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1.
Cell ; 187(4): 945-961.e18, 2024 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-38320550

RESUMO

DNA double-strand breaks (DSBs) are repaired at DSB sites. How DSB sites assemble and how broken DNA is prevented from separating is not understood. Here we uncover that the synapsis of broken DNA is mediated by the DSB sensor protein poly(ADP-ribose) (PAR) polymerase 1 (PARP1). Using bottom-up biochemistry, we reconstitute functional DSB sites and show that DSB sites form through co-condensation of PARP1 multimers with DNA. The co-condensates exert mechanical forces to keep DNA ends together and become enzymatically active for PAR synthesis. PARylation promotes release of PARP1 from DNA ends and the recruitment of effectors, such as Fused in Sarcoma, which stabilizes broken DNA ends against separation, revealing a finely orchestrated order of events that primes broken DNA for repair. We provide a comprehensive model for the hierarchical assembly of DSB condensates to explain DNA end synapsis and the recruitment of effector proteins for DNA damage repair.


Assuntos
Reparo do DNA , Poli(ADP-Ribose) Polimerase-1 , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Humanos
2.
Elife ; 92020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32396063

RESUMO

Loop extrusion by structural maintenance of chromosomes (SMC) complexes has been proposed as a mechanism to organize chromatin in interphase and metaphase. However, the requirements for chromatin organization in these cell cycle phases are different, and it is unknown whether loop extrusion dynamics and the complexes that extrude DNA also differ. Here, we used Xenopus egg extracts to reconstitute and image loop extrusion of single DNA molecules during the cell cycle. We show that loops form in both metaphase and interphase, but with distinct dynamic properties. Condensin extrudes DNA loops non-symmetrically in metaphase, whereas cohesin extrudes loops symmetrically in interphase. Our data show that loop extrusion is a general mechanism underlying DNA organization, with dynamic and structural properties that are biochemically regulated during the cell cycle.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/química , DNA/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Interfase , Metáfase , Conformação de Ácido Nucleico , Xenopus laevis , Coesinas
3.
Proc Natl Acad Sci U S A ; 109(46): 18827-32, 2012 Nov 13.
Artigo em Inglês | MEDLINE | ID: mdl-23112173

RESUMO

Vitamin D signaling regulates cell proliferation and differentiation, and epidemiological data suggest that it functions as a cancer chemopreventive agent, although the underlying mechanisms are poorly understood. Vitamin D signaling can suppress expression of genes regulated by c-MYC, a transcription factor that controls epidermal differentiation and cell proliferation and whose activity is frequently elevated in cancer. We show through cell- and animal-based studies and mathematical modeling that hormonal 1,25-dihydroxyvitamin D (1,25D) and the vitamin D receptor (VDR) profoundly alter, through multiple mechanisms, the balance in function of c-MYC and its antagonist the transcriptional repressor MAD1/MXD1. 1,25D inhibited transcription of c-MYC-regulated genes in vitro, and topical 1,25D suppressed expression of c-MYC and its target setd8 in mouse skin, whereas MXD1 levels increased. 1,25D inhibited MYC gene expression and accelerated its protein turnover. In contrast, it enhanced MXD1 expression and stability, dramatically altering ratios of DNA-bound c-MYC and MXD1. Remarkably, F-box protein FBW7, an E3-ubiquitin ligase, controlled stability of both arms of the c-MYC/MXD1 push-pull network, and FBW7 ablation attenuated 1,25D regulation of c-MYC and MXD1 turnover. Additionally, c-MYC expression increased upon VDR knockdown, an effect abrogated by ablation of MYC regulator ß-catenin. c-MYC levels were widely elevated in vdr(-/-) mice, including in intestinal epithelium, where hyperproliferation has been reported, and in skin epithelia, where phenotypes of VDR-deficient mice and those overexpressing epidermal c-MYC are similar. Thus, 1,25D and the VDR regulate the c-MYC/MXD1 network to suppress c-MYC function, providing a molecular basis for cancer preventive actions of vitamin D.


Assuntos
Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Calcitriol/metabolismo , Regulação da Expressão Gênica/fisiologia , Proteínas Proto-Oncogênicas c-myc/biossíntese , Receptores de Calcitriol/metabolismo , Proteínas Repressoras/metabolismo , Transdução de Sinais/fisiologia , Transcrição Gênica/fisiologia , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Calcitriol/farmacologia , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Proteína 7 com Repetições F-Box-WD , Regulação da Expressão Gênica/efeitos dos fármacos , Histona-Lisina N-Metiltransferase/genética , Histona-Lisina N-Metiltransferase/metabolismo , Mucosa Intestinal/metabolismo , Camundongos , Camundongos Knockout , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/prevenção & controle , Estabilidade Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/genética , Receptores de Calcitriol/genética , Proteínas Repressoras/genética , Transdução de Sinais/efeitos dos fármacos , Pele/metabolismo , Transcrição Gênica/efeitos dos fármacos , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
4.
Chaos ; 22(3): 033140, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23020479

RESUMO

Chaotic rhythms in deterministic models can arise as a consequence of changes in model parameters. We carried out experimental studies in which we induced a variety of complex rhythms in aggregates of embryonic chick cardiac cells using E-4031 (1.0-2.5 µM), a drug that blocks the hERG potassium channel. Following the addition of the drug, the regular rhythm evolved to display a spectrum of complex dynamics: irregular rhythms, bursting oscillations, doublets, and accelerated rhythms. The interbeat intervals of the irregular rhythms can be described by one-dimensional return maps consistent with chaotic dynamics. A Hodgkin-Huxley-style cardiac ionic model captured the different types of complex dynamics following blockage of the hERG mediated potassium current.


Assuntos
Ventrículos do Coração/citologia , Dinâmica não Linear , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/metabolismo , Função Ventricular/efeitos dos fármacos , Animais , Agregação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Embrião de Galinha , Ventrículos do Coração/efeitos dos fármacos , Espaço Intracelular/fisiologia , Potenciais da Membrana/efeitos dos fármacos , Modelos Cardiovasculares , Piperidinas/farmacologia , Piridinas/farmacologia
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