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1.
Cells ; 12(22)2023 11 11.
Artigo em Inglês | MEDLINE | ID: mdl-37998342

RESUMO

Duplication of the genome requires the replication apparatus to overcome a variety of impediments, including covalent DNA adducts, the most challenging of which is on the leading template strand. Replisomes consist of two functional units, a helicase to unwind DNA and polymerases to synthesize it. The helicase is a multi-protein complex that encircles the leading template strand and makes the first contact with a leading strand adduct. The size of the channel in the helicase would appear to preclude transit by large adducts such as DNA: protein complexes (DPC). Here we discuss some of the extensively studied pathways that support replication restart after replisome encounters with leading template strand adducts. We also call attention to recent work that highlights the tolerance of the helicase for adducts ostensibly too large to pass through the central channel.


Assuntos
DNA Helicases , Replicação do DNA , DNA Helicases/metabolismo , DNA/metabolismo
2.
DNA Repair (Amst) ; 128: 103525, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37320956

RESUMO

Immunofluorescence imaging is a standard experimental tool for monitoring the response of cellular factors to DNA damage. Visualizing the recruitment of DNA Damage Response (DDR) components requires high affinity antibodies, which are generally available. In contrast, reagents for the display of the lesions that induce the response are far more limited. Consequently, DDR factor accumulation often serves as a surrogate for damage, without reporting the actual inducing structure. This limitation has practical implications given the importance of the response to DNA reactive drugs such as those used in cancer therapy. These include interstrand crosslink (ICL) forming compounds which are frequently employed clinically. Among them are the psoralens, natural products that form ICLs upon photoactivation and applied therapeutically since antiquity. However, despite multiple attempts, antibodies against psoralen ICLs have not been developed. To overcome this limitation, we developed a psoralen tagged with an antigen for which there are commercial antibodies. In this report we describe our application of the tagged psoralen in imaging experiments, and the unexpected discoveries they revealed.


Assuntos
Reparo do DNA , Ficusina , Ficusina/farmacologia , Reagentes de Ligações Cruzadas/farmacologia , Dano ao DNA , DNA
3.
Methods Enzymol ; 661: 53-75, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34776223

RESUMO

Replication forks encounter numerous challenges as they move through eu- and hetero-chromatin during S phase in mammalian cells. These include a variety of impediments to the unwinding of DNA by the replicative helicase such as alternate DNA structures, transcription complexes and R-loops, DNA-protein complexes, and DNA chemical adducts. Much of our knowledge of these events is based on analysis of markers of the replication stress and DNA Damage Response that follow stalling of replisomes. To examine consequences for the replisomes more directly, we developed an approach for imaging collisions of replication forks with the potent block presented by an interstrand crosslink (ICL). The strategy is based on the visualization on DNA fibers of the encounter of replication tracts and an antigen tagged ICL. Our studies revealed an unexpected restart of DNA synthesis past an intact ICL. In addition, and also unexpected, we found two distinct versions of the replisome, one biased toward euchromatin and the other more prominent in heterochromatin. Here, we present details of our experimental procedures that led to these observations.


Assuntos
DNA Helicases , Replicação do DNA , Animais , DNA/química , Dano ao DNA , DNA Helicases/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Mamíferos/genética
4.
Front Cell Dev Biol ; 9: 729265, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34532320

RESUMO

Replisomes follow a schedule in which replication of DNA in euchromatin is early in S phase while sequences in heterochromatin replicate late. Impediments to DNA replication, referred to as replication stress, can stall replication forks triggering activation of the ATR kinase and downstream pathways. While there is substantial literature on the local consequences of replisome stalling-double strand breaks, reversed forks, or genomic rearrangements-there is limited understanding of the determinants of replisome stalling vs. continued progression. Although many proteins are recruited to stalled replisomes, current models assume a single species of "stressed" replisome, independent of genomic location. Here we describe our approach to visualizing replication fork encounters with the potent block imposed by a DNA interstrand crosslink (ICL) and our discovery of an unexpected pathway of replication restart (traverse) past an intact ICL. Additionally, we found two biochemically distinct replisomes distinguished by activity in different stages of S phase and chromatin environment. Each contains different proteins that contribute to ICL traverse.

5.
J Vis Exp ; (173)2021 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-34398140

RESUMO

Considerable insight is present into the cellular response to double strand breaks (DSBs), induced by nucleases, radiation, and other DNA breakers. In part, this reflects the availability of methods for the identification of break sites, and characterization of factors recruited to DSBs at those sequences. However, DSBs also appear as intermediates during the processing of DNA adducts formed by compounds that do not directly cause breaks, and do not react at specific sequence sites. Consequently, for most of these agents, technologies that permit the analysis of binding interactions with response factors and repair proteins are unknown. For example, DNA interstrand crosslinks (ICLs) can provoke breaks following replication fork encounters. Although formed by drugs widely used as cancer chemotherapeutics, there has been no methodology for monitoring their interactions with replication proteins. Here, we describe our strategy for following the cellular response to fork collisions with these challenging adducts. We linked a steroid antigen to psoralen, which forms photoactivation dependent ICLs in nuclei of living cells. The ICLs were visualized by immunofluorescence against the antigen tag. The tag can also be a partner in the Proximity Ligation Assay (PLA) which reports the close association of two antigens. The PLA was exploited to distinguish proteins that were closely associated with the tagged ICLs from those that were not. It was possible to define replisome proteins that were retained after encounters with ICLs and identify others that were lost. This approach is applicable to any structure or DNA adduct that can be detected immunologically.


Assuntos
Dano ao DNA , Reparo do DNA , Reagentes de Ligações Cruzadas , Adutos de DNA , Replicação do DNA , Ficusina
6.
Nucleic Acids Res ; 48(16): 9161-9180, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32797166

RESUMO

FANCJ, a DNA helicase and interacting partner of the tumor suppressor BRCA1, is crucial for the repair of DNA interstrand crosslinks (ICL), a highly toxic lesion that leads to chromosomal instability and perturbs normal transcription. In diploid cells, FANCJ is believed to operate in homologous recombination (HR) repair of DNA double-strand breaks (DSB); however, its precise role and molecular mechanism is poorly understood. Moreover, compensatory mechanisms of ICL resistance when FANCJ is deficient have not been explored. In this work, we conducted a siRNA screen to identify genes of the DNA damage response/DNA repair regime that when acutely depleted sensitize FANCJ CRISPR knockout cells to a low concentration of the DNA cross-linking agent mitomycin C (MMC). One of the top hits from the screen was RAP80, a protein that recruits repair machinery to broken DNA ends and regulates DNA end-processing. Concomitant loss of FANCJ and RAP80 not only accentuates DNA damage levels in human cells but also adversely affects the cell cycle checkpoint, resulting in profound chromosomal instability. Genetic complementation experiments demonstrated that both FANCJ's catalytic activity and interaction with BRCA1 are important for ICL resistance when RAP80 is deficient. The elevated RPA and RAD51 foci in cells co-deficient of FANCJ and RAP80 exposed to MMC are attributed to single-stranded DNA created by Mre11 and CtIP nucleases. Altogether, our cell-based findings together with biochemical studies suggest a critical function of FANCJ to suppress incompletely processed and toxic joint DNA molecules during repair of ICL-induced DNA damage.


Assuntos
Proteína BRCA1/genética , Proteínas de Ligação a DNA/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Instabilidade Genômica/genética , Chaperonas de Histonas/genética , RNA Helicases/genética , Rad51 Recombinase/genética , Instabilidade Cromossômica/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Dano ao DNA/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/deficiência , Técnicas de Inativação de Genes , Células HeLa , Chaperonas de Histonas/deficiência , Humanos , Mitomicina/farmacologia , Reparo de DNA por Recombinação/genética
7.
Nat Commun ; 11(1): 3951, 2020 08 07.
Artigo em Inglês | MEDLINE | ID: mdl-32769987

RESUMO

Duplication of mammalian genomes requires replisomes to overcome numerous impediments during passage through open (eu) and condensed (hetero) chromatin. Typically, studies of replication stress characterize mixed populations of challenged and unchallenged replication forks, averaged across S phase, and model a single species of "stressed" replisome. Here, in cells containing potent obstacles to replication, we find two different lesion proximal replisomes. One is bound by the DONSON protein and is more frequent in early S phase, in regions marked by euchromatin. The other interacts with the FANCM DNA translocase, is more prominent in late S phase, and favors heterochromatin. The two forms can also be detected in unstressed cells. ChIP-seq of DNA associated with DONSON or FANCM confirms the bias of the former towards regions that replicate early and the skew of the latter towards regions that replicate late.


Assuntos
Proteínas de Ciclo Celular/metabolismo , DNA Helicases/metabolismo , Período de Replicação do DNA , Eucromatina/metabolismo , Heterocromatina/metabolismo , Proteínas Nucleares/metabolismo , Sequenciamento de Cromatina por Imunoprecipitação , Células HeLa , Humanos , Fase S
8.
Mol Cell ; 75(3): 605-619.e6, 2019 08 08.
Artigo em Inglês | MEDLINE | ID: mdl-31255466

RESUMO

Accurate DNA replication is essential to preserve genomic integrity and prevent chromosomal instability-associated diseases including cancer. Key to this process is the cells' ability to stabilize and restart stalled replication forks. Here, we show that the EXD2 nuclease is essential to this process. EXD2 recruitment to stressed forks suppresses their degradation by restraining excessive fork regression. Accordingly, EXD2 deficiency leads to fork collapse, hypersensitivity to replication inhibitors, and genomic instability. Impeding fork regression by inactivation of SMARCAL1 or removal of RECQ1's inhibition in EXD2-/- cells restores efficient fork restart and genome stability. Moreover, purified EXD2 efficiently processes substrates mimicking regressed forks. Thus, this work identifies a mechanism underpinned by EXD2's nuclease activity, by which cells balance fork regression with fork restoration to maintain genome stability. Interestingly, from a clinical perspective, we discover that EXD2's depletion is synthetic lethal with mutations in BRCA1/2, implying a non-redundant role in replication fork protection.


Assuntos
DNA Helicases/genética , Replicação do DNA/genética , Exodesoxirribonucleases/genética , RecQ Helicases/genética , Proteína BRCA1/genética , Proteína BRCA2/genética , Instabilidade Genômica/genética , Células HeLa , Humanos , Neoplasias/genética , Mutações Sintéticas Letais/genética
9.
Clin Cancer Res ; 25(18): 5584-5594, 2019 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-31196855

RESUMO

PURPOSE: Somatic inactivating mutations in ARID1A, a component of the SWI/SNF chromatin remodeling complex, are detected in various types of human malignancies. Loss of ARID1A compromises DNA damage repair. The induced DNA damage burden may increase reliance on PARP-dependent DNA repair of cancer cells to maintain genome integrity and render susceptibility to PARP inhibitor therapy.Experimental Design: Isogenic ARID1A-/- and wild-type cell lines were used for assessing DNA damage response, DNA compactness, and profiling global serine/threonine phosphoproteomic in vivo. A panel of inhibitors targeting DNA repair pathways was screened for a synergistic antitumor effect with irradiation in ARID1A-/- tumors. RESULTS: ARID1A-deficient endometrial cells exhibit sustained levels in DNA damage response, a result further supported by in vivo phosphoproteomic analysis. Our results show that ARID1A is essential for establishing an open chromatin state upon DNA damage, a process required for recruitment of 53BP1 and RIF1, key mediators of non-homologous end-joining (NHEJ) machinery, to DNA lesions. The inability of ARID1A-/- cells to mount NHEJ repair results in a partial cytotoxic response to radiation. Small-molecule compound screens revealed that PARP inhibitors act synergistically with radiation to potentiate cytotoxicity in ARID1A-/- cells. Combination treatment with low-dose radiation and olaparib greatly improved antitumor efficacy, resulting in long-term remission in mice bearing ARID1A-deficient tumors. CONCLUSIONS: ARID1A-deficient cells acquire high sensitivity to PARP inhibition after exposure to exogenously induced DNA breaks such as ionizing radiation. Our findings suggest a novel biologically informed strategy for treating ARID1A-deficient malignancies.


Assuntos
Proteínas de Ligação a DNA/deficiência , Resistencia a Medicamentos Antineoplásicos/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Tolerância a Radiação/genética , Fatores de Transcrição/deficiência , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Reparo do DNA por Junção de Extremidades , Reparo do DNA , Modelos Animais de Doenças , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Transgênicos , Modelos Biológicos
10.
Cell Rep ; 27(6): 1794-1808.e5, 2019 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-31067464

RESUMO

Eukaryotic replisomes are driven by the mini chromosome maintenance (MCM [M]) helicase complex, an offset ring locked around the template for leading strand synthesis by CDC45 (C) and GINS (G) proteins. Although the CDC45 MCM GINS (CMG) structure implies that interstrand crosslinks (ICLs) are absolute blocks to replisomes, recent studies indicate that cells can restart DNA synthesis on the side of the ICL distal to the initial encounter. Here, we report that restart requires ATR and is promoted by FANCD2 and phosphorylated FANCM. Following introduction of genomic ICLs and dependent on ATR and FANCD2 but not on the Fanconi anemia core proteins or FAAP24, FANCM binds the replisome complex, with concomitant release of the GINS proteins. In situ analysis of replisomes proximal to ICLs confirms the ATR-dependent release of GINS proteins while CDC45 is retained on the remodeled replisome. The results demonstrate the plasticity of CMG composition in response to replication stress.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , DNA Helicases/metabolismo , DNA Polimerase Dirigida por DNA , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Complexos Multienzimáticos , Animais , Galinhas , Replicação do DNA , Epistasia Genética , Feminino , Células HeLa , Humanos , Masculino , Camundongos , Complexos Multiproteicos/metabolismo , Fosforilação , Ligação Proteica
11.
DNA Repair (Amst) ; 71: 183-189, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30166246

RESUMO

Repair pathways of covalent DNA damage are understood in considerable detail due to decades of brilliant biochemical studies by many investigators. An important feature of these experiments is the defined adduct location on oligonucleotide or plasmid substrates that are incubated with purified proteins or cell free extracts. With some exceptions, this certainty is lost when the inquiry shifts to the response of living mammalian cells to the same adducts in genomic DNA. This reflects the limitation of assays, such as those based on immunofluorescence, that are widely used to follow responding proteins in cells exposed to a DNA reactive compound. The lack of effective reagents for adduct detection means that the proximity between responding proteins and an adduct must be assumed. Since these assumptions can be incorrect, models based on in vitro systems may fail to account for observations made in vivo. Here we discuss the use of a detection tag to address the problem of lesion location, as illustrated by our recent work on replication dependent and independent responses to interstrand crosslinks.


Assuntos
Adutos de DNA/metabolismo , Reparo do DNA , Replicação do DNA , Imuno-Histoquímica/métodos , Testes de Mutagenicidade/métodos , Reagentes de Ligações Cruzadas/farmacologia , Reagentes de Ligações Cruzadas/toxicidade , DNA/efeitos dos fármacos , Humanos
12.
Nucleic Acids Res ; 46(12): 6238-6256, 2018 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-29788478

RESUMO

Fanconi Anemia (FA) is characterized by bone marrow failure, congenital abnormalities, and cancer. Of over 20 FA-linked genes, FANCJ uniquely encodes a DNA helicase and mutations are also associated with breast and ovarian cancer. fancj-/- cells are sensitive to DNA interstrand cross-linking (ICL) and replication fork stalling drugs. We delineated the molecular defects of two FA patient-derived FANCJ helicase domain mutations. FANCJ-R707C was compromised in dimerization and helicase processivity, whereas DNA unwinding by FANCJ-H396D was barely detectable. DNA binding and ATP hydrolysis was defective for both FANCJ-R707C and FANCJ-H396D, the latter showing greater reduction. Expression of FANCJ-R707C or FANCJ-H396D in fancj-/- cells failed to rescue cisplatin or mitomycin sensitivity. Live-cell imaging demonstrated a significantly compromised recruitment of FANCJ-R707C to laser-induced DNA damage. However, FANCJ-R707C expressed in fancj-/- cells conferred resistance to the DNA polymerase inhibitor aphidicolin, G-quadruplex ligand telomestatin, or DNA strand-breaker bleomycin, whereas FANCJ-H396D failed. Thus, a minimal threshold of FANCJ catalytic activity is required to overcome replication stress induced by aphidicolin or telomestatin, or to repair bleomycin-induced DNA breakage. These findings have implications for therapeutic strategies relying on DNA cross-link sensitivity or heightened replication stress characteristic of cancer cells.


Assuntos
Quebras de DNA de Cadeia Dupla , DNA Helicases/genética , DNA Helicases/metabolismo , Reparo do DNA , Replicação do DNA , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , RNA Helicases/genética , RNA Helicases/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Animais , Afidicolina/toxicidade , Linhagem Celular , Quinase 1 do Ponto de Checagem/metabolismo , Galinhas , Cisplatino/toxicidade , DNA de Cadeia Simples , Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/química , Quadruplex G , Mutação de Sentido Incorreto , Oxazóis/toxicidade , RNA Helicases/química , Rad51 Recombinase/análise , Recombinases/genética , Recombinases/metabolismo , Proteína de Replicação A/metabolismo , Estresse Fisiológico
13.
Cancer Cell ; 31(5): 653-668.e7, 2017 05 08.
Artigo em Inglês | MEDLINE | ID: mdl-28486105

RESUMO

An oncogenic role for CHD4, a NuRD component, is defined for initiating and supporting tumor suppressor gene (TSG) silencing in human colorectal cancer. CHD4 recruits repressive chromatin proteins to sites of DNA damage repair, including DNA methyltransferases where it imposes de novo DNA methylation. At TSGs, CHD4 retention helps maintain DNA hypermethylation-associated transcriptional silencing. CHD4 is recruited by the excision repair protein OGG1 for oxidative damage to interact with the damage-induced base 8-hydroxydeoxyguanosine (8-OHdG), while ZMYND8 recruits it to double-strand breaks. CHD4 knockdown activates silenced TSGs, revealing their role for blunting colorectal cancer cell proliferation, invasion, and metastases. High CHD4 and 8-OHdG levels plus low expression of TSGs strongly correlates with early disease recurrence and decreased overall survival.


Assuntos
Autoantígenos/genética , Neoplasias Colorretais/genética , Metilação de DNA , Repressão Epigenética , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Genes Supressores de Tumor , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/genética , 8-Hidroxi-2'-Desoxiguanosina , Animais , Autoantígenos/metabolismo , Movimento Celular , Proliferação de Células , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/patologia , Neoplasias Colorretais/cirurgia , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Dano ao DNA , DNA Glicosilases/genética , DNA Glicosilases/metabolismo , Desoxiguanosina/análogos & derivados , Desoxiguanosina/metabolismo , Intervalo Livre de Doença , Regulação para Baixo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Células HCT116 , Antígenos de Histocompatibilidade/genética , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Humanos , Estimativa de Kaplan-Meier , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Nus , Metástase Neoplásica , Estresse Oxidativo , Modelos de Riscos Proporcionais , Interferência de RNA , Receptores de Quinase C Ativada , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Fatores de Tempo , Transcrição Gênica , Transfecção , Proteínas Supressoras de Tumor
14.
ACS Chem Biol ; 12(7): 1858-1866, 2017 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-28535027

RESUMO

Human exposure to arsenic in drinking water is known to be associated with the development of bladder, lung, kidney, and skin cancers. The molecular mechanisms underlying the carcinogenic effects of arsenic species remain incompletely understood. DNA interstrand cross-links (ICLs) are among the most cytotoxic type of DNA lesions that block DNA replication and transcription, and these lesions can be induced by endogenous metabolism and by exposure to exogenous agents. Fanconi anemia (FA) is a congenital disorder manifested with elevated sensitivity toward DNA interstrand cross-linking agents, and monoubiquitination of FANCD2 by FANCL is a crucial step in FA-mediated DNA repair. Here, we demonstrated that As3+ could bind to the PHD/RING finger domain of FANCL in vitro and in cells. This binding led to compromised ubiquitination of FANCD2 in cells and diminished recruitment of FANCD2 to chromatin and DNA damage sites induced by 4,5',8-trimethylpsoralen plus UVA irradiation. Furthermore, clonogenic survival assay results showed that arsenite coexposure rendered cells more sensitive toward DNA interstrand cross-linking agents. Together, our study suggested that arsenite may compromise genomic stability via perturbation of the Fanconi anemia pathway, thereby conferring its carcinogenic effect.


Assuntos
Arsenitos/metabolismo , Arsenitos/toxicidade , Reparo do DNA , Proteína do Grupo de Complementação L da Anemia de Fanconi/metabolismo , Instabilidade Genômica/efeitos dos fármacos , Domínios RING Finger , Ubiquitina-Proteína Ligases/química , Arsenitos/química , Sítios de Ligação , Linhagem Celular Tumoral , Dano ao DNA , Proteína do Grupo de Complementação L da Anemia de Fanconi/química , Humanos , Ubiquitinação
15.
J Vis Exp ; (122)2017 04 20.
Artigo em Inglês | MEDLINE | ID: mdl-28448050

RESUMO

The DNA Damage Response (DDR) has been extensively characterized in studies of double strand breaks (DSBs) induced by laser micro beam irradiation in live cells. The DDR to helix distorting covalent DNA modifications, including interstrand DNA crosslinks (ICLs), is not as well defined. We have studied the DDR stimulated by ICLs, localized by laser photoactivation of immunotagged psoralens, in the nuclei of live cells. In order to address fundamental questions about adduct distribution and replication fork encounters, we combined laser localization with two other technologies. DNA fibers are often used to display the progress of replication forks by immunofluorescence of nucleoside analogues incorporated during short pulses. Immunoquantum dots have been widely employed for single molecule imaging. In the new approach, DNA fibers from cells carrying laser localized ICLs are spread onto microscope slides. The tagged ICLs are displayed with immunoquantum dots and the inter-lesion distances determined. Replication fork collisions with ICLs can be visualized and different encounter patterns identified and quantitated.


Assuntos
Adutos de DNA/análise , Furocumarinas/análise , Lasers , Imagem Individual de Molécula/métodos , Linhagem Celular , DNA/química , Adutos de DNA/química , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Imunofluorescência/métodos , Furocumarinas/química , Humanos , Microscopia Confocal , Microscopia de Fluorescência/instrumentação , Microscopia de Fluorescência/métodos , Pontos Quânticos , Imagem Individual de Molécula/instrumentação
16.
Ageing Res Rev ; 33: 67-75, 2017 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-27223997

RESUMO

Fanconi Anemia (FA) is a rare autosomal genetic disorder characterized by progressive bone marrow failure (BMF), endocrine dysfunction, cancer, and other clinical features commonly associated with normal aging. The anemia stems directly from an accelerated decline of the hematopoietic stem cell compartment. Although FA is a complex heterogeneous disease linked to mutations in 19 currently identified genes, there has been much progress in understanding the molecular pathology involved. FA is broadly considered a DNA repair disorder and the FA gene products, together with other DNA repair factors, have been implicated in interstrand cross-link (ICL) repair. However, in addition to the defective DNA damage response, altered epigenetic regulation, and telomere defects, FA is also marked by elevated levels of inflammatory mediators in circulation, a hallmark of faster decline in not only other hereditary aging disorders but also normal aging. In this review, we offer a perspective of FA as a monogenic accelerated aging disorder, citing the latest evidence for its multi-factorial deficiencies underlying its unique clinical and cellular features.


Assuntos
Envelhecimento/fisiologia , Reparo do DNA/fisiologia , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Anemia de Fanconi , Dano ao DNA , Epigênese Genética , Anemia de Fanconi/diagnóstico , Anemia de Fanconi/genética , Anemia de Fanconi/fisiopatologia , Células-Tronco Hematopoéticas/fisiologia , Humanos , Telômero/genética
17.
Cancer Cell ; 30(4): 637-650, 2016 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-27728808

RESUMO

Poly (ADP-ribose) polymerase inhibitors (PARPis) are clinically effective predominantly for BRCA-mutant tumors. We introduce a mechanism-based strategy to enhance PARPi efficacy based on DNA damage-related binding between DNA methyltransferases (DNMTs) and PARP1. In acute myeloid leukemia (AML) and breast cancer cells, DNMT inhibitors (DNMTis) alone covalently bind DNMTs into DNA and increase PARP1 tightly bound into chromatin. Low doses of DNMTis plus PARPis, versus each drug alone, increase PARPi efficacy, increasing amplitude and retention of PARP1 directly at laser-induced DNA damage sites. This correlates with increased DNA damage, synergistic tumor cytotoxicity, blunting of self-renewal, and strong anti-tumor responses, in vivo in unfavorable AML subtypes and BRCA wild-type breast cancer cells. Our combinatorial approach introduces a strategy to enhance efficacy of PARPis in treating cancer.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Metilação de DNA/efeitos dos fármacos , Leucemia Mieloide Aguda/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Cromatina/metabolismo , Quebras de DNA de Cadeia Dupla , Sinergismo Farmacológico , Feminino , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos Nus , Ftalazinas/farmacologia , Poli(ADP-Ribose) Polimerase-1/antagonistas & inibidores , Poli(ADP-Ribose) Polimerase-1/metabolismo , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Front Genet ; 7: 84, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27242893

RESUMO

DNA interstrand crosslinks (ICLs) block unwinding of the double helix, and have always been regarded as major challenges to replication and transcription. Compounds that form these lesions are very toxic and are frequently used in cancer chemotherapy. We have developed two strategies, both based on immunofluorescence (IF), for studying cellular responses to ICLs. The basis of each is psoralen, a photoactive (by long wave ultraviolet light, UVA) DNA crosslinking agent, to which we have linked an antigen tag. In the one approach, we have taken advantage of DNA fiber and immuno-quantum dot technologies for visualizing the encounter of replication forks with ICLs induced by exposure to UVA lamps. In the other, psoralen ICLs are introduced into nuclei in live cells in regions of interest defined by a UVA laser. The antigen tag can be displayed by conventional IF, as can the recruitment and accumulation of DNA damage response proteins to the laser localized ICLs. However, substantial difference between the technologies creates considerable uncertainty as to whether conclusions from one approach are applicable to those of the other. In this report, we have employed the fiber/quantum dot methodology to determine lesion density and spacing on individual DNA molecules carrying laser localized ICLs. We have performed the same measurements on DNA fibers with ICLs induced by exposure of psoralen to UVA lamps. Remarkably, we find little difference in the adduct distribution on fibers prepared from cells exposed to the different treatment protocols. Furthermore, there is considerable similarity in patterns of replication in the vicinity of the ICLs introduced by the two techniques.

19.
Genes Dev ; 30(6): 645-59, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26980189

RESUMO

Deficiency of FANCD2/FANCI-associated nuclease 1 (FAN1) in humans leads to karyomegalic interstitial nephritis (KIN), a rare hereditary kidney disease characterized by chronic renal fibrosis, tubular degeneration, and characteristic polyploid nuclei in multiple tissues. The mechanism of how FAN1 protects cells is largely unknown but is thought to involve FAN1's function in DNA interstrand cross-link (ICL) repair. Here, we describe a Fan1-deficient mouse and show that FAN1 is required for cellular and organismal resistance to ICLs. We show that the ubiquitin-binding zinc finger (UBZ) domain of FAN1, which is needed for interaction with FANCD2, is not required for the initial rapid recruitment of FAN1 to ICLs or for its role in DNA ICL resistance. Epistasis analyses reveal that FAN1 has cross-link repair activities that are independent of the Fanconi anemia proteins and that this activity is redundant with the 5'-3' exonuclease SNM1A. Karyomegaly becomes prominent in kidneys and livers of Fan1-deficient mice with age, and mice develop liver dysfunction. Treatment of Fan1-deficient mice with ICL-inducing agents results in pronounced thymic and bone marrow hypocellularity and the disappearance of c-kit(+) cells. Our results provide insight into the mechanism of FAN1 in ICL repair and demonstrate that the Fan1 mouse model effectively recapitulates the pathological features of human FAN1 deficiency.


Assuntos
Endodesoxirribonucleases/deficiência , Endodesoxirribonucleases/genética , Rim/patologia , Hepatopatias/genética , Animais , Medula Óssea/efeitos dos fármacos , Reagentes de Ligações Cruzadas/farmacologia , Dano ao DNA/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Endodesoxirribonucleases/metabolismo , Endonucleases/metabolismo , Epistasia Genética , Exodesoxirribonucleases/metabolismo , Fígado/patologia , Camundongos , Enzimas Multifuncionais , Estrutura Terciária de Proteína , Transporte Proteico
20.
Genes Dev ; 29(18): 1955-68, 2015 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-26338419

RESUMO

MERIT40 is an essential component of the RAP80 ubiquitin recognition complex that targets BRCA1 to DNA damage sites. Although this complex is required for BRCA1 foci formation, its physiologic role in DNA repair has remained enigmatic, as has its relationship to canonical DNA repair mechanisms. Surprisingly, we found that Merit40(-/-) mice displayed marked hypersensitivity to DNA interstrand cross-links (ICLs) but not whole-body irradiation. MERIT40 was rapidly recruited to ICL lesions prior to FANCD2, and Merit40-null cells exhibited delayed ICL unhooking coupled with reduced end resection and homologous recombination at ICL damage. Interestingly, Merit40 mutation exacerbated ICL-induced chromosome instability in the context of concomitant Brca2 deficiency but not in conjunction with Fancd2 mutation. These findings implicate MERIT40 in the earliest stages of ICL repair and define specific functional interactions between RAP80 complex-dependent ubiquitin recognition and the Fanconi anemia (FA)-BRCA ICL repair network.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína BRCA2/metabolismo , Reparo do DNA/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Instabilidade Cromossômica/genética , Dano ao DNA , DNA Helicases/metabolismo , Proteínas de Ligação a DNA , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/genética , Proteína do Grupo de Complementação D2 da Anemia de Fanconi/metabolismo , Chaperonas de Histonas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mutação , Transporte Proteico , Fatores de Transcrição/metabolismo , Ubiquitinação
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