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1.
Nat Commun ; 11(1): 3907, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32764578

RESUMO

Nucleic acids can fold into G-quadruplex (G4) structures that can fine-tune biological processes. Proteins are required to recognize G4 structures and coordinate their function. Here we identify Zuo1 as a novel G4-binding protein in vitro and in vivo. In vivo in the absence of Zuo1 fewer G4 structures form, cell growth slows and cells become UV sensitive. Subsequent experiments reveal that these cellular changes are due to reduced levels of G4 structures. Zuo1 function at G4 structures results in the recruitment of nucleotide excision repair (NER) factors, which has a positive effect on genome stability. Cells lacking functional NER, as well as Zuo1, accumulate G4 structures, which become accessible to translesion synthesis. Our results suggest a model in which Zuo1 supports NER function and regulates the choice of the DNA repair pathway nearby G4 structures.


Assuntos
Reparo do DNA/fisiologia , Quadruplex G , Chaperonas Moleculares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Sítios de Ligação/genética , Dano ao DNA , Reparo do DNA/genética , DNA Fúngico/química , DNA Fúngico/genética , DNA Fúngico/metabolismo , Deleção de Genes , Aptidão Genética , Genoma Fúngico , Instabilidade Genômica , Modelos Biológicos , Chaperonas Moleculares/genética , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética
2.
Nat Commun ; 11(1): 4124, 2020 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-32807787

RESUMO

In response to DNA damage, a synthetic lethal relationship exists between the cell cycle checkpoint kinase MK2 and the tumor suppressor p53. Here, we describe the concept of augmented synthetic lethality (ASL): depletion of a third gene product enhances a pre-existing synthetic lethal combination. We show that loss of the DNA repair protein XPA markedly augments the synthetic lethality between MK2 and p53, enhancing anti-tumor responses alone and in combination with cisplatin chemotherapy. Delivery of siRNA-peptide nanoplexes co-targeting MK2 and XPA to pre-existing p53-deficient tumors in a highly aggressive, immunocompetent mouse model of lung adenocarcinoma improves long-term survival and cisplatin response beyond those of the synthetic lethal p53 mutant/MK2 combination alone. These findings establish a mechanism for co-targeting DNA damage-induced cell cycle checkpoints in combination with repair of cisplatin-DNA lesions in vivo using RNAi nanocarriers, and motivate further exploration of ASL as a generalized strategy to improve cancer treatment.


Assuntos
Pontos de Checagem do Ciclo Celular/fisiologia , Reparo do DNA/fisiologia , Animais , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular Tumoral , Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo do DNA/genética , Células HCT116 , Humanos , Immunoblotting , Camundongos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Nanomedicina/métodos , Interferência de RNA , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo
3.
Nat Commun ; 11(1): 3181, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32576832

RESUMO

The DNA damage checkpoint (DDC) is often robustly activated during the homologous recombination (HR) repair of DNA double strand breaks (DSBs). DDC activation controls several HR repair factors by phosphorylation, preventing premature segregation of entangled chromosomes formed during HR repair. The DDC mediator 53BP1/Rad9 limits the nucleolytic processing (resection) of a DSB, controlling the formation of the 3' single-stranded DNA (ssDNA) filament needed for recombination, from yeast to human. Here we show that Rad9 promotes stable annealing between the recombinogenic filament and the donor template in yeast, limiting strand rejection by the Sgs1 and Mph1 helicases. This regulation allows repair by long tract gene conversion, crossover recombination and break-induced replication (BIR), only after DDC activation. These findings shed light on how cells couple DDC with the choice and effectiveness of HR sub-pathways, with implications for genome instability and cancer.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Reparo do DNA/genética , Reparo do DNA/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Proteínas de Ciclo Celular/genética , Sobrevivência Celular , RNA Helicases DEAD-box/metabolismo , Quebras de DNA de Cadeia Dupla , DNA de Cadeia Simples , Proteínas de Ligação a DNA/metabolismo , Conversão Gênica , Instabilidade Genômica , Recombinação Homóloga , Humanos , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , RecQ Helicases/metabolismo , Reparo de DNA por Recombinação , Proteínas de Saccharomyces cerevisiae/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética
4.
Nat Commun ; 11(1): 2950, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32528002

RESUMO

During homologous recombination, Rad51 forms a nucleoprotein filament on single-stranded DNA to promote DNA strand exchange. This filament binds to double-stranded DNA (dsDNA), searches for homology, and promotes transfer of the complementary strand, producing a new heteroduplex. Strand exchange proceeds via two distinct three-strand intermediates, C1 and C2. C1 contains the intact donor dsDNA whereas C2 contains newly formed heteroduplex DNA. Here, we show that the conserved DNA binding motifs, loop 1 (L1) and loop 2 (L2) in site I of Rad51, play distinct roles in this process. L1 is involved in formation of the C1 complex whereas L2 mediates the C1-C2 transition, producing the heteroduplex. Another DNA binding motif, site II, serves as the DNA entry position for initial Rad51 filament formation, as well as for donor dsDNA incorporation. Our study provides a comprehensive molecular model for the catalytic process of strand exchange mediated by eukaryotic RecA-family recombinases.


Assuntos
DNA/metabolismo , Rad51 Recombinase/química , Rad51 Recombinase/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação/genética , DNA/genética , Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo do DNA/genética , Reparo do DNA/fisiologia , DNA de Cadeia Simples/genética , Recombinação Homóloga/genética , Recombinação Homóloga/fisiologia , Humanos , Mutação/genética , Ácidos Nucleicos Heteroduplexes/genética , Ácidos Nucleicos Heteroduplexes/metabolismo , Estrutura Secundária de Proteína , Rad51 Recombinase/genética , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética
5.
Mol Cell ; 78(6): 1070-1085, 2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32459988

RESUMO

Anti-cancer drugs targeting the DNA damage response (DDR) exploit genetic or functional defects in this pathway through synthetic lethal mechanisms. For example, defects in homologous recombination (HR) repair arise in cancer cells through inherited or acquired mutations in BRCA1, BRCA2, or other genes in the Fanconi anemia/BRCA pathway, and these tumors have been shown to be particularly sensitive to inhibitors of the base excision repair (BER) protein poly (ADP-ribose) polymerase (PARP). Recent work has identified additional genomic and functional assays of DNA repair that provide new predictive and pharmacodynamic biomarkers for these targeted therapies. Here, we examine the development of selective agents targeting DNA repair, including PARP inhibitors; inhibitors of the DNA damage kinases ataxia-telangiectasia and Rad3 related (ATR), CHK1, WEE1, and ataxia-telangiectasia mutated (ATM); and inhibitors of classical non-homologous end joining (cNHEJ) and alternative end joining (Alt EJ). We also review the biomarkers that guide the use of these agents and current clinical trials with these therapies.


Assuntos
Reparo do DNA/efeitos dos fármacos , Reparo do DNA/fisiologia , Neoplasias/tratamento farmacológico , Animais , Antineoplásicos/uso terapêutico , Biomarcadores Farmacológicos , Dano ao DNA/efeitos dos fármacos , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , Reparo do DNA/genética , Genes BRCA1/efeitos dos fármacos , Recombinação Homóloga , Humanos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo
6.
Nat Commun ; 11(1): 2147, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358495

RESUMO

Upon genotoxic stress, PCNA ubiquitination allows for replication of damaged DNA by recruiting lesion-bypass DNA polymerases. However, PCNA is also ubiquitinated during normal S-phase progression. By employing 293T and RPE1 cells deficient in PCNA ubiquitination, generated through CRISPR/Cas9 gene editing, here, we show that this modification promotes cellular proliferation and suppression of genomic instability under normal growth conditions. Loss of PCNA-ubiquitination results in DNA2-dependent but MRE11-independent nucleolytic degradation of nascent DNA at stalled replication forks. This degradation is linked to defective gap-filling in the wake of the replication fork and incomplete Okazaki fragment maturation, which interferes with efficient PCNA unloading by ATAD5 and subsequent nucleosome deposition by CAF-1. Moreover, concomitant loss of PCNA-ubiquitination and the BRCA pathway results in increased nascent DNA degradation and PARP inhibitor sensitivity. In conclusion, we show that by ensuring efficient Okazaki fragment maturation, PCNA-ubiquitination protects fork integrity and promotes the resistance of BRCA-deficient cells to PARP-inhibitors.


Assuntos
Antígeno Nuclear de Célula em Proliferação/metabolismo , Linhagem Celular Tumoral , Montagem e Desmontagem da Cromatina/genética , Montagem e Desmontagem da Cromatina/fisiologia , Ensaio Cometa , DNA/genética , Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo do DNA/genética , Reparo do DNA/fisiologia , Replicação do DNA/genética , Replicação do DNA/fisiologia , Imunofluorescência , Instabilidade Genômica/genética , Instabilidade Genômica/fisiologia , Células HEK293 , Células HeLa , Humanos , Antígeno Nuclear de Célula em Proliferação/genética , Ligação Proteica , Ubiquitinação/genética , Ubiquitinação/fisiologia
7.
Nat Commun ; 11(1): 2169, 2020 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-32358516

RESUMO

Cells possess an armamentarium of DNA repair pathways to counter DNA damage and prevent mutation. Here we use C. elegans whole genome sequencing to systematically quantify the contributions of these factors to mutational signatures. We analyse 2,717 genomes from wild-type and 53 DNA repair defective backgrounds, exposed to 11 genotoxins, including UV-B and ionizing radiation, alkylating compounds, aristolochic acid, aflatoxin B1, and cisplatin. Combined genotoxic exposure and DNA repair deficiency alters mutation rates or signatures in 41% of experiments, revealing how different DNA alterations induced by the same genotoxin are mended by separate repair pathways. Error-prone translesion synthesis causes the majority of genotoxin-induced base substitutions, but averts larger deletions. Nucleotide excision repair prevents up to 99% of point mutations, almost uniformly across the mutation spectrum. Our data show that mutational signatures are joint products of DNA damage and repair and suggest that multiple factors underlie signatures observed in cancer genomes.


Assuntos
Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Animais , Caenorhabditis elegans/genética , Dano ao DNA/genética , Reparo do DNA/genética , Genômica/métodos , Humanos , Mutação/genética , Mutação Puntual/genética
8.
Proc Natl Acad Sci U S A ; 117(21): 11513-11522, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32381741

RESUMO

Female fertility and offspring health are critically dependent on an adequate supply of high-quality oocytes, the majority of which are maintained in the ovaries in a unique state of meiotic prophase arrest. While mechanisms of DNA repair during meiotic recombination are well characterized, the same is not true for prophase-arrested oocytes. Here we show that prophase-arrested oocytes rapidly respond to γ-irradiation-induced DNA double-strand breaks by activating Ataxia Telangiectasia Mutated, phosphorylating histone H2AX, and localizing RAD51 to the sites of DNA damage. Despite mobilizing the DNA repair response, even very low levels of DNA damage result in the apoptosis of prophase-arrested oocytes. However, we show that, when apoptosis is inhibited, severe DNA damage is corrected via homologous recombination repair. The repair is sufficient to support fertility and maintain health and genetic fidelity in offspring. Thus, despite the preferential induction of apoptosis following exogenously induced genotoxic stress, prophase-arrested oocytes are highly capable of functionally efficient DNA repair. These data implicate DNA repair as a key quality control mechanism in the female germ line and a critical determinant of fertility and genetic integrity.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA/fisiologia , Fertilidade/fisiologia , Oócitos/fisiologia , Animais , Apoptose/fisiologia , Feminino , Masculino , Camundongos Endogâmicos C57BL , Prófase/fisiologia
9.
Proc Natl Acad Sci U S A ; 117(21): 11409-11420, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32404420

RESUMO

Formation of G-quadruplex (G4) DNA structures in key regulatory regions in the genome has emerged as a secondary structure-based epigenetic mechanism for regulating multiple biological processes including transcription, replication, and telomere maintenance. G4 formation (folding), stabilization, and unfolding must be regulated to coordinate G4-mediated biological functions; however, how cells regulate the spatiotemporal formation of G4 structures in the genome is largely unknown. Here, we demonstrate that endogenous oxidized guanine bases in G4 sequences and the subsequent activation of the base excision repair (BER) pathway drive the spatiotemporal formation of G4 structures in the genome. Genome-wide mapping of occurrence of Apurinic/apyrimidinic (AP) site damage, binding of BER proteins, and G4 structures revealed that oxidized base-derived AP site damage and binding of OGG1 and APE1 are predominant in G4 sequences. Loss of APE1 abrogated G4 structure formation in cells, which suggests an essential role of APE1 in regulating the formation of G4 structures in the genome. Binding of APE1 to G4 sequences promotes G4 folding, and acetylation of APE1, which enhances its residence time, stabilizes G4 structures in cells. APE1 subsequently facilitates transcription factor loading to the promoter, providing mechanistic insight into the role of APE1 in G4-mediated gene expression. Our study unravels a role of endogenous oxidized DNA bases and APE1 in controlling the formation of higher-order DNA secondary structures to regulate transcription beyond its well-established role in safeguarding the genomic integrity.


Assuntos
Dano ao DNA , Reparo do DNA/fisiologia , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Quadruplex G , Células A549 , Acetilação , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Expressão Gênica , Genes myc , Genoma Humano , Guanina/química , Guanina/metabolismo , Células HCT116 , Humanos , Oxirredução , Estresse Oxidativo/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas p21(ras)/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
10.
Proc Natl Acad Sci U S A ; 117(17): 9338-9348, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32284409

RESUMO

Oxidation of guanine generates several types of DNA lesions, such as 8-oxoguanine (8OG), 5-guanidinohydantoin (Gh), and spiroiminodihydantoin (Sp). These guanine-derived oxidative DNA lesions interfere with both replication and transcription. However, the molecular mechanism of transcription processing of Gh and Sp remains unknown. In this study, by combining biochemical and structural analysis, we revealed distinct transcriptional processing of these chemically related oxidized lesions: 8OG allows both error-free and error-prone bypass, whereas Gh or Sp causes strong stalling and only allows slow error-prone incorporation of purines. Our structural studies provide snapshots of how polymerase II (Pol II) is stalled by a nonbulky Gh lesion in a stepwise manner, including the initial lesion encounter, ATP binding, ATP incorporation, jammed translocation, and arrested states. We show that while Gh can form hydrogen bonds with adenosine monophosphate (AMP) during incorporation, this base pair hydrogen bonding is not sufficient to hold an ATP substrate in the addition site and is not stable during Pol II translocation after the chemistry step. Intriguingly, we reveal a unique structural reconfiguration of the Gh lesion in which the hydantoin ring rotates ∼90° and is perpendicular to the upstream base pair planes. The perpendicular hydantoin ring of Gh is stabilized by noncanonical lone pair-π and CH-π interactions, as well as hydrogen bonds. As a result, the Gh lesion, as a functional mimic of a 1,2-intrastrand crosslink, occupies canonical -1 and +1 template positions and compromises the loading of the downstream template base. Furthermore, we suggest Gh and Sp lesions are potential targets of transcription-coupled repair.


Assuntos
Guanidinas/química , Guanosina/análogos & derivados , Hidantoínas/química , RNA Polimerase II/metabolismo , Compostos de Espiro/química , Pareamento de Bases , DNA/química , DNA/metabolismo , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Guanidinas/metabolismo , Guanina/metabolismo , Guanosina/química , Guanosina/metabolismo , Hidantoínas/metabolismo , Oxirredução , Estresse Oxidativo/fisiologia , Purinas/metabolismo , RNA Polimerase II/fisiologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Compostos de Espiro/metabolismo , Transcrição Genética/fisiologia , Ativação Transcricional/fisiologia
11.
Proc Natl Acad Sci U S A ; 117(17): 9318-9328, 2020 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-32273391

RESUMO

Alkylation of guanine bases in DNA is detrimental to cells due to its high mutagenic and cytotoxic potential and is repaired by the alkyltransferase AGT. Additionally, alkyltransferase-like proteins (ATLs), which are structurally similar to AGTs, have been identified in many organisms. While ATLs are per se catalytically inactive, strong evidence has suggested that ATLs target alkyl lesions to the nucleotide excision repair system (NER). Using a combination of single-molecule and ensemble approaches, we show here recruitment of UvrA, the initiating enzyme of prokaryotic NER, to an alkyl lesion by ATL. We further characterize lesion recognition by ATL and directly visualize DNA lesion search by highly motile ATL and ATL-UvrA complexes on DNA at the molecular level. Based on the high similarity of ATLs and the DNA-interacting domain of AGTs, our results provide important insight in the lesion search mechanism, not only by ATL but also by AGT, thus opening opportunities for controlling the action of AGT for therapeutic benefit during chemotherapy.


Assuntos
Adenosina Trifosfatases/metabolismo , Alquil e Aril Transferases/metabolismo , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Escherichia coli/metabolismo , Alquil e Aril Transferases/genética , Alquil e Aril Transferases/fisiologia , Alquilação/fisiologia , DNA/metabolismo , Dano ao DNA , Escherichia coli/metabolismo , Proteínas de Escherichia coli/fisiologia , Guanina/metabolismo , Microscopia de Força Atômica/métodos , Mutagênese , O(6)-Metilguanina-DNA Metiltransferase/genética , Pinças Ópticas
12.
Nat Commun ; 11(1): 1478, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32198374

RESUMO

The Escherichia coli transcription-repair coupling factor Mfd displaces stalled RNA polymerase and delivers the stall site to the nucleotide excision repair factors UvrAB for damage detection. Whether this handoff from RNA polymerase to UvrA occurs via the Mfd-UvrA2-UvrB complex or alternate reaction intermediates in cells remains unclear. Here, we visualise Mfd in actively growing cells and determine the catalytic requirements for faithful recruitment of nucleotide excision repair proteins. We find that ATP hydrolysis by UvrA governs formation and disassembly of the Mfd-UvrA2 complex. Further, Mfd-UvrA2-UvrB complexes formed by UvrB mutants deficient in DNA loading and damage recognition are impaired in successful handoff. Our single-molecule dissection of interactions of Mfd with its partner proteins inside live cells shows that the dissociation of Mfd is tightly coupled to successful loading of UvrB, providing a mechanism via which loading of UvrB occurs in a strand-specific manner.


Assuntos
Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA/fisiologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Imagem Individual de Molécula/métodos , Fatores de Transcrição/metabolismo , Adenosina Trifosfatases , Proteínas de Bactérias , ATPases Bacterianas Próton-Translocadoras , DNA Helicases/genética , DNA Helicases/metabolismo , Proteínas de Ligação a DNA , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Modelos Moleculares , Complexos Multienzimáticos/metabolismo , Conformação Proteica , Dedos de Zinco/genética , Dedos de Zinco/fisiologia
13.
Nat Commun ; 11(1): 1477, 2020 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-32198385

RESUMO

In the model organism Escherichia coli, helix distorting lesions are recognized by the UvrAB damage surveillance complex in the global genomic nucleotide excision repair pathway (GGR). Alternately, during transcription-coupled repair (TCR), UvrA is recruited to Mfd at sites of RNA polymerases stalled by lesions. Ultimately, damage recognition is mediated by UvrA, followed by verification by UvrB. Here we characterize the differences in the kinetics of interactions of UvrA with Mfd and UvrB by following functional, fluorescently tagged UvrA molecules in live TCR-deficient or wild-type cells. The lifetimes of UvrA in Mfd-dependent or Mfd-independent interactions in the absence of exogenous DNA damage are comparable in live cells, and are governed by UvrB. Upon UV irradiation, the lifetimes of UvrA strongly depended on, and matched those of Mfd. Overall, we illustrate a non-perturbative, imaging-based approach to quantify the kinetic signatures of damage recognition enzymes participating in multiple pathways in cells.


Assuntos
Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , DNA Bacteriano/metabolismo , Escherichia coli/metabolismo , Imagem Óptica/métodos , Células Procarióticas/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Biofísica , Dano ao DNA/efeitos da radiação , DNA Helicases/genética , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA , DNA Bacteriano/efeitos da radiação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Cinética , Fatores de Transcrição/metabolismo , Raios Ultravioleta
14.
Nat Commun ; 11(1): 1356, 2020 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-32170071

RESUMO

Nucleotide excision repair (NER) removes a wide range of DNA lesions, including UV-induced photoproducts and bulky base adducts. XPA is an essential protein in eukaryotic NER, although reports about its stoichiometry and role in damage recognition are controversial. Here, by PeakForce Tapping atomic force microscopy, we show that human XPA binds and bends DNA by ∼60° as a monomer. Furthermore, we observe XPA specificity for the helix-distorting base adduct N-(2'-deoxyguanosin-8-yl)-2-acetylaminofluorene over non-damaged dsDNA. Moreover, single molecule fluorescence microscopy reveals that DNA-bound XPA exhibits multiple modes of linear diffusion between paused phases. The presence of DNA damage increases the frequency of pausing. Truncated XPA, lacking the intrinsically disordered N- and C-termini, loses specificity for DNA lesions and shows less pausing on damaged DNA. Our data are consistent with a working model in which monomeric XPA bends DNA, displays episodic phases of linear diffusion along DNA, and pauses in response to DNA damage.


Assuntos
DNA/química , DNA/metabolismo , Imagem Individual de Molécula/métodos , Proteína de Xeroderma Pigmentoso Grupo A/química , Proteína de Xeroderma Pigmentoso Grupo A/metabolismo , Biofísica/métodos , Adutos de DNA/química , Adutos de DNA/metabolismo , Dano ao DNA/fisiologia , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Humanos , Microscopia de Força Atômica , Ligação Proteica , Raios Ultravioleta
15.
Nat Commun ; 11(1): 786, 2020 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-32034146

RESUMO

The XPF-ERCC1 heterodimer is a structure-specific endonuclease that is essential for nucleotide excision repair (NER) and interstrand crosslink (ICL) repair in mammalian cells. However, whether and how XPF binding to ERCC1 is regulated has not yet been established. Here, we show that TIP60, also known as KAT5, a haplo-insufficient tumor suppressor, directly acetylates XPF at Lys911 following UV irradiation or treatment with mitomycin C and that this acetylation is required for XPF-ERCC1 complex assembly and subsequent activation. Mechanistically, acetylation of XPF at Lys911 disrupts the Glu907-Lys911 salt bridge, thereby leading to exposure of a previously unidentified second binding site for ERCC1. Accordingly, loss of XPF acetylation impairs the damage-induced XPF-ERCC1 interaction, resulting in defects in both NER and ICL repair. Our results not only reveal a mechanism that regulates XPF-ERCC1 complex assembly and activation, but also provide important insight into the role of TIP60 in the maintenance of genome stability.


Assuntos
Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Lisina Acetiltransferase 5/metabolismo , Acetilação , Sítios de Ligação , Dano ao DNA , Reparo do DNA/efeitos dos fármacos , Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA/genética , Endonucleases/genética , Células HEK293 , Células HeLa , Humanos , Lisina/metabolismo , Lisina Acetiltransferase 5/genética , Mitomicina/farmacologia , Complexos Multiproteicos , Sirtuína 1/metabolismo , Raios Ultravioleta
16.
Oxid Med Cell Longev ; 2020: 5367102, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32104534

RESUMO

Treatments on neoplastic diseases and cancer using genotoxic drugs often cause long-term health problems related to premature aging. The underlying mechanism is poorly understood. Based on the study of a long-lasting senescence-like growth arrest (10-12 weeks) of human dermal fibroblasts induced by psoralen plus UVA (PUVA) treatment, we here revealed that slowly repaired bulky DNA damages can serve as a "molecular scar" leading to reduced cell proliferation through persistent endogenous production of reactive oxygen species (ROS) that caused accelerated telomere erosion. The elevated levels of ROS were the results of mitochondrial dysfunction and the activation of NADPH oxidase (NOX). A combined inhibition of DNA-PK and PARP1 could suppress the level of ROS. Together with a reduced expression level of BRCA1 as well as the upregulation of PP2A and 53BP1, these data suggest that the NHEJ repair of DNA double-strand breaks may be the initial trigger of metabolic changes leading to ROS production. Further study showed that stimulation of the pentose phosphate pathway played an important role for NOX activation, and ROS could be efficiently suppressed by modulating the NADP/NADPH ratio. Interestingly, feeding cells with ribose-5-phosphate, a precursor for nucleotide biosynthesis that produced through the PPP, could evidently suppress the ROS level and prevent the cell enlargement related to mitochondrial biogenesis. Taken together, these results revealed an important signaling pathway between DNA damage repair and the cell metabolism, which contributed to the premature aging effects of PUVA, and may be generally applicable for a large category of chemotherapeutic reagents including many cancer drugs.


Assuntos
Senescência Celular/fisiologia , Dano ao DNA/fisiologia , Estresse Oxidativo/fisiologia , Células Cultivadas , Senescência Celular/genética , Dano ao DNA/genética , Reparo do DNA/genética , Reparo do DNA/fisiologia , Humanos , NADP/genética , NADP/metabolismo , Oxirredução , Estresse Oxidativo/genética , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ribosemonofosfatos/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
17.
Gene ; 730: 144323, 2020 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-31917230

RESUMO

The subjection of DNA to numerous lethal damages is threatening for the stability and integrity of the whole body genome. DNA damage response (DDR) is a critical phosphorylation-based signaling pathway developed for the maintaining of the genome against these threatens. Recent studies showed that various targets of DDR are involved in the activation of autophagy, as one of the important effectors of this signaling. The interplay between DDR and autophagy may have a critical role in the pathogenesis of various malignancies such as colorectal cancer, which can be a basement for the designing novel therapeutic strategies for combating this cancer type. On the other hand, autophagy is also demonstrated to be contributed to the regulation of DDR components. Therefore, in this review article, we will discuss the crosstalk between DDR and autophagy and their exact function in the pathogenesis of various human cancer types, with special attention on colorectal cancer.


Assuntos
Autofagia/genética , Neoplasias Colorretais/metabolismo , Reparo do DNA/fisiologia , Neoplasias do Colo/genética , Neoplasias Colorretais/genética , Dano ao DNA/fisiologia , Instabilidade Genômica/genética , Humanos , Transdução de Sinais/genética
18.
J Cancer Res Clin Oncol ; 146(2): 343-356, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31932908

RESUMO

PURPOSE: We set out to determine whether clinically tested epigenetic drugs against class I histone deacetylases (HDACs) affect hallmarks of the metastatic process. METHODS: We treated permanent and primary renal, lung, and breast cancer cells with the class I histone deacetylase inhibitors (HDACi) entinostat (MS-275) and valproic acid (VPA), the replicative stress inducer hydroxyurea (HU), the DNA-damaging agent cis-platinum (L-OHP), and the cytokine transforming growth factor-ß (TGFß). We used proteomics, quantitative PCR, immunoblot, single cell DNA damage assays, and flow cytometry to analyze cell fate after drug exposure. RESULTS: We show that HDACi interfere with DNA repair protein expression and trigger DNA damage and apoptosis alone and in combination with established chemotherapeutics. Furthermore, HDACi disrupt the balance of cell adhesion protein expression and abrogate TGFß-induced cellular plasticity of transformed cells. CONCLUSION: HDACi suppress the epithelial-mesenchymal transition (EMT) and compromise the DNA integrity of cancer cells. These data encourage further testing of HDACi against tumor cells.


Assuntos
Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Neoplasias/tratamento farmacológico , Animais , Benzamidas/farmacologia , Plasticidade Celular/efeitos dos fármacos , Cisplatino/farmacologia , Enzimas Reparadoras do DNA/metabolismo , Resistencia a Medicamentos Antineoplásicos , Humanos , Hidroxiureia/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Metástase Neoplásica , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Piridinas/farmacologia , Fator de Crescimento Transformador beta/farmacologia , Ácido Valproico/farmacologia
19.
FASEB J ; 34(2): 2812-2820, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31908056

RESUMO

The Mre11A/RAD50/NBN complex (MRN) is an essential regulator of the cellular damage response after DNA double-strand breaks (DSBs). More recent work has indicated that MRN may also impact on the duration of mitosis. We show here that RAD50-deficient fibroblasts exhibit a marked delay in mitotic progression that can be rescued by lentiviral transduction of RAD50. The delay was observed throughout all mitotic phases in live cell imaging using GFP-labeled H2B as a fluorescent marker. In complementation assays with RAD50 phosphorylation mutants, modifications at Ser635 had little effect on mitotic progression. By contrast with RAD50, fibroblast strains deficient in ATM or NBN did not show a significant slowing of mitotic progression. Ataxia-telangiectasia-like disorder (ATLD) fibroblasts with nuclease-deficient MRE11A (p.W210C) tended to show slower mitosis, though by far not as significant as RAD50-deficient cells. Inhibitor studies indicated that ATM kinase activity might not grossly impact on mitotic progression, while treatment with MRE11A inhibitor PFM39 modestly prolonged mitosis. Inhibition of ATR kinase significantly prolonged mitosis but this effect was mostly independent of RAD50 status. Taken together, our data unravel a mitotic role of RAD50 that can be separated from its known functions in DNA repair.


Assuntos
Hidrolases Anidrido Ácido/metabolismo , Reparo do DNA/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteína Homóloga a MRE11/genética , Mitose , Ataxia Telangiectasia/genética , Quebras de DNA de Cadeia Dupla , Humanos
20.
Int J Mol Sci ; 21(2)2020 Jan 10.
Artigo em Inglês | MEDLINE | ID: mdl-31936707

RESUMO

Cells are constantly suffering genotoxic stresses that affect the integrity of our genetic material. Genotoxic insults must be repaired to avoid the loss or inappropriate transmission of the genetic information, a situation that could lead to the appearance of developmental abnormalities and tumorigenesis. To combat this threat, eukaryotic cells have evolved a set of sophisticated molecular mechanisms that are collectively known as the DNA damage response (DDR). This surveillance system controls several aspects of the cellular response, including the detection of lesions, a temporary cell cycle arrest, and the repair of the broken DNA. While the regulation of the DDR by numerous kinases has been well documented over the last decade, the complex roles of protein dephosphorylation have only recently begun to be investigated. Here, we review recent progress in the characterization of DDR-related protein phosphatases during the response to a DNA lesion, focusing mainly on their ability to modulate the DNA damage checkpoint and the repair of the damaged DNA. We also discuss their protein composition and structure, target specificity, and biochemical regulation along the different stages encompassed in the DDR. The compilation of this information will allow us to better comprehend the physiological significance of protein dephosphorylation in the maintenance of genome integrity and cell viability in response to genotoxic stress.


Assuntos
Ciclo Celular/genética , Reparo do DNA/fisiologia , Fosfoproteínas Fosfatases/metabolismo , Animais , Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA , Humanos , Proteínas Nucleares , Proteína Fosfatase 1 , Proteína Fosfatase 2 , Proteína Fosfatase 2C , Proteínas Tirosina Fosfatases
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