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1.
Genes (Basel) ; 12(12)2021 12 09.
Artigo em Inglês | MEDLINE | ID: mdl-34946909

RESUMO

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.


Assuntos
Dano ao DNA , Reparo do DNA , Replicação do DNA , Instabilidade Genômica , Recombinação Homóloga , Animais , Humanos
2.
Life (Basel) ; 11(4)2021 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-33917253

RESUMO

The diagnostics of prostate cancer are currently based on three pillars: prostate biomarker panel, imaging techniques, and histological verification. This paper presents a diagnostic algorithm that can serve as a "road map": from initial patient stratification to the final decision regarding treatment. The algorithm is based on a review of the current literature combined with our own experience. Diagnostic algorithms are a feature of an advanced healthcare system in which all steps are consciously coordinated and optimized to ensure the proper individualization of the treatment process. The prostate cancer diagnostic algorithm was created using the prostate specific antigen and in particular the Prostate Health Index in the first line of patient stratification. It then continued on the diagnostic pathway via imaging techniques, biopsy, or active surveillance, and then on to the treatment decision itself. In conclusion, the prostate cancer diagnostic algorithm presented here is a functional tool for initial patient stratification, comprehensive staging, and aggressiveness assessment. Above all, emphasis is placed on the use of the Prostate Health Index (PHI) in the first stratification of the patients as a predictor of aggressiveness and clinical stage of prostrate cancer (PCa). The inclusion of PHI in the algorithm significantly increases the accuracy and speed of the diagnostic procedure and allows to choose the optimal pathway just from the beginning. The use of advanced diagnostic techniques allows us to move towards to a more advanced level of cancer care. This diagnostics algorithm has become a standard of care in our hospital. The algorithm is continuously validated and modified based on our results.

3.
World J Urol ; 39(6): 1889-1895, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32761380

RESUMO

PURPOSE: To compare the ability of Prostate Health Index (PHI) to diagnose csPCa, with that of total PSA, PSA density (PSAD) and the multiparametric magnetic resonance (mpMRI) of the prostate. METHODS: We analysed a group of 395 men planned for a prostate biopsy who underwent a mpMRI of the prostate evaluated using the PIRADS v1 criteria. All patients had their PHI measured before prostate biopsy. In patients with an mpMRI suspicious lesions, an mpMRI/ultrasound software fusion-guided biopsy was performed first, with 12 core systematic biopsy performed in all patients. A ROC analysis was performed for PCa detection for total PSA, PSAD, PIRADS score and PHI; with an AUC curve calculated for all criteria and a combination of PIRADS score and PHI. Subsequent sub-analyses included patients undergoing first and repeat biopsy. RESULTS: The AUC for predicting the presence of csPCa in all patients was 59.5 for total PSA, 69.7 for PHI, 64.9 for PSAD and 62.5 for PIRADS. In biopsy naive patients it was 61.6 for total PSA, 68.9 for PHI, 64.6 for PSAD and 63.1 for PIRADS. In patients with previous negative biopsy the AUC for total PSA, PHI, PSAD and PIRADS was 55.4, 71.2, 64.4 and 69.3, respectively. Adding of PHI to PIRADS increased significantly (p = 0.007) the accuracy for prediction of csPCa. CONCLUSION: Prostate Health Index could serve as a tool in predicting csPCa. When compared to the mpMRI, it shows comparable results. The PHI cannot, however, help us guide prostate biopsies in any way, and its main use may, therefore, be in pre-MRI or pre-biopsy triage.


Assuntos
Imageamento por Ressonância Magnética Multiparamétrica , Neoplasias da Próstata/diagnóstico por imagem , Neoplasias da Próstata/patologia , Idoso , Humanos , Biópsia Guiada por Imagem , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Estudos Retrospectivos
4.
Nature ; 587(7833): 297-302, 2020 11.
Artigo em Inglês | MEDLINE | ID: mdl-33087936

RESUMO

Minichromosome maintenance proteins (MCMs) are DNA-dependent ATPases that bind to replication origins and license them to support a single round of DNA replication. A large excess of MCM2-7 assembles on chromatin in G1 phase as pre-replication complexes (pre-RCs), of which only a fraction become the productive CDC45-MCM-GINS (CMG) helicases that are required for genome duplication1-4. It remains unclear why cells generate this surplus of MCMs, how they manage to sustain it across multiple generations, and why even a mild reduction in the MCM pool compromises the integrity of replicating genomes5,6. Here we show that, for daughter cells to sustain error-free DNA replication, their mother cells build up a nuclear pool of MCMs both by recycling chromatin-bound (parental) MCMs and by synthesizing new (nascent) MCMs. Although all MCMs can form pre-RCs, it is the parental pool that is inherently stable and preferentially matures into CMGs. By contrast, nascent MCM3-7 (but not MCM2) undergo rapid proteolysis in the cytoplasm, and their stabilization and nuclear translocation require interaction with minichromosome-maintenance complex-binding protein (MCMBP), a distant MCM paralogue7,8. By chaperoning nascent MCMs, MCMBP safeguards replicating genomes by increasing chromatin coverage with pre-RCs that do not participate on replication origins but adjust the pace of replisome movement to minimize errors during DNA replication. Consequently, although the paucity of pre-RCs in MCMBP-deficient cells does not alter DNA synthesis overall, it increases the speed and asymmetry of individual replisomes, which leads to DNA damage. The surplus of MCMs therefore increases the robustness of genome duplication by restraining the speed at which eukaryotic cells replicate their DNA. Alterations in physiological fork speed might thus explain why even a minor reduction in MCM levels destabilizes the genome and predisposes to increased incidence of tumour formation.


Assuntos
Replicação do DNA/genética , Genoma Humano/genética , Proteínas de Manutenção de Minicromossomo/biossíntese , Proteínas de Manutenção de Minicromossomo/metabolismo , Transporte Ativo do Núcleo Celular , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Dano ao DNA , Humanos , Proteínas de Manutenção de Minicromossomo/análise , Chaperonas Moleculares/química , Chaperonas Moleculares/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Estabilidade Proteica , Transporte Proteico
5.
BMC Urol ; 20(1): 144, 2020 Sep 07.
Artigo em Inglês | MEDLINE | ID: mdl-32894109

RESUMO

BACKGROUND: We aimed to explore the utility of prostate specific antigen (PSA) isoform [- 2] proPSA and its derivatives for prediction of pathological outcome after radical prostatectomy (RP). METHODS: Preoperative blood samples were prospectively and consecutivelyanalyzed from 472 patients treated with RP for clinically localized prostate cancerat four medical centers. Measured parameters were PSA, free PSA (fPSA), fPSA/PSA ratio, [- 2] proPSA (p2PSA), p2PSA/fPSA ratio and Prostate Health Index (PHI)(p2PSA/fPSA)*√PSA]. Logistic regression models were fitted to determine the accuracy of markers for prediction of pathological Gleason score (GS) ≥7, Gleason score upgrading, extracapsular extension of the tumor (pT3) and the presence of positive surgical margin (PSM). The accuracy of predictive models was compared using area under the receiver operating curve (AUC). RESULTS: Of 472 patients undergoing RP, 339 (72%) were found to have pathologic GS ≥ 7, out of them 178 (53%) experienced an upgrade from their preoperative GS = 6. The findings of pT3 and PSM were present in 132 (28%) and 133 (28%) cases, respectively. At univariable analysis of all the preoperative parameters, PHI was the most accurate predictor of pathological GS ≥7 (OR 1.02, 95% CI 1.01-1.03, p<0.001), GS upgrading (OR 1.02, 95% CI 1.01-1.03, p<0.003), pT3 disease (OR 1.01, 95% CI 1.00-1.02, p<0.007) and the presence of PSM (OR 1.01, 95% CI 1.00-1.02, p<0.002). Adding of PHI into the base multivariable model increased significantly the accuracy for prediction of pathological GS by 4.4% to AUC = 66.6 (p = 0.015) and GS upgrading by 5.0% to AUC = 65.9 (p = 0.025), respectively. CONCLUSIONS: Preoperative PHI levels may contribute significantly to prediction of prostate cancer aggressiveness and expansion of the tumor detected at final pathology.


Assuntos
Prostatectomia , Neoplasias da Próstata/patologia , Neoplasias da Próstata/cirurgia , Idoso , Humanos , Masculino , Pessoa de Meia-Idade , Gradação de Tumores , Valor Preditivo dos Testes , Período Pré-Operatório , Estudos Prospectivos , Antígeno Prostático Específico/sangue , Prostatectomia/métodos , Neoplasias da Próstata/sangue
6.
Nature ; 574(7779): 571-574, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31645724

RESUMO

To safeguard genome integrity in response to DNA double-strand breaks (DSBs), mammalian cells mobilize the neighbouring chromatin to shield DNA ends against excessive resection that could undermine repair fidelity and cause damage to healthy chromosomes1. This form of genome surveillance is orchestrated by 53BP1, whose accumulation at DSBs triggers sequential recruitment of RIF1 and the shieldin-CST-POLα complex2. How this pathway reflects and influences the three-dimensional nuclear architecture is not known. Here we use super-resolution microscopy to show that 53BP1 and RIF1 form an autonomous functional module that stabilizes three-dimensional chromatin topology at sites of DNA breakage. This process is initiated by accumulation of 53BP1 at regions of compact chromatin that colocalize with topologically associating domain (TAD) sequences, followed by recruitment of RIF1 to the boundaries between such domains. The alternating distribution of 53BP1 and RIF1 stabilizes several neighbouring TAD-sized structures at a single DBS site into an ordered, circular arrangement. Depletion of 53BP1 or RIF1 (but not shieldin) disrupts this arrangement and leads to decompaction of DSB-flanking chromatin, reduction in interchromatin space, aberrant spreading of DNA repair proteins, and hyper-resection of DNA ends. Similar topological distortions are triggered by depletion of cohesin, which suggests that the maintenance of chromatin structure after DNA breakage involves basic mechanisms that shape three-dimensional nuclear organization. As topological stabilization of DSB-flanking chromatin is independent of DNA repair, we propose that, besides providing a structural scaffold to protect DNA ends against aberrant processing, 53BP1 and RIF1 safeguard epigenetic integrity at loci that are disrupted by DNA breakage.


Assuntos
Cromatina/genética , Cromatina/metabolismo , Instabilidade Genômica , Conformação de Ácido Nucleico , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Cromatina/química , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteínas de Ligação a Telômeros/deficiência , Proteínas de Ligação a Telômeros/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/deficiência , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
7.
Science ; 358(6364): 797-802, 2017 11 10.
Artigo em Inglês | MEDLINE | ID: mdl-29123070

RESUMO

DNA replication requires coordination between replication fork progression and deoxynucleotide triphosphate (dNTP)-generating metabolic pathways. We find that perturbation of ribonucleotide reductase (RNR) in humans elevates reactive oxygen species (ROS) that are detected by peroxiredoxin 2 (PRDX2). In the oligomeric state, PRDX2 forms a replisome-associated ROS sensor, which binds the fork accelerator TIMELESS when exposed to low levels of ROS. Elevated ROS levels generated by RNR attenuation disrupt oligomerized PRDX2 to smaller subunits, whose dissociation from chromatin enforces the displacement of TIMELESS from the replisome. This process instantly slows replication fork progression, which mitigates pathological consequences of replication stress. Thus, redox signaling couples fluctuations of dNTP biogenesis with replisome activity to reduce stress during genome duplication. We propose that cancer cells exploit this pathway to increase their adaptability to adverse metabolic conditions.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Replicação do DNA , Instabilidade Genômica , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Neoplasias/genética , Peroxirredoxinas/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ribonucleotídeo Redutases/metabolismo , Adaptação Biológica , Cromatina/metabolismo , Desoxirribonucleotídeos/metabolismo , Humanos , Redes e Vias Metabólicas , Oxirredução , Transdução de Sinais
8.
Mol Cell ; 66(5): 658-671.e8, 2017 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-28575661

RESUMO

The MUS81-EME1 endonuclease cleaves late replication intermediates at common fragile sites (CFSs) during early mitosis to trigger DNA-repair synthesis that ensures faithful chromosome segregation. Here, we show that these DNA transactions are promoted by RECQ5 DNA helicase in a manner dependent on its Ser727 phosphorylation by CDK1. Upon replication stress, RECQ5 associates with CFSs in early mitosis through its physical interaction with MUS81 and promotes MUS81-dependent mitotic DNA synthesis. RECQ5 depletion or mutational inactivation of its ATP-binding site, RAD51-interacting domain, or phosphorylation site causes excessive binding of RAD51 to CFS loci and impairs CFS expression. This leads to defective chromosome segregation and accumulation of CFS-associated DNA damage in G1 cells. Biochemically, RECQ5 alleviates the inhibitory effect of RAD51 on 3'-flap DNA cleavage by MUS81-EME1 through its RAD51 filament disruption activity. These data suggest that RECQ5 removes RAD51 filaments stabilizing stalled replication forks at CFSs and hence facilitates CFS cleavage by MUS81-EME1.


Assuntos
Sítios Frágeis do Cromossomo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , DNA/biossíntese , Endonucleases/metabolismo , Mitose , RecQ Helicases/metabolismo , Origem de Replicação , Sítios de Ligação , Proteína Quinase CDC2 , Instabilidade Cromossômica , Segregação de Cromossomos , Quinases Ciclina-Dependentes/metabolismo , DNA/genética , Dano ao DNA , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/metabolismo , Endonucleases/genética , Células HEK293 , Células HeLa , Humanos , Fosforilação , Ligação Proteica , Interferência de RNA , Rad51 Recombinase/metabolismo , RecQ Helicases/genética , Fatores de Tempo , Transfecção
9.
Oncotarget ; 6(38): 40464-79, 2015 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-26588054

RESUMO

DNA replication is a highly coordinated process that is initiated at multiple replication origins in eukaryotes. These origins are bound by the origin recognition complex (ORC), which subsequently recruits the Mcm2-7 replicative helicase in a Cdt1/Cdc6-dependent manner. In budding yeast, two essential replication factors, Sld2 and Mcm10, are then important for the activation of replication origins. In humans, the putative Sld2 homolog, RECQ4, interacts with MCM10. Here, we have identified two mutants of human RECQ4 that are deficient in binding to MCM10. We show that these RECQ4 variants are able to complement the lethality of an avian cell RECQ4 deletion mutant, indicating that the essential function of RECQ4 in vertebrates is unlikely to require binding to MCM10. Nevertheless, we show that the RECQ4-MCM10 interaction is important for efficient replication origin firing.


Assuntos
Neoplasias Ósseas/genética , Replicação do DNA , Proteínas de Manutenção de Minicromossomo/metabolismo , Osteossarcoma/genética , RecQ Helicases/metabolismo , Origem de Replicação/genética , Sequência de Aminoácidos , Animais , Apoptose , Western Blotting , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Proliferação de Células , Galinhas/genética , Cromatina/genética , Citometria de Fluxo , Humanos , Técnicas Imunoenzimáticas , Imunoprecipitação , Componente 2 do Complexo de Manutenção de Minicromossomo/genética , Componente 2 do Complexo de Manutenção de Minicromossomo/metabolismo , Componente 7 do Complexo de Manutenção de Minicromossomo/genética , Componente 7 do Complexo de Manutenção de Minicromossomo/metabolismo , Proteínas de Manutenção de Minicromossomo/genética , Dados de Sequência Molecular , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Domínios e Motivos de Interação entre Proteínas , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , RecQ Helicases/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Ressonância de Plasmônio de Superfície , Células Tumorais Cultivadas
10.
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
11.
DNA Repair (Amst) ; 30: 80-9, 2015 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-25769792

RESUMO

The RECQ4 protein belongs to the RecQ helicase family, which plays crucial roles in genome maintenance. Mutations in the RECQ4 gene are associated with three insidious hereditary disorders: Rothmund-Thomson, Baller-Gerold, and RAPADILINO syndromes. These syndromes are characterized by growth deficiency, radial ray defects, red rashes, and higher predisposition to malignancy, especially osteosarcomas. Within the RecQ family, RECQ4 is the least characterized, and its role in DNA replication and repair remains unknown. We have identified several DNA binding sites within RECQ4. Two are located at the N-terminus and one is located within the conserved helicase domain. N-terminal domains probably cooperate with one another and promote the strong annealing activity of RECQ4. Surprisingly, the region spanning 322-400aa shows a very high affinity for branched DNA substrates, especially Holliday junctions. This study demonstrates biochemical activities of RECQ4 that could be involved in genome maintenance and suggest its possible role in processing replication and recombination intermediates.


Assuntos
DNA Cruciforme/metabolismo , RecQ Helicases/metabolismo , Sequência de Bases , Sítios de Ligação , Replicação do DNA , Recombinação Homóloga , Humanos , Multimerização Proteica , Estrutura Terciária de Proteína , RecQ Helicases/química
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