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1.
EMBO J ; 42(20): e110844, 2023 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-37661798

RESUMO

Homologous recombination (HR) is a prominent DNA repair pathway maintaining genome integrity. Mutations in many HR genes lead to cancer predisposition. Paradoxically, the implication of the pivotal HR factor RAD51 on cancer development remains puzzling. Particularly, no RAD51 mouse models are available to address the role of RAD51 in aging and carcinogenesis in vivo. We engineered a mouse model with an inducible dominant-negative form of RAD51 (SMRad51) that suppresses RAD51-mediated HR without stimulating alternative mutagenic repair pathways. We found that in vivo expression of SMRad51 led to replicative stress, systemic inflammation, progenitor exhaustion, premature aging and reduced lifespan, but did not trigger tumorigenesis. Expressing SMRAD51 in a breast cancer predisposition mouse model (PyMT) decreased the number and the size of tumors, revealing an anti-tumor activity of SMRAD51. We propose that these in vivo phenotypes result from chronic endogenous replication stress caused by HR decrease, which preferentially targets progenitors and tumor cells. Our work underlines the importance of RAD51 activity for progenitor cell homeostasis, preventing aging and more generally for the balance between cancer and aging.


Assuntos
Neoplasias , Rad51 Recombinase , Animais , Camundongos , Envelhecimento/genética , Carcinogênese/genética , Transformação Celular Neoplásica , Dano ao DNA , Reparo do DNA , Recombinação Homóloga , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo
2.
Nat Med ; 29(8): 2110-2120, 2023 08.
Artigo em Inglês | MEDLINE | ID: mdl-37488289

RESUMO

The mechanisms of action of and resistance to trastuzumab deruxtecan (T-DXd), an anti-HER2-drug conjugate for breast cancer treatment, remain unclear. The phase 2 DAISY trial evaluated the efficacy of T-DXd in patients with HER2-overexpressing (n = 72, cohort 1), HER2-low (n = 74, cohort 2) and HER2 non-expressing (n = 40, cohort 3) metastatic breast cancer. In the full analysis set population (n = 177), the confirmed objective response rate (primary endpoint) was 70.6% (95% confidence interval (CI) 58.3-81) in cohort 1, 37.5% (95% CI 26.4-49.7) in cohort 2 and 29.7% (95% CI 15.9-47) in cohort 3. The primary endpoint was met in cohorts 1 and 2. Secondary endpoints included safety. No new safety signals were observed. During treatment, HER2-expressing tumors (n = 4) presented strong T-DXd staining. Conversely, HER2 immunohistochemistry 0 samples (n = 3) presented no or very few T-DXd staining (Pearson correlation coefficient r = 0.75, P = 0.053). Among patients with HER2 immunohistochemistry 0 metastatic breast cancer, 5 of 14 (35.7%, 95% CI 12.8-64.9) with ERBB2 expression below the median presented a confirmed objective response as compared to 3 of 10 (30%, 95% CI 6.7-65.2) with ERBB2 expression above the median. Although HER2 expression is a determinant of T-DXd efficacy, our study suggests that additional mechanisms may also be involved. (ClinicalTrials.gov identifier NCT04132960 .).


Assuntos
Neoplasias da Mama , Imunoconjugados , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Trastuzumab/uso terapêutico , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Camptotecina/uso terapêutico
3.
Nat Commun ; 14(1): 2561, 2023 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-37142601

RESUMO

Xeroderma pigmentosum (XP) is a genetic disorder caused by mutations in genes of the Nucleotide Excision Repair (NER) pathway (groups A-G) or in Translesion Synthesis DNA polymerase η (V). XP is associated with an increased skin cancer risk, reaching, for some groups, several thousand-fold compared to the general population. Here, we analyze 38 skin cancer genomes from five XP groups. We find that the activity of NER determines heterogeneity of the mutation rates across skin cancer genomes and that transcription-coupled NER extends beyond the gene boundaries reducing the intergenic mutation rate. Mutational profile in XP-V tumors and experiments with POLH knockout cell line reveal the role of polymerase η in the error-free bypass of (i) rare TpG and TpA DNA lesions, (ii) 3' nucleotides in pyrimidine dimers, and (iii) TpT photodimers. Our study unravels the genetic basis of skin cancer risk in XP and provides insights into the mechanisms reducing UV-induced mutagenesis in the general population.


Assuntos
Neoplasias Cutâneas , Xeroderma Pigmentoso , Humanos , Xeroderma Pigmentoso/patologia , Raios Ultravioleta/efeitos adversos , Reparo do DNA/genética , Mutação , Neoplasias Cutâneas/genética , Genômica
4.
Front Oncol ; 12: 982790, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36387101

RESUMO

Background: Anti-PD-(L)1 treatment is indicated for patients with mismatch repair-deficient (MMRD) tumors, regardless of tumor origin. However, the response rate is highly heterogeneous across MMRD tumors. The objective of the study is to find a score that predicts anti-PD-(L)1 response in patients with MMRD tumors. Methods: Sixty-one patients with various origin of MMRD tumors and treated with anti-PD-(L)1 were retrospectively included in this study. An expert radiologist annotated all tumors present at the baseline and first evaluation CT-scans for all the patients by circumscribing them on their largest axial axis (single slice), allowing us to compute an approximation of their tumor volume. In total, 2120 lesions were annotated, which led to the computation of the total tumor volume for each patient. The RECIST sum of target lesions' diameters and neutrophile-to-lymphocyte (NLR) were also reported at both examinations. These parameters were determined at baseline and first evaluation and the variation between the first evaluation and baseline was calculated, to determine a comprehensive score for overall survival (OS) and progression-free survival (PFS). Results: Total tumor volume at baseline was found to be significantly correlated to the OS (p-value: 0.005) and to the PFS (p-value:<0.001). The variation of the RECIST sum of target lesions' diameters, total tumor volume and NLR were found to be significantly associated to the OS (p-values:<0.001, 0.006,<0.001 respectively) and to the PFS (<0.001,<0.001, 0.007 respectively). The concordance score combining total tumor volume and NLR variation was better at stratifying patients compared to the tumor volume or NLR taken individually according to the OS (pairwise log-rank test p-values: 0.033,<0.001, 0.002) and PFS (pairwise log-rank test p-values: 0.041,<0.001, 0.003). Conclusion: Total tumor volume appears to be a prognostic biomarker of anti-PD-(L)1 response to immunotherapy in metastatic patients with MMRD tumors. Combining tumor volume and NLR with a simple concordance score stratifies patients well according to their survival and offers a good predictive measure of response to immunotherapy.

5.
Nucleic Acids Res ; 50(17): 9909-9929, 2022 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-36107774

RESUMO

DNA lesions in S phase threaten genome stability. The DNA damage tolerance (DDT) pathways overcome these obstacles and allow completion of DNA synthesis by the use of specialised translesion (TLS) DNA polymerases or through recombination-related processes. However, how these mechanisms coordinate with each other and with bulk replication remains elusive. To address these issues, we monitored the variation of replication intermediate architecture in response to ultraviolet irradiation using transmission electron microscopy. We show that the TLS polymerase η, able to accurately bypass the major UV lesion and mutated in the skin cancer-prone xeroderma pigmentosum variant (XPV) syndrome, acts at the replication fork to resolve uncoupling and prevent post-replicative gap accumulation. Repriming occurs as a compensatory mechanism when this on-the-fly mechanism cannot operate, and is therefore predominant in XPV cells. Interestingly, our data support a recombination-independent function of RAD51 at the replication fork to sustain repriming. Finally, we provide evidence for the post-replicative commitment of recombination in gap repair and for pioneering observations of in vivo recombination intermediates. Altogether, we propose a chronology of UV damage tolerance in human cells that highlights the key role of polη in shaping this response and ensuring the continuity of DNA synthesis.


Assuntos
Reparo do DNA , Xeroderma Pigmentoso , Dano ao DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Humanos , Raios Ultravioleta , Xeroderma Pigmentoso/genética
6.
J Immunother Cancer ; 10(7)2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35896284

RESUMO

BACKGROUND: Clinical studies have highlighted the efficacy of anti-programmed death 1 (αPD-1) monoclonal antibodies in patients with DNA mismatch repair-deficient (MMRD) tumors. However, the responsiveness of MMRD cancers to αPD-1 therapy is highly heterogeneous, and the origins of this variability remain not fully understood. METHODS: 4T1 and CT26 mouse tumor cell lines were inactivated for the MMRD gene Msh2, leading to a massive accumulation of mutations after serial passages of cells. Insertions/deletion events and mutation load were evaluated by whole exome sequencing. Mice bearing highly mutated MMRD tumor or parental tumors were treated with αPD-1 and tumor volume was monitored. Immune cell type abundance was dynamically assessed in the tumor microenvironment and the blood by flow cytometry. Neutrophils were depleted in mice using αLY6G antibody, and regulatory T (Treg) cell population was reduced with αCD25 or anti-cytotoxic T-lymphocytes-associated protein 4 (αCTLA-4) antibodies. Patients with MMRD tumors treated with immune checkpoint blockade-based therapy were retrospectively identified and neutrophil-to-lymphocyte ratio (NLR) was evaluated and examined for correlation with clinical benefit. RESULTS: By recapitulating mismatch repair deficiency in different mouse tumor models, we revealed that elevated circulating tumor-induced neutrophils (TIN) in hypermutated MMRD tumors hampered response to αPD-1 monotherapy. Importantly, depletion of TIN using αLy-6G antibody reduced Treg cells and restored αPD-1 response. Conversely, targeting Treg cells by αCD25 or αCTLA-4 antibodies limited peripheral TIN accumulation and elicited response in αPD-1-resistant MMRD tumors, highlighting a crosstalk between TIN and Treg cells. Thus, αPD-1+αCTLA-4 combination overcomes TIN-induced resistance to αPD-1 in mice bearing MMRD tumors. Finally, in a cohort of human (high microsatellite instability)/MMRD tumors we revealed that early on-treatment change in the NLR ratio may predict resistance to αPD-1 therapy. CONCLUSIONS: TIN countered αPD-1 efficacy in MMRD tumors. Since αCTLA-4 could restrict TIN accumulation, αPD-1+αCTLA-4 combination overcomes αPD-1 resistance in hosts with hypermutated MMRD tumors displaying abnormal neutrophil accumulation.


Assuntos
Neutrófilos , Animais , Humanos , Camundongos , Neoplasias Encefálicas , Neoplasias Colorretais , Instabilidade de Microssatélites , Síndromes Neoplásicas Hereditárias , Estudos Retrospectivos , Microambiente Tumoral
7.
Hum Mol Genet ; 31(18): 3083-3094, 2022 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-35512351

RESUMO

BACKGROUND: TASP1 encodes an endopeptidase activating histone methyltransferases of the KMT2 family. Homozygous loss-of-function variants in TASP1 have recently been associated with Suleiman-El-Hattab syndrome. We report six individuals with Suleiman-El-Hattab syndrome and provide functional characterization of this novel histone modification disorder in a multi-omics approach. METHODS: Chromosomal microarray/exome sequencing in all individuals. Western blotting from fibroblasts in two individuals. RNA sequencing and proteomics from fibroblasts in one individual. Methylome analysis from blood in two individuals. Knock-out of tasp1 orthologue in zebrafish and phenotyping. RESULTS: All individuals had biallelic TASP1 loss-of-function variants and a phenotype including developmental delay, multiple congenital anomalies (including cardiovascular and posterior fossa malformations), a distinct facial appearance and happy demeanor. Western blot revealed absence of TASP1. RNA sequencing/proteomics showed HOX gene downregulation (HOXA4, HOXA7, HOXA1 and HOXB2) and dysregulation of transcription factor TFIIA. A distinct methylation profile intermediate between control and Kabuki syndrome (KMT2D) profiles could be produced. Zebrafish tasp1 knock-out revealed smaller head size and abnormal cranial cartilage formation in tasp1 crispants. CONCLUSION: This work further delineates Suleiman-El-Hattab syndrome, a recognizable neurodevelopmental syndrome. Possible downstream mechanisms of TASP1 deficiency include perturbed HOX gene expression and dysregulated TFIIA complex. Methylation pattern suggests that Suleiman-El-Hattab syndrome can be categorized into the group of histone modification disorders including Wiedemann-Steiner and Kabuki syndrome.


Assuntos
Código das Histonas , Peixe-Zebra , Anormalidades Múltiplas , Animais , Endopeptidases/genética , Face/anormalidades , Doenças Hematológicas , Histona Metiltransferases/genética , Fenótipo , Fator de Transcrição TFIIA/genética , Doenças Vestibulares , Peixe-Zebra/genética
8.
JCI Insight ; 7(11)2022 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-35511434

RESUMO

DNA damage and genomic instability contribute to non-small cell lung cancer (NSCLC) etiology and progression. However, their therapeutic exploitation is disappointing. CTC-derived explants (CDX) offer systems for mechanistic investigation of CTC metastatic potency and may provide rationale for biology-driven therapeutics. Four CDX models and 3 CDX-derived cell lines were established from NSCLC CTCs and recapitulated patient tumor histology and response to platinum-based chemotherapy. CDX (GR-CDXL1, GR-CDXL2, GR-CDXL3, GR-CDXL4) demonstrated considerable mutational landscape similarity with patient tumor biopsy and/or single CTCs. Truncal alterations in key DNA damage response (DDR) and genome integrity-related genes were prevalent across models and assessed as therapeutic targets in vitro, in ovo, and in vivo. GR-CDXL1 presented homologous recombination deficiency linked to biallelic BRCA2 mutation and FANCA deletion, unrepaired DNA lesions after mitosis, and olaparib sensitivity, despite resistance to chemotherapy. SLFN11 overexpression in GR-CDXL4 led to olaparib sensitivity and was in coherence with neuroendocrine marker expression in patient tumor biopsy, suggesting a predictive value of SLFN11 in NSCLC histological transformation into small cell lung cancer (SCLC). Centrosome clustering promoted targetable chromosomal instability in GR-CDXL3 cells. These CDX unravel DDR and genome integrity-related defects as a central mechanism underpinning metastatic potency of CTCs and provide rationale for their therapeutic targeting in metastatic NSCLC.


Assuntos
Carcinoma Pulmonar de Células não Pequenas , Neoplasias Pulmonares , Células Neoplásicas Circulantes , Carcinoma de Pequenas Células do Pulmão , Biomarcadores Tumorais/genética , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Humanos , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Células Neoplásicas Circulantes/metabolismo , Proteínas Nucleares , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/genética , Carcinoma de Pequenas Células do Pulmão/patologia
9.
EMBO J ; 40(21): e104543, 2021 11 02.
Artigo em Inglês | MEDLINE | ID: mdl-34533226

RESUMO

The DNA polymerase zeta (Polζ) plays a critical role in bypassing DNA damage. REV3L, the catalytic subunit of Polζ, is also essential in mouse embryonic development and cell proliferation for reasons that remain incompletely understood. In this study, we reveal that REV3L protein interacts with heterochromatin components including repressive histone marks and localizes in pericentromeric regions through direct interaction with HP1 dimer. We demonstrate that Polζ/REV3L ensures progression of replication forks through difficult-to-replicate pericentromeric heterochromatin, thereby preventing spontaneous chromosome break formation. We also find that Rev3l-deficient cells are compromised in the repair of heterochromatin-associated double-stranded breaks, eliciting deletions in late-replicating regions. Lack of REV3L leads to further consequences that may be ascribed to heterochromatin replication and repair-associated functions of Polζ, with a disruption of the temporal replication program at specific loci. This is correlated with changes in epigenetic landscape and transcriptional control of developmentally regulated genes. These results reveal a new function of Polζ in preventing chromosome instability during replication of heterochromatic regions.


Assuntos
Replicação do DNA , Proteínas de Ligação a DNA/genética , DNA Polimerase Dirigida por DNA/genética , DNA/genética , Células-Tronco Embrionárias/metabolismo , Epigênese Genética , Heterocromatina/metabolismo , Animais , Linhagem Celular , Linhagem Celular Transformada , Proliferação de Células , Homólogo 5 da Proteína Cromobox/genética , Homólogo 5 da Proteína Cromobox/metabolismo , Instabilidade Cromossômica , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Embrião de Mamíferos , Células-Tronco Embrionárias/citologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Células HeLa , Heterocromatina/química , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células NIH 3T3 , Transdução de Sinais
10.
Front Genet ; 12: 784963, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-35111200

RESUMO

Xeroderma pigmentosum (XP) is a rare genetic condition in which exposure to sunlight leads to a high tumor incidence due to defective DNA repair machinery. Herein, we investigated seven patients clinically diagnosed with XP living in a small city, Montanhas (Rio Grande do Norte), in the Northeast region of Brazil. We performed high-throughput sequencing and, surprisingly, identified two different mutated genes. Six patients carry a novel homozygote mutation in the POLH/XPV gene, c.672_673insT (p.Leu225Serfs*33), while one patient carries a homozygote mutation in the XPC gene, c.2251-1G>C. This latter mutation was previously described in Southeastern Africa (Comoro Island and Mozambique), Pakistan, and in a high incidence in Brazil. The XP-C patient had the first symptoms before the first year of life with aggressive ophthalmologic tumor progression and a melanoma onset at 7 years of age. The XP-V patients presented a milder phenotype with later onset of the disorder (mean age of 16 years old), and one of the six XP-V patients developed melanoma at 72 years. The photoprotection is minimal among them, mainly for the XP-V patients. The differences in the disease severity between XP-C (more aggressive) and XP-V (milder) patients are obvious and point to the major role of photoprotection in the XPs. We estimate that the incidence of XP patients at Montanhas can be higher, but with no diagnosis, due to poor health assistance. Patients still suffer from the stigmatization of the condition, impairing diagnosis, education for sun protection, and medical care.

11.
Genet Mol Biol ; 43(4): e20200100, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33001133

RESUMO

Endometrial cancer (EC) harboring heterozygous POLE proofreading inactivating mutations (POLE-exo*) is associated with an increased number of somatic mutations that result in a distinctive anti-tumor immune response. However, the consequences of such POLE mutations in the context of the missing wild-type allele have not yet been described in endometrial tumors. A 72-year-old woman harboring a germline monoallelic frameshift mutation (p.Pro269fsTer26) in POLE was diagnosed with an EC having a somatic heterozygous mutation in the exonuclease domain of POLE (S459F). Targeted gene sequencing revealed an ultramutated phenotype (381 mutations/Mb) in the tumor and a 2-fold excess of mutations on the DNA leading strand. Additionally, we observed a mutational signature similar to the COSMIC signature 10, a higher mutation rate in this tumor than in endometrial tumors with heterozygous POLE-exo*, and an increased number of T lymphocytes. This is the first report of an ultramutated EC harboring a somatic POLE-exo* mutation in association with a germline loss-of-function mutation in this gene. The absence of a wild type POLE allele led to a particularly high mutational burden.

13.
Bull Cancer ; 106(2): 105-113, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30342749

RESUMO

In the last few years, immunotherapy has revolutionized the oncology landscape by targeting the host immune system. Blocking immune checkpoints such as cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed cell death-1 (PD-1) and its ligand (PD-L1 or B7-H1), has proven its efficacy in several solid cancers. Recently, several clinical studies have demonstrated a significant improvement in clinical response to the anti-PD-1-based immunotherapy in a subset of patients with microsatellite instability-high (MSI-H)/mismatch repair (MMR)-deficient tumors that accumulate short insertion/deletion mutations notably in coding microsatellites regions of the genome. Thus, the responsiveness of MSI cancers to immune checkpoint inhibitors can be explained by the increased rate of putative frameshift peptide neoantigens and the immunogenic tumor microenvironment. However, not all MSI tumors respond to immunotherapy. The current review will summarize how and why MMR deficiency has emerged as an important predictor of sensitivity for immunotherapy-based strategies. We will also discuss tumor-cell intrinsic genetic and immune-related features of MSI tumors that can modulate immune checkpoint blockade response and explain primary and/or acquired resistance to anti-PD-1 therapy. Finally, we will also discuss about emerging scores which can define more precisely the immune context of the tumor microenvironment and thus better evaluate prognosis and predict response to Immune Checkpoint Blockade.


Assuntos
Antígeno B7-H1/antagonistas & inibidores , Antígeno CTLA-4/antagonistas & inibidores , Reparo de Erro de Pareamento de DNA/genética , Imunoterapia/métodos , Instabilidade de Microssatélites , Neoplasias/genética , Neoplasias/terapia , Receptor de Morte Celular Programada 1/antagonistas & inibidores , Humanos , Acúmulo de Mutações , Neoplasias/imunologia , Prognóstico , Microambiente Tumoral/imunologia
14.
Eur J Cancer ; 84: 290-303, 2017 10.
Artigo em Inglês | MEDLINE | ID: mdl-28846956

RESUMO

Immune checkpoint inhibitors have demonstrated unprecedented clinical activity in a wide range of cancers. Significant therapeutic responses have recently been observed in patients presenting mismatch repair-deficient (MMRD) tumours. MMRD cancers exhibit a remarkably high rate of mutations, which can result in the formation of neoantigens, hypothesised to enhance the antitumour immune response. In addition to MMRD tumours, cancers mutated in the exonuclease domain of the catalytic subunit of the DNA polymerase epsilon (POLE) also exhibit an ultramutated genome and are thus likely to benefit from immunotherapy. In this review, we provide an overview of recent data on hypermutated tumours, including MMRD and POLE-mutated cancers, with a focus on their distinctive clinicopathological and molecular characteristics as well as their immune environment. We also discuss the emergence of immune therapy to treat these hypermutated cancers, and we comment on the recent Food and Drug Administration approval of an immune checkpoint inhibitor, the programmed cell death 1 antibody (pembrolizumab, Keytruda), for the treatment of patients with metastatic MMRD cancers regardless of the tumour type. This breakthrough represents a turning point in the management of these hypermutated tumours and paves the way for broader strategies in immunoprecision medicine.


Assuntos
Antígenos de Neoplasias/genética , Biomarcadores Tumorais/genética , Imunoterapia/métodos , Mutação , Neoplasias/genética , Neoplasias/terapia , Medicina de Precisão/métodos , Antígenos de Neoplasias/imunologia , Biomarcadores Tumorais/imunologia , Reparo de Erro de Pareamento de DNA , Análise Mutacional de DNA , DNA Polimerase II/genética , DNA Polimerase II/metabolismo , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , Predisposição Genética para Doença , Humanos , Instabilidade de Microssatélites , Terapia de Alvo Molecular , Neoplasias/imunologia , Neoplasias/patologia , Fenótipo , Proteínas de Ligação a Poli-ADP-Ribose , Valor Preditivo dos Testes , Evasão Tumoral , Microambiente Tumoral
15.
Nat Commun ; 7: 13326, 2016 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-27811911

RESUMO

Translesion polymerase eta (polη) was characterized for its ability to replicate ultraviolet-induced DNA lesions that stall replicative polymerases, a process promoted by Rad18-dependent PCNA mono-ubiquitination. Recent findings have shown that polη also acts at intrinsically difficult to replicate sequences. However, the molecular mechanisms that regulate its access to these loci remain elusive. Here, we uncover that polη travels with replication forks during unchallenged S phase and this requires its SUMOylation on K163. Abrogation of polη SUMOylation results in replication defects in response to mild replication stress, leading to chromosome fragments in mitosis and damage transmission to daughter cells. Rad18 plays a pivotal role, independently of its ubiquitin ligase activity, acting as a molecular bridge between polη and the PIAS1 SUMO ligase to promote polη SUMOylation. Our results provide the first evidence that SUMOylation represents a new way to target polη to replication forks, independent of the Rad18-mediated PCNA ubiquitination, thereby preventing under-replicated DNA.


Assuntos
Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/genética , DNA Polimerase Dirigida por DNA/genética , Humanos , Antígeno Nuclear de Célula em Proliferação/genética , Proteínas Inibidoras de STAT Ativados/genética , Fase S/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Sumoilação/genética , Ubiquitina-Proteína Ligases/genética
16.
DNA Repair (Amst) ; 29: 154-65, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25766642

RESUMO

Xeroderma pigmentosum variant (XP-V) is a rare genetic disease, characterized by sunlight sensitivity and predisposition to cutaneous malignancies. XP-V is caused by a deficiency in DNA polymerase eta (Polη) that plays a pivotal role in translesion synthesis by bypassing UV-induced pyrimidine dimers. Previously we identified a new Polη variant containing two missense mutations, one mutation within the bipartite NLS (T692A) and a second mutation on the stop codon (X714W) leading to a longer protein with an extra 8 amino acids (721 instead of 713 AA). First biochemical analysis revealed that this Polη missense variant was barely detectable by western blot. As this mutant is extremely unstable and is nearly undetectable, a definitive measure of its functional deficit in cells has not been explored. Here we report the molecular and cellular characterization of this missense variant. In cell free extracts, the extra 8 amino acids in the C-terminal of Polη(721) only slightly reduce the bypass efficiency through CPD lesions. In vivo, Polη(721) accumulates in replication factories and interacts with mUb-PCNA albeit at lower level than Polη(wt). XP-V cells overexpressing Polη(721) were only slightly UV-sensitive. Altogether, our data strongly suggest that Polη(721) is functional and that the patient displays a XP-V phenotype because the mutant protein is excessively unstable. We then investigated the molecular mechanisms involved in this excessive proteolysis. We showed that Polη(721) is degraded by the proteasome in an ubiquitin-dependent manner and that this proteolysis is independent of the E3 ligases, CRL4(cdt2) and Pirh2, reported to promote Polη degradation. We then demonstrated that the extra 8 amino acids of Polη(721) do not act as a degron but rather induce a conformational change of the Polη C-terminus exposing its bipartite NLS as well as a sequence close to its UBZ to the ubiquitin/proteasome system. Interestingly we showed that the clinically approved proteasome inhibitor, Bortezomib restores the levels of Polη(721) suggesting that this might be a therapeutic approach to preventing tumor development in certain XP-V patients harboring missense mutations.


Assuntos
DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Mutação de Sentido Incorreto , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Ubiquitina/metabolismo , Códon de Terminação , Expressão Gênica , Humanos , Proteínas Nucleares/metabolismo , Estabilidade Proteica , Estrutura Terciária de Proteína , Ubiquitina-Proteína Ligases/metabolismo , Xeroderma Pigmentoso/genética , Xeroderma Pigmentoso/metabolismo
17.
Mol Cell ; 57(1): 123-37, 2015 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-25533188

RESUMO

The SLX4 Fanconi anemia protein is a tumor suppressor that may act as a key regulator that engages the cell into specific genome maintenance pathways. Here, we show that the SLX4 complex is a SUMO E3 ligase that SUMOylates SLX4 itself and the XPF subunit of the DNA repair/recombination XPF-ERCC1 endonuclease. This SLX4-dependent activity is mediated by a remarkably specific interaction between SLX4 and the SUMO-charged E2 conjugating enzyme UBC9 and relies not only on newly identified SUMO-interacting motifs (SIMs) in SLX4 but also on its BTB domain. In contrast to its ubiquitin-binding UBZ4 motifs, SLX4 SIMs are dispensable for its DNA interstrand crosslink repair functions. Instead, while detrimental in response to global replication stress, the SUMO E3 ligase activity of the SLX4 complex is critical to prevent mitotic catastrophe following common fragile site expression.


Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Genoma , Subunidades Proteicas/metabolismo , Recombinases/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Sequência de Aminoácidos , Linhagem Celular Tumoral , Replicação do DNA , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Instabilidade Genômica , Humanos , Dados de Sequência Molecular , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Subunidades Proteicas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Recombinases/genética , Alinhamento de Sequência , Transdução de Sinais , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Sumoilação , Enzimas de Conjugação de Ubiquitina/genética , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética
18.
Hum Mutat ; 35(1): 117-28, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24130121

RESUMO

Xeroderma pigmentosum variant (XP-V) is a rare genetic disease, characterized by some sunlight sensitivity and predisposition to cutaneous malignancies. We described clinical and genetic features of the largest collection ever published of 23 XPV patients (ages between 21 and 86) from 20 unrelated families. Primary fibroblasts from patients showed normal nucleotide excision repair but UV-hypersensitivity in the presence of caffeine, a signature of the XP-V syndrome. 87% of patients developed skin tumors with a median age of 21 for the first occurrence. The median numbers of basal-cell carcinoma was 13 per patient, six for squamous-cell carcinoma, and five for melanoma. XP-V is due to defects in the translesion-synthesis DNA polymerase Polη coded by the POLH gene. DNA sequencing of POLH revealed 29 mutations, where 12 have not been previously identified, leading to truncated polymerases in 69% of patients. Four missense mutations are correlated with the protein stability by structural modeling of the Polη polymerase domain. There is a clear relationship between the types of missense mutations and clinical severity. For truncating mutations, which lead to an absence of or to inactive proteins, the life-cumulated UV exposure is probably the best predictor of cancer incidence, reinforcing the necessity to protect XP-Vs from sun exposure.


Assuntos
DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Neoplasias Cutâneas/genética , Xeroderma Pigmentoso/genética , Xeroderma Pigmentoso/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Cafeína , Carcinoma Basocelular/epidemiologia , Carcinoma Basocelular/genética , Carcinoma Basocelular/patologia , Carcinoma de Células Escamosas/epidemiologia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patologia , Células Cultivadas , Reparo do DNA , Feminino , Fibroblastos/metabolismo , Variação Genética , Genótipo , Humanos , Masculino , Melanoma/epidemiologia , Melanoma/genética , Melanoma/patologia , Pessoa de Meia-Idade , Modelos Moleculares , Mutação de Sentido Incorreto , Fenótipo , Estabilidade Proteica , Estudos Retrospectivos , Neoplasias Cutâneas/epidemiologia , Neoplasias Cutâneas/patologia , Raios Ultravioleta , Xeroderma Pigmentoso/complicações , Adulto Jovem
19.
Nucleic Acids Res ; 41(13): 6501-13, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23677613

RESUMO

During replication, DNA damage can challenge replication fork progression and cell viability. Homologous Recombination (HR) and Translesion Synthesis (TLS) pathways appear as major players involved in the resumption and completion of DNA replication. How both pathways are coordinated in human cells to maintain genome stability is unclear. Numerous helicases are involved in HR regulation. Among them, the helicase FBH1 accumulates at sites of DNA damage and potentially constrains HR via its anti-recombinase activity. However, little is known about its regulation in vivo. Here, we report a mechanism that controls the degradation of FBH1 after DNA damage. Firstly, we found that the sliding clamp Proliferating Cell Nuclear Antigen (PCNA) is critical for FBH1 recruitment to replication factories or DNA damage sites. We then showed the anti-recombinase activity of FBH1 is partially dependent on its interaction with PCNA. Intriguingly, after its re-localization, FBH1 is targeted for degradation by the Cullin-ring ligase 4-Cdt2 (CRL4(Cdt2))-PCNA pathway via a PCNA-interacting peptide (PIP) degron. Importantly, expression of non-degradable FBH1 mutant impairs the recruitment of the TLS polymerase eta to chromatin in UV-irradiated cells. Thus, we propose that after DNA damage, FBH1 might be required to restrict HR and then degraded by the Cdt2-proteasome pathway to facilitate TLS pathway.


Assuntos
Dano ao DNA , DNA Helicases/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas Nucleares/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Linhagem Celular , Cromatina/enzimologia , DNA Helicases/química , Proteínas de Ligação a DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Recombinação Homóloga , Humanos , Dados de Sequência Molecular , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise , Raios Ultravioleta
20.
Genome Res ; 23(2): 331-40, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23038767

RESUMO

We have developed an approach termed PUB-NChIP (proximity utilizing biotinylation with native ChIP) to purify and study the protein composition of chromatin in proximity to a nuclear protein of interest. It is based on coexpression of (1) a protein of interest, fused with the bacterial biotin ligase BirA, together with (2) a histone fused to a biotin acceptor peptide (BAP), which is specifically biotinylated by BirA-fusion in the proximity of the protein of interest. Using the RAD18 protein as a model, we demonstrate that the RAD18-proximal chromatin is enriched in some H4 acetylated species. Moreover, the RAD18-proximal chromatin containing a replacement histone H2AZ has a different pattern of H4 acetylation. Finally, biotin pulse-chase experiments show that the H4 acetylation pattern starts to resemble the acetylation pattern of total H4 after the proximity of chromatin to RAD18 has been lost.


Assuntos
Imunoprecipitação da Cromatina/métodos , Cromatina/metabolismo , Proteínas Nucleares/metabolismo , Acetilação , Biotinilação/métodos , Linhagem Celular , Histonas , Humanos , Processamento de Proteína Pós-Traducional
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