fármacos , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/fisiologia , Endonucleases/fisiologia , Conformação de Ácido Nucleico/efeitos dos fármacos , Rad51 Recombinase/fisiologia , Inibidores da Topoisomerase I/farmacologia , Helicase da Síndrome de Werner/deficiência , Proteína BRCA2/fisiologia , Linhagem Celular Transformada , Quinase 1 do Ponto de Checagem/metabolismo , Quebras de DNA de Cadeia Dupla , Ativação Enzimática , Fibroblastos , Humanos , Mitocôndrias/efeitos dos fármacos , Mitose/efeitos dos fármacos , Complexos Multiproteicos/metabolismo , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Interferência de RNA , Síndrome de Werner/metabolismo , Helicase da Síndrome de Werner/fisiologia
3.
Microb Cell Fact ; 18(1): 60, 2019 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-30909908

RESUMO

BACKGROUND: Corynebacterium glutamicum is an important industrial strain for the production of a diverse range of chemicals. Cpf1 nucleases are highly specific and programmable, with efficiencies comparable to those of Cas9. Although the Francisella novicida (Fn) CRISPR-Cpf1 system has been adapted for genome editing in C. glutamicum, the editing efficiency is currently less than 15%, due to false positives caused by the poor targeting efficiency of the crRNA. RESULTS: To address this limitation, a screening strategy was developed in this study to systematically evaluate crRNA targeting efficiency in C. glutamicum. We quantitatively examined various parameters of the C. glutamicum CRISPR-Cpf1 system, including the protospacer adjacent motif (PAM) sequence, the length of the spacer sequence, and the type of repair template. We found that the most efficient C. glutamicum crRNA contained a 5'-NYTV-3' PAM and a 21 bp spacer sequence. Moreover, we observed that linear DNA could be used to repair double strand breaks. CONCLUSIONS: Here, we identified optimized PAM-related parameters for the CRISPR-Cpf1 system in C. glutamicum. Our study sheds light on the function of the FnCpf1 endonuclease and Cpf1-based genome editing. This optimized system, with higher editing efficiency, could be used to increase the production of bulk chemicals, such as isobutyrate, in C. glutamicum.


Assuntos
Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Corynebacterium glutamicum/enzimologia , Corynebacterium glutamicum/genética , RNA/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/fisiologia , Endonucleases/fisiologia , Edição de Genes
4.
Cell ; 175(2): 558-570.e11, 2018 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-30245011

RESUMO

Given that genomic DNA exerts its function by being transcribed, it is critical for the maintenance of homeostasis that DNA damage, such as double-strand breaks (DSBs), within transcriptionally active regions undergoes accurate repair. However, it remains unclear how this is achieved. Here, we describe a mechanism for transcription-associated homologous recombination repair (TA-HRR) in human cells. The process is initiated by R-loops formed upon DSB induction. We identify Rad52, which is recruited to the DSB site in a DNA-RNA-hybrid-dependent manner, as playing pivotal roles in promoting XPG-mediated R-loop processing and initiating subsequent repair by HRR. Importantly, dysfunction of TA-HRR promotes DSB repair via non-homologous end joining, leading to a striking increase in genomic aberrations. Thus, our data suggest that the presence of R-loops around DSBs within transcriptionally active regions promotes accurate repair of DSBs via processing by Rad52 and XPG to protect genomic information in these critical regions from gene alterations.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Proteínas Nucleares/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Reparo de DNA por Recombinação/fisiologia , Fatores de Transcrição/metabolismo , Linhagem Celular , DNA/genética , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Reparo do DNA por Junção de Extremidades , Reparo do DNA , Proteínas de Ligação a DNA/fisiologia , Endonucleases/fisiologia , Recombinação Homóloga , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/fisiologia , RNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Fatores de Transcrição/fisiologia
5.
Nucleic Acids Res ; 46(19): 10272-10285, 2018 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-30239882

RESUMO

Cpf1s, the RNA-guided nucleases of the class II clustered regularly interspaced short palindromic repeats system require a short motive called protospacer adjacent motif (PAM) to be present next to the targeted sequence for their activity. The TTTV PAM sequence of As- and LbCpf1 nucleases is relatively rare in the genome of higher eukaryotic organisms. Here, we show that two other Cpf1 nucleases, Fn- and MbCpf1, which have been reported to utilize a shorter, more frequently occurring PAM sequence (TTN) when tested in vitro, carry out efficient genome modification in mammalian cells. We found that all four Cpf1 nucleases showed similar activities and TTTV PAM preferences. Our approach also revealed that besides their activities their PAM preferences are also target dependent. To increase the number of the available targets for Fn- and MbCpf1 we generated their RVR and RR mutants with altered PAM specificity and compared them to the wild-type and analogous As- and LbCpf1 variants. The mutants gained new PAM specificities but retained their activity on targets with TTTV PAMs, redefining RR-Cpf1's PAM-specificities as TTYV/TCCV, respectively. These variants may become versatile substitutes for wild-type Cpf1s by providing an expanded range of targets for genome engineering applications.


Assuntos
Proteínas Associadas a CRISPR/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endonucleases/fisiologia , Francisella/enzimologia , Moraxella/enzimologia , Animais , Sequência de Bases , Sítios de Ligação/genética , Sistemas CRISPR-Cas/genética , Endonucleases/metabolismo , Células HEK293 , Humanos , Mamíferos , Camundongos , Ligação Proteica , Especificidade por Substrato , Células Tumorais Cultivadas
6.
Plant Cell ; 30(9): 1954-1970, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-30135084

RESUMO

Base excision repair (BER) is essential for active DNA demethylation and DNA damage repair in mammals and plants. Here, we provide genetic and biochemical evidence that APURINIC/APYRIMIDINIC ENDONUCLEASE2 (APE2) plays overlapping roles with ZINC FINGER DNA 3'-PHOSPHOESTERASE (ZDP) in active DNA demethylation and DNA damage repair in Arabidopsis thaliana Simultaneous mutation of APE2 and ZDP causes DNA hypermethylation at more than 2000 loci, most of which are not hypermethylated in ape2 or zdp single mutants. The zdp and ape2 single mutants exhibit normal development, but the zdp ape2 double mutants display pleiotropic developmental defects and are supersensitive to the DNA alkylating reagent methyl methanesulfonate. The gradual accumulation of DNA lesions in the zdp ape2 seedlings is accompanied by constitutive activation of the DNA damage response and alteration of the cell cycle. Interestingly, knockout of the key DNA demethylase REPRESSOR OF SILENCING1 reduces the magnitude of DNA lesion accumulation and the DNA damage response in the zdp ape2 mutants, suggesting that a proportion of the DNA damage in the zdp ape2 mutants arises from incomplete active DNA demethylation. Lastly, we find that APE2 has 3'-phosphatase activity and strong 3'-5' exonuclease activity in vitro. Together, our results suggest that APE2 and ZDP, two BER proteins, play overlapping roles in the maintenance of epigenome and genome stability in plants.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Reparo do DNA/genética , Endonucleases/fisiologia , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/fisiologia , Dano ao DNA/genética , Desmetilação do DNA , Endonucleases/genética , Epigenômica , Instabilidade Genômica/genética , Mutação/genética
7.
Dev Cell ; 45(6): 785-800.e6, 2018 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-29920281

RESUMO

During meiosis, crossover recombination promotes the establishment of physical connections between homologous chromosomes, enabling their bipolar segregation. To ensure that persistent recombination intermediates are disengaged prior to the completion of meiosis, the Yen1(GEN1) resolvase is strictly activated at the onset of anaphase II. Whether controlled activation of Yen1 is important for meiotic crossing-over is unknown. Here, we show that CDK-mediated phosphorylation of Yen1 averts its pervasive recruitment to recombination intermediates during prophase I. Yen1 mutants that are refractory to phosphorylation resolve DNA joint molecules prematurely and form crossovers independently of MutLγ, the central crossover resolvase during meiosis. Despite bypassing the requirement for MutLγ in joint molecule processing and promoting crossover-specific resolution, unrestrained Yen1 impairs the spatial distribution of crossover events, genome-wide. Thus, active suppression of Yen1 function, and by inference also of Mus81-Mms4(EME1) and Slx1-Slx4(BTBD12) resolvases, avoids precocious resolution of recombination intermediates to enable meiotic crossover patterning.


Assuntos
Resolvases de Junção Holliday/genética , Resolvases de Junção Holliday/metabolismo , Prófase Meiótica I/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Cromossomos Fúngicos , Troca Genética , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Endonucleases/fisiologia , Prófase Meiótica I/genética , Fosforilação , Saccharomyces cerevisiae/citologia
8.
Free Radic Biol Med ; 124: 79-96, 2018 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-29860127

RESUMO

Lipid peroxidation (LPO) products are relatively stable and abundant metabolites, which accumulate in tissues of mammals with aging, being able to modify all cellular nucleophiles, creating protein and DNA adducts including crosslinks. Here, we used cells and mice deficient in the ERCC1-XPF endonuclease required for nucleotide excision repair and the repair of DNA interstrand crosslinks to ask if specifically LPO-induced DNA damage contributes to loss of cell and tissue homeostasis. Ercc1-/- mouse embryonic fibroblasts were more sensitive than wild-type (WT) cells to the LPO products: 4-hydroxy-2-nonenal (HNE), crotonaldehyde and malondialdehyde. ERCC1-XPF hypomorphic mice were hypersensitive to CCl4 and a diet rich in polyunsaturated fatty acids, two potent inducers of endogenous LPO. To gain insight into the mechanism of how LPO influences DNA repair-deficient cells, we measured the impact of the major endogenous LPO product, HNE, on WT and Ercc1-/- cells. HNE inhibited proliferation, stimulated ROS and LPO formation, induced DNA base damage, strand breaks, error-prone translesion DNA synthesis and cellular senescence much more potently in Ercc1-/- cells than in DNA repair-competent control cells. HNE also deregulated base excision repair and energy production pathways. Our observations that ERCC1-deficient cells and mice are hypersensitive to LPO implicates LPO-induced DNA damage in contributing to cellular demise and tissue degeneration, notably even when the source of LPO is dietary polyunsaturated fats.


Assuntos
Senescência Celular , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/fisiologia , Endonucleases/fisiologia , Peroxidação de Lipídeos , Estresse Oxidativo , Animais , Proliferação de Células , Camundongos , Camundongos Knockout , Espécies Reativas de Oxigênio/metabolismo
9.
Bioessays ; 38 Suppl 1: S136-45, 2016 07.
Artigo em Inglês | MEDLINE | ID: mdl-27417117

RESUMO

Depletion of mitochondrial endo/exonuclease G-like (EXOG) in cultured neonatal cardiomyocytes stimulates mitochondrial oxygen consumption rate (OCR) and induces hypertrophy via reactive oxygen species (ROS). Here, we show that neurohormonal stress triggers cell death in endo/exonuclease G-like-depleted cells, and this is marked by a decrease in mitochondrial reserve capacity. Neurohormonal stimulation with phenylephrine (PE) did not have an additive effect on the hypertrophic response induced by endo/exonuclease G-like depletion. Interestingly, PE-induced atrial natriuretic peptide (ANP) gene expression was completely abolished in endo/exonuclease G-like-depleted cells, suggesting a reverse signaling function of endo/exonuclease G-like. Endo/exonuclease G-like depletion initially resulted in increased mitochondrial OCR, but this declined upon PE stimulation. In particular, the reserve capacity of the mitochondrial respiratory chain and maximal respiration were the first indicators of perturbations in mitochondrial respiration, and these marked the subsequent decline in mitochondrial function. Although pathological stimulation accelerated these processes, prolonged EXOG depletion also resulted in a decline in mitochondrial function. At early stages of endo/exonuclease G-like depletion, mitochondrial ROS production was increased, but this did not affect mitochondrial DNA (mtDNA) integrity. After prolonged depletion, ROS levels returned to control values, despite hyperpolarization of the mitochondrial membrane. The mitochondrial dysfunction finally resulted in cell death, which appears to be mainly a form of necrosis. In conclusion, endo/exonuclease G-like plays an essential role in cardiomyocyte physiology. Loss of endo/exonuclease G-like results in diminished adaptation to pathological stress. The decline in maximal respiration and reserve capacity is the first sign of mitochondrial dysfunction that determines subsequent cell death.


Assuntos
Morte Celular , Endonucleases/fisiologia , Mitocôndrias/metabolismo , Miócitos Cardíacos/metabolismo , Animais , DNA Mitocondrial/metabolismo , Transporte de Elétrons , Endonucleases/metabolismo , Miócitos Cardíacos/patologia , Estresse Oxidativo , Consumo de Oxigênio , Ratos
10.
Nucleic Acids Res ; 44(9): 4243-51, 2016 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-27060147

RESUMO

The RNA-guided Cas9 nuclease from CRISPR-Cas systems has emerged as a powerful biotechnological tool. The specificity of Cas9 can be reprogrammed to cleave desired sequences in a cell's chromosome simply by changing the sequence of a small guide RNA. Unlike in most eukaryotes, Cas9 cleavage in the chromosome of bacteria has been reported to kill the cell. However, the mechanism of cell death remains to be investigated. Bacteria mainly rely on homologous recombination (HR) with sister chromosomes to repair double strand breaks. Here, we show that the simultaneous cleavage of all copies of the Escherichia coli chromosome at the same position cannot be repaired, leading to cell death. However, inefficient cleavage can be tolerated through continuous repair by the HR pathway. In order to kill cells reliably, HR can be blocked using the Mu phage Gam protein. Finally, the introduction of the non-homologous end joining (NHEJ) pathway from Mycobacterium tuberculosis was not able to rescue the cells from Cas9-mediated killing, but did introduce small deletions at a low frequency. This work provides a better understanding of the consequences of Cas9 cleavage in bacterial chromosomes which will be instrumental in the development of future CRISPR tools.


Assuntos
Cromossomos Bacterianos/genética , Escherichia coli/genética , Sistemas CRISPR-Cas , Clivagem do DNA , Endonucleases/fisiologia , Viabilidade Microbiana , Reparo de DNA por Recombinação
11.
PLoS One ; 11(3): e0152278, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27010503

RESUMO

Ankyrin repeat and LEM-domain containing protein 1 (ANKLE1) is a GIY-YIG endonuclease with unknown functions, mainly expressed in mouse hematopoietic tissues. To test its potential role in hematopoiesis we generated Ankle1-deficient mice. Ankle1Δ/Δ mice are viable without any detectable phenotype in hematopoiesis. Neither hematopoietic progenitor cells, myeloid and lymphoid progenitors, nor B and T cell development in bone marrow, spleen and thymus, are affected in Ankle1Δ/Δ-mice. Similarly embryonic stress erythropoiesis in liver and adult erythropoiesis in bone marrow and spleen appear normal. To test whether ANKLE1, like the only other known GIY-YIG endonuclease in mammals, SLX1, may contribute to Holliday junction resolution during DNA repair, Ankle1-deficient cells were exposed to various DNA-damage inducing agents. However, lack of Ankle1 did not affect cell viability and, unlike depletion of Slx1, Ankle1-deficiency did not increase sister chromatid exchange in Bloom helicase-depleted cells. Altogether, we show that lack of Ankle1 does neither affect mouse hematopoiesis nor DNA damage repair in mouse embryonic fibroblasts, indicating a redundant or non-essential function of ANKLE1 in mouse.


Assuntos
Endonucleases/fisiologia , Hematopoese/fisiologia , Animais , Endonucleases/genética , Citometria de Fluxo , Técnicas de Silenciamento de Genes , Camundongos , Camundongos Endogâmicos C57BL , Polimorfismo de Nucleotídeo Único , RNA Mensageiro/genética
12.
Cancer Lett ; 373(2): 214-21, 2016 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-26805762

RESUMO

Cellular response to DNA damage, including ionizing radiation (IR) and UV radiation, is critical for the maintenance of genomic fidelity. Defects of DNA repair often result in genomic instability and malignant cell transformation. Centrosomal protein Nlp (ninein-like protein) has been characterized as an important cell cycle regulator that is required for proper mitotic progression. In this study, we demonstrate that Nlp is able to improve nucleotide excision repair (NER) activity and protects cells against UV radiation. Upon exposure of cells to UVC, Nlp is translocated into the nucleus. The C-terminus (1030-1382) of Nlp is necessary and sufficient for its nuclear import. Upon UVC radiation, Nlp interacts with XPA and ERCC1, and enhances their association. Interestingly, down-regulated expression of Nlp is found to be associated with human skin cancers, indicating that dysregulated Nlp might be related to the development of human skin cancers. Taken together, this study identifies mitotic protein Nlp as a new and important member of NER pathway and thus provides novel insights into understanding of regulatory machinery involved in NER.


Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/fisiologia , Endonucleases/fisiologia , Proteínas Associadas aos Microtúbulos/fisiologia , Proteínas Nucleares/fisiologia , Proteína de Xeroderma Pigmentoso Grupo A/fisiologia , Apoptose/efeitos da radiação , Linhagem Celular Tumoral , Humanos , Neoplasias Induzidas por Radiação/etiologia , Neoplasias Cutâneas/etiologia , Raios Ultravioleta
13.
Cancer Lett ; 370(1): 19-26, 2016 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-26476415

RESUMO

Previously, we demonstrated the association between autophagy and gossypol-induced growth inhibition of mutant BRAF melanoma cells. Here, we investigate the role of autophagy in ATG5 knockout cell lines generated by the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas-mediated genome editing. The MTT assay revealed that the inhibitory effect of gossypol was weaker on ATG5 knockout cells than that on the wild type (WT) cells. The conversion of non-autophagic LC3-I to autophagic LC3-II and RT-PCR confirmed the functional gene knockout. However, Cyto-ID autophagy assay revealed that gossypol induced ATG5- and LC3-independent autophagy in ATG5 knockout cells. Moreover, gossypol acts as an autophagy inducer in ATG5 knockout cells while blocking the later stages of the autophagy process in WT cells, which was determined by measuring autophagic flux after co-treatment of gossypol with chloroquine (late-stage autophagy inhibitor). On the other hand, inhibition of autophagy with 3-MA or Beclin-1 siRNA caused a partial increase in the sensitivity to gossypol in ATG5 knockout cells, but not in the WT cells. Together, our findings suggest that the resistance to gossypol in ATG5 knockout cells is associated with increased cytoprotective autophagy, independent of ATG5.


Assuntos
Autofagia/fisiologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/fisiologia , Citoproteção , Endonucleases/fisiologia , Gossipol/farmacologia , Proteínas Associadas aos Microtúbulos/fisiologia , Proteína 5 Relacionada à Autofagia , Linhagem Celular Tumoral , Cloroquina/farmacologia , Humanos
14.
Artigo em Inglês | MEDLINE | ID: mdl-26511629

RESUMO

The study of homologous recombination has its historical roots in meiosis. In this context, recombination occurs as a programmed event that culminates in the formation of crossovers, which are essential for accurate chromosome segregation and create new combinations of parental alleles. Thus, meiotic recombination underlies both the independent assortment of parental chromosomes and genetic linkage. This review highlights the features of meiotic recombination that distinguish it from recombinational repair in somatic cells, and how the molecular processes of meiotic recombination are embedded and interdependent with the chromosome structures that characterize meiotic prophase. A more in-depth review presents our understanding of how crossover and noncrossover pathways of meiotic recombination are differentiated and regulated. The final section of this review summarizes the studies that have defined defective recombination as a leading cause of pregnancy loss and congenital disease in humans.


Assuntos
Meiose , Modelos Genéticos , Recombinação Genética , Cromossomos/metabolismo , Cromossomos/fisiologia , Troca Genética , DNA/biossíntese , Quebras de DNA de Cadeia Dupla , Endonucleases/fisiologia , Feminino , Humanos , Idade Materna , Reprodução/genética
15.
Hum Gene Ther ; 26(7): 425-31, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26068112

RESUMO

Monomeric clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated 9 (Cas9) nucleases have been widely adopted for simple and robust targeted genome editing but also have the potential to induce high-frequency off-target mutations. In principle, two orthogonal strategies for reducing off-target cleavage, truncated guide RNAs (tru-gRNAs) and dimerization-dependent RNA-guided FokI-dCas9 nucleases (RFNs), could be combined as tru-RFNs to further improve genome editing specificity. Here we identify a robust tru-RFN architecture that shows high activity in human cancer cell lines and embryonic stem cells. Additionally, we demonstrate that tru-gRNAs reduce the undesirable mutagenic effects of monomeric FokI-dCas9. Tru-RFNs combine the advantages of two orthogonal strategies for improving the specificity of CRISPR-Cas nucleases and therefore provide a highly specific platform for performing genome editing.


Assuntos
Proteínas de Bactérias/fisiologia , Sistemas CRISPR-Cas , Endodesoxirribonucleases/fisiologia , Endonucleases/fisiologia , Engenharia Genética/métodos , RNA Guia/fisiologia , DNA Metiltransferases Sítio Específica (Adenina-Específica)/fisiologia , Proteína 9 Associada à CRISPR , Linhagem Celular Tumoral , Células-Tronco Embrionárias/fisiologia , Humanos
16.
J Biomol Screen ; 20(7): 829-41, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-25877151

RESUMO

Genomic DNA is constantly exposed to endogenous and exogenous damaging agents. To overcome these damaging effects and maintain genomic stability, cells have robust coping mechanisms in place, including repair of the damaged DNA. There are a number of DNA repair pathways available to cells dependent on the type of damage induced. The removal of damaged DNA is essential to allow successful repair. Removal of DNA strands is achieved by nucleases. Exonucleases are those that progressively cut from DNA ends, and endonucleases make single incisions within strands of DNA. This review focuses on the group of endonucleases involved in DNA repair pathways, their mechanistic functions, roles in cancer development, and how targeting these enzymes is proving to be an exciting new strategy for personalized therapy in cancer.


Assuntos
Reparo do DNA/fisiologia , Endonucleases/fisiologia , Biomarcadores , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Endonucleases/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Humanos , Terapia de Alvo Molecular , Neoplasias/diagnóstico , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Prognóstico
17.
Wiley Interdiscip Rev RNA ; 6(3): 337-49, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25755220

RESUMO

All organisms encode transfer RNAs (tRNAs) that are synthesized as precursor molecules bearing extra sequences at their 5' and 3' ends; some tRNAs also contain introns, which are removed by splicing. Despite commonality in what the ultimate goal is (i.e., producing a mature tRNA), mechanistically, tRNA splicing differs between Bacteria and Archaea or Eukarya. The number and position of tRNA introns varies between organisms and even between different tRNAs within the same organism, suggesting a degree of plasticity in both the evolution and persistence of modern tRNA splicing systems. Here we will review recent findings that not only highlight nuances in splicing pathways but also provide potential reasons for the maintenance of introns in tRNA. Recently, connections between defects in the components of the tRNA splicing machinery and medically relevant phenotypes in humans have been reported. These differences will be discussed in terms of the importance of splicing for tRNA function and in a broader context on how tRNA splicing defects can often have unpredictable consequences.


Assuntos
Modelos Genéticos , Processamento de RNA , RNA de Transferência/química , Animais , Archaea/genética , Bactérias/genética , Endonucleases/fisiologia , Íntrons/fisiologia , Fosfotransferases/fisiologia , RNA de Transferência/metabolismo , RNA de Transferência/fisiologia , Vertebrados/genética
18.
Nucleic Acids Res ; 43(7): 3626-42, 2015 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-25765656

RESUMO

A variety of DNA lesions, secondary DNA structures or topological stress within the DNA template may lead to stalling of the replication fork. Recovery of such forks is essential for the maintenance of genomic stability. The structure-specific endonuclease Mus81-Mms4 has been implicated in processing DNA intermediates that arise from collapsed forks and homologous recombination. According to previous genetic studies, the Srs2 helicase may play a role in the repair of double-strand breaks and ssDNA gaps together with Mus81-Mms4. In this study, we show that the Srs2 and Mus81-Mms4 proteins physically interact in vitro and in vivo and we map the interaction domains within the Srs2 and Mus81 proteins. Further, we show that Srs2 plays a dual role in the stimulation of the Mus81-Mms4 nuclease activity on a variety of DNA substrates. First, Srs2 directly stimulates Mus81-Mms4 nuclease activity independent of its helicase activity. Second, Srs2 removes Rad51 from DNA to allow access of Mus81-Mms4 to cleave DNA. Concomitantly, Mus81-Mms4 inhibits the helicase activity of Srs2. Taken together, our data point to a coordinated role of Mus81-Mms4 and Srs2 in processing of recombination as well as replication intermediates.


Assuntos
DNA Helicases/fisiologia , Proteínas de Ligação a DNA/fisiologia , Endonucleases/fisiologia , Endonucleases Flap/fisiologia , Recombinação Genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Sequência de Bases , Primers do DNA , Microscopia de Fluorescência , Reação em Cadeia da Polimerase , Técnicas do Sistema de Duplo-Híbrido
19.
Mol Cell ; 57(4): 583-594, 2015 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-25699707

RESUMO

The topoisomerase III (Top3)-Rmi1 heterodimer, which catalyzes DNA single-strand passage, forms a conserved complex with the Bloom's helicase (BLM, Sgs1 in budding yeast). This complex has been proposed to regulate recombination by disassembling double Holliday junctions in a process called dissolution. Top3-Rmi1 has been suggested to act at the end of this process, resolving hemicatenanes produced by earlier BLM/Sgs1 activity. We show here that, to the contrary, Top3-Rmi1 acts in all meiotic recombination functions previously associated with Sgs1, most notably as an early recombination intermediate chaperone, promoting regulated crossover and noncrossover recombination and preventing aberrant recombination intermediate accumulation. In addition, we show that Top3-Rmi1 has important Sgs1-independent functions that ensure complete recombination intermediate resolution and chromosome segregation. These findings indicate that Top3-Rmi1 activity is important throughout recombination to resolve strand crossings that would otherwise impede progression through both early steps of pathway choice and late steps of intermediate resolution.


Assuntos
DNA Topoisomerases Tipo I/fisiologia , Proteínas de Ligação a DNA/fisiologia , Recombinação Homóloga/fisiologia , Meiose/genética , Modelos Genéticos , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Segregação de Cromossomos , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Endonucleases/fisiologia , Endonucleases Flap/metabolismo , Endonucleases Flap/fisiologia , Resolvases de Junção Holliday/metabolismo , Resolvases de Junção Holliday/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo
20.
Cancer Invest ; 33(4): 89-97, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25723812

RESUMO

Increased expression of excision repair cross-complementing 1 (ERCC1) in mCRC patients could be related to their response to Oxaliplatin based chemotherapy. We evaluated ERCC1 mRNA expression levels in primary bowel and liver metastases of 51 patients, and correlated with pathologic parameters and clinical outcomes. A significant negative correlation was detected between primary tumor ERCC1 and both the extent of clear surgical margins (P = 0.0011) and the percent of liver metastasis necrosis (P = 0.0167). No relationship was observed between ERCC1 expression and survival. Further study is needed to assess the promising role of ERCC1 expression as a predictive marker benefiting subgroups for Oxaliplatin.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Neoplasias Colorretais/patologia , Proteínas de Ligação a DNA/fisiologia , Endonucleases/fisiologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/secundário , Terapia Neoadjuvante , Compostos Organoplatínicos/administração & dosagem , Adulto , Idoso , Idoso de 80 Anos ou mais , Proteínas de Ligação a DNA/análise , Endonucleases/análise , Feminino , Humanos , Fígado/patologia , Neoplasias Hepáticas/mortalidade , Masculino , Pessoa de Meia-Idade , Oxaliplatina
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