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Genes (Basel) ; 12(12)2021 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-34946909


Accurate and complete genome replication is a fundamental cellular process for the proper transfer of genetic material to cell progenies, normal cell growth, and genome stability. However, a plethora of extrinsic and intrinsic factors challenge individual DNA replication forks and cause replication stress (RS), a hallmark of cancer. When challenged by RS, cells deploy an extensive range of mechanisms to safeguard replicating genomes and limit the burden of DNA damage. Prominent among those is homologous recombination (HR). Although fundamental to cell division, evidence suggests that cancer cells exploit and manipulate these RS responses to fuel their evolution and gain resistance to therapeutic interventions. In this review, we focused on recent insights into HR-mediated protection of stress-induced DNA replication intermediates, particularly the repair and protection of daughter strand gaps (DSGs) that arise from discontinuous replication across a damaged DNA template. Besides mechanistic underpinnings of this process, which markedly differ depending on the extent and duration of RS, we highlight the pathophysiological scenarios where DSG repair is naturally silenced. Finally, we discuss how such pathophysiological events fuel rampant mutagenesis, promoting cancer evolution, but also manifest in adaptative responses that can be targeted for cancer therapy.

Dano ao DNA , Reparo do DNA , Replicação do DNA , Instabilidade Genômica , Recombinação Homóloga , Animais , Humanos
Dev Cell ; 56(4): 461-477.e7, 2021 02 22.
Artigo em Inglês | MEDLINE | ID: mdl-33621493


Homology-directed repair (HDR) safeguards DNA integrity under various forms of stress, but how HDR protects replicating genomes under extensive metabolic alterations remains unclear. Here, we report that besides stalling replication forks, inhibition of ribonucleotide reductase (RNR) triggers metabolic imbalance manifested by the accumulation of increased reactive oxygen species (ROS) in cell nuclei. This leads to a redox-sensitive activation of the ATM kinase followed by phosphorylation of the MRE11 nuclease, which in HDR-deficient settings degrades stalled replication forks. Intriguingly, nascent DNA degradation by the ROS-ATM-MRE11 cascade is also triggered by hypoxia, which elevates signaling-competent ROS and attenuates functional HDR without arresting replication forks. Under these conditions, MRE11 degrades daughter-strand DNA gaps, which accumulate behind active replisomes and attract error-prone DNA polymerases to escalate mutation rates. Thus, HDR safeguards replicating genomes against metabolic assaults by restraining mutagenic repair at aberrantly processed nascent DNA. These findings have implications for cancer evolution and tumor therapy.

Replicação do DNA , Genoma Humano , Metabolismo , Reparo de DNA por Recombinação , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA2/deficiência , Proteína BRCA2/metabolismo , Hipóxia Celular , Linhagem Celular Tumoral , DNA/metabolismo , Humanos , Proteína Homóloga a MRE11/metabolismo , Modelos Biológicos , Mutação/genética , Neoplasias/genética , Neoplasias/patologia , Polimerização , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais
Biophys J ; 116(8): 1406-1419, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30961891


The repair of DNA double-strand breaks by homologous recombination is of crucial importance for maintaining genomic stability. Two major players during this repair pathway are Rad51 and Rad54. Previously, it was shown that Rad54 exists as a monomer or oligomer when bound to DNA and drives the displacement of Rad51 by translocating along the DNA. Moreover, phosphorylation of Rad54 was reported to stimulate this clearance of Rad51 from DNA. However, it is currently unclear how phosphorylation of Rad54 modulates its molecular-structural function and how it affects the activity of monomeric or oligomeric Rad54 during the removal of Rad51. To examine the impact of Rad54 phosphorylation on a molecular-structural level, we applied molecular dynamics simulations of Rad54 monomers and hexamers in the absence or presence of DNA. Our results suggest that 1) phosphorylation of Rad54 stabilizes the monomeric form by reducing the interlobe movement of Rad54 monomers and might therefore facilitate multimer formation around DNA and 2) phosphorylation of Rad54 in a higher-order hexamer reduces its binding strength to DNA, which is a requirement for efficient mobility on DNA. To further address the relationship between the mobility of Rad54 and its phosphorylation state, we performed fluorescence recovery after photobleaching experiments in living cells, which expressed different versions of the Rad54 protein. Here, we could measure that the phosphomimetic version of Rad54 was highly mobile on DNA, whereas a nonphosphorylatable mutant displayed a mobility defect. Taken together, these data show that the phosphorylation of Rad54 is a critical event in balancing the DNA binding strength and mobility of Rad54 and might therefore provide optimal conditions for DNA translocation and subsequent removal of Rad51 during homologous recombination repair.

DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , DNA/genética , DNA/metabolismo , Movimento , Adenosina Trifosfatases/metabolismo , Animais , DNA Helicases/química , Proteínas de Ligação a DNA/química , Recombinação Homóloga , Humanos , Modelos Moleculares , Fosforilação , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Estrutura Quaternária de Proteína , Rad51 Recombinase/metabolismo , Peixe-Zebra
Nat Cell Biol ; 21(4): 487-497, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30804506


Failure to complete DNA replication is a stochastic by-product of genome doubling in almost every cell cycle. During mitosis, under-replicated DNA (UR-DNA) is converted into DNA lesions, which are inherited by daughter cells and sequestered in 53BP1 nuclear bodies (53BP1-NBs). The fate of such cells remains unknown. Here, we show that the formation of 53BP1-NBs interrupts the chain of iterative damage intrinsically embedded in UR-DNA. Unlike clastogen-induced 53BP1 foci that are repaired throughout interphase, 53BP1-NBs restrain replication of the embedded genomic loci until late S phase, thus enabling the dedicated RAD52-mediated repair of UR-DNA lesions. The absence or malfunction of 53BP1-NBs causes premature replication of the affected loci, accompanied by genotoxic RAD51-mediated recombination. Thus, through adjusting replication timing and repair pathway choice at under-replicated loci, 53BP1-NBs enable the completion of genome duplication of inherited UR-DNA and prevent the conversion of stochastic under-replications into genome instability.

Estruturas do Núcleo Celular/fisiologia , Dano ao DNA , Período de Replicação do DNA , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/fisiologia , Linhagem Celular , Segregação de Cromossomos , Reparo do DNA , Replicação do DNA , Humanos , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Recombinação Genética , Fase S/genética , Proteínas de Ligação a Telômeros/fisiologia
Mol Cell ; 65(4): 671-684.e5, 2017 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-28132842


Canonical non-homologous end joining (c-NHEJ) repairs DNA double-strand breaks (DSBs) in G1 cells with biphasic kinetics. We show that DSBs repaired with slow kinetics, including those localizing to heterochromatic regions or harboring additional lesions at the DSB site, undergo resection prior to repair by c-NHEJ and not alt-NHEJ. Resection-dependent c-NHEJ represents an inducible process during which Plk3 phosphorylates CtIP, mediating its interaction with Brca1 and promoting the initiation of resection. Mre11 exonuclease, EXD2, and Exo1 execute resection, and Artemis endonuclease functions to complete the process. If resection does not commence, then repair can ensue by c-NHEJ, but when executed, Artemis is essential to complete resection-dependent c-NHEJ. Additionally, Mre11 endonuclease activity is dispensable for resection in G1. Thus, resection in G1 differs from the process in G2 that leads to homologous recombination. Resection-dependent c-NHEJ significantly contributes to the formation of deletions and translocations in G1, which represent important initiating events in carcinogenesis.

Núcleo Celular/efeitos da radiação , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/efeitos da radiação , Fase G1/efeitos da radiação , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Núcleo Celular/enzimologia , Núcleo Celular/patologia , Enzimas Reparadoras do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases , Endonucleases , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Fase G2 , Deleção de Genes , Células HeLa , Humanos , Cinética , Proteína Homóloga a MRE11 , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilação , /metabolismo , Fatores de Tempo , Transfecção , Translocação Genética , Proteínas Supressoras de Tumor , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
Mol Cell ; 62(6): 903-917, 2016 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-27264870


Never-in-mitosis A-related kinase 1 (Nek1) has established roles in apoptosis and cell cycle regulation. We show that human Nek1 regulates homologous recombination (HR) by phosphorylating Rad54 at Ser572 in late G2 phase. Nek1 deficiency as well as expression of unphosphorylatable Rad54 (Rad54-S572A) cause unresolved Rad51 foci and confer a defect in HR. Phospho-mimic Rad54 (Rad54-S572E), in contrast, promotes HR and rescues the HR defect associated with Nek1 loss. Although expression of phospho-mimic Rad54 is beneficial for HR, it causes Rad51 removal from chromatin and degradation of stalled replication forks in S phase. Thus, G2-specific phosphorylation of Rad54 by Nek1 promotes Rad51 chromatin removal during HR in G2 phase, and its absence in S phase is required for replication fork stability. In summary, Nek1 regulates Rad51 removal to orchestrate HR and replication fork stability.

Quebras de DNA de Cadeia Dupla , DNA Helicases/metabolismo , Reparo do DNA , Replicação do DNA , Recombinação Homóloga , Quinase 1 Relacionada a NIMA/metabolismo , Proteínas Nucleares/metabolismo , Origem de Replicação , Pontos de Checagem da Fase S do Ciclo Celular , DNA Helicases/genética , Proteínas de Ligação a DNA , Fibroblastos/enzimologia , Pontos de Checagem da Fase G2 do Ciclo Celular , Regulação da Expressão Gênica , Células HEK293 , Células HeLa , Humanos , Mutação , Quinase 1 Relacionada a NIMA/genética , Proteínas Nucleares/genética , Fosforilação , Interferência de RNA , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Serina , Transdução de Sinais , Fatores de Tempo , Transfecção