RESUMO
The high incidence of whole-arm chromosome aneuploidy and translocations in tumors suggests instability of centromeres, unique loci built on repetitive sequences and essential for chromosome separation. The causes behind this fragility and the mechanisms preserving centromere integrity remain elusive. We show that replication stress, hallmark of pre-cancerous lesions, promotes centromeric breakage in mitosis, due to spindle forces and endonuclease activities. Mechanistically, we unveil unique dynamics of the centromeric replisome distinct from the rest of the genome. Locus-specific proteomics identifies specialized DNA replication and repair proteins at centromeres, highlighting them as difficult-to-replicate regions. The translesion synthesis pathway, along with other factors, acts to sustain centromere replication and integrity. Prolonged stress causes centromeric alterations like ruptures and translocations, as observed in ovarian cancer models experiencing replication stress. This study provides unprecedented insights into centromere replication and integrity, proposing mechanistic insights into the origins of centromere alterations leading to abnormal cancerous karyotypes.
Assuntos
Centrômero , Sequências Repetitivas de Ácido Nucleico , Humanos , Centrômero/genética , Mitose/genética , Instabilidade GenômicaRESUMO
Centromeres are essential for chromosome segregation in most animals and plants yet are among the most rapidly evolving genome elements. The mechanisms underlying this paradoxical phenomenon remain enigmatic. Here, we report that human centromeres innately harbor a striking enrichment of DNA breaks within functionally active centromere regions. Establishing a single-cell imaging strategy that enables comparative assessment of DNA breaks at repetitive regions, we show that centromeric DNA breaks are induced not only during active cellular proliferation but also de novo during quiescence. Markedly, centromere DNA breaks in quiescent cells are resolved enzymatically by the evolutionarily conserved RAD51 recombinase, which in turn safeguards the specification of functional centromeres. This study highlights the innate fragility of centromeres, which may have been co-opted over time to reinforce centromere specification while driving rapid evolution. The findings also provide insights into how fragile centromeres are likely to contribute to human disease.
Assuntos
Centrômero , DNA , Animais , Humanos , Centrômero/genética , Centrômero/metabolismo , Proteína Centromérica A , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Recombinação GenéticaRESUMO
Centromeres are large structural regions in the genomic DNA, which are essential for accurately transmitting a complete set of chromosomes to daughter cells during cell division. In humans, centromeres consist of highly repetitive α-satellite DNA sequences and unique epigenetic components, forming large proteinaceous structures required for chromosome segregation. Despite their biological importance, there is a growing body of evidence for centromere breakage across the cell cycle, including periods of quiescence. In this review, we provide an up-to-date examination of the distinct centromere environments at different stages of the cell cycle, highlighting their plausible contribution to centromere breakage. Additionally, we explore the implications of these breaks on centromere function, both in terms of negative consequences and potential positive effects.
Assuntos
Centrômero , DNA , Humanos , Centrômero/genética , Segregação de Cromossomos , Evolução BiológicaRESUMO
The timely activation of homologous recombination is essential for the maintenance of genome stability, in which the RAD51 recombinase plays a central role. Biochemically, human RAD51 polymerises faster on single-stranded DNA (ssDNA) compared to double-stranded DNA (dsDNA), raising a key conceptual question: how does it discriminate between them? In this study, we tackled this problem by systematically assessing RAD51 binding kinetics on ssDNA and dsDNA differing in length and flexibility using surface plasmon resonance. By directly fitting a mechanistic model to our experimental data, we demonstrate that the RAD51 polymerisation rate positively correlates with the flexibility of DNA. Once the RAD51-DNA complex is formed, however, RAD51 remains stably bound independent of DNA flexibility, but rapidly dissociates from flexible DNA when RAD51 self-association is perturbed. This model presents a new general framework suggesting that the flexibility of DNA, which may increase locally as a result of DNA damage, plays an important role in rapidly recruiting repair factors that multimerise at sites of DNA damage.
Assuntos
DNA/química , DNA/metabolismo , Rad51 Recombinase/química , Rad51 Recombinase/metabolismo , Reparo do DNA , Humanos , Modelos Teóricos , Mutação Puntual , Ligação Proteica , Rad51 Recombinase/genética , Espalhamento a Baixo Ângulo , Ressonância de Plasmônio de Superfície , Difração de Raios XRESUMO
As the primary catalyst of homologous recombination (HR) in vertebrates, RAD51 has been extensively studied in the context of repair of double-stranded DNA breaks (DSBs). With recent advances in the understanding of RAD51 function extending beyond DSBs, the importance of RAD51 throughout DNA metabolism has become increasingly clear. Here we review the suggested roles of RAD51 beyond HR, specifically focusing on their interplay with DNA replication and the maintenance of genomic stability, in which RAD51 function emerges as a double-edged sword.
Assuntos
Instabilidade Genômica/genética , Rad51 Recombinase/metabolismo , Quebras de DNA de Cadeia Dupla , HumanosRESUMO
Homologous recombination (HR) plays an important role in the maintenance of genome integrity. HR repairs broken DNA during S and G2 phases of the cell cycle but its regulatory mechanisms remain elusive. Here, we report that Polo-like kinase 1 (Plk1), which is vital for cell proliferation and is frequently upregulated in cancer cells, phosphorylates the essential Rad51 recombinase at serine 14 (S14) during the cell cycle and in response to DNA damage. Strikingly, S14 phosphorylation licenses subsequent Rad51 phosphorylation at threonine 13 (T13) by casein kinase 2 (CK2), which in turn triggers direct binding to the Nijmegen breakage syndrome gene product, Nbs1. This mechanism facilitates Rad51 recruitment to damage sites, thus enhancing cellular resistance to genotoxic stresses. Our results uncover a role of Plk1 in linking DNA damage recognition with HR repair and suggest a molecular mechanism for cancer development associated with elevated activity of Plk1.
Assuntos
Caseína Quinase II/metabolismo , Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Rad51 Recombinase/metabolismo , Reparo de DNA por Recombinação , Sequência de Aminoácidos , Proteína BRCA2/metabolismo , Pontos de Checagem do Ciclo Celular , Linhagem Celular , Sequência Conservada , Instabilidade Genômica , Humanos , Dados de Sequência Molecular , Proteínas Nucleares/metabolismo , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Rad51 Recombinase/química , Quinase 1 Polo-LikeRESUMO
The partner and localiser of BRCA2 (PALB2) plays important roles in the maintenance of genome integrity and protection against cancer. Although PALB2 is commonly described as a repair factor recruited to sites of DNA breaks, recent studies provide evidence that PALB2 also associates with unperturbed chromatin. Here, we investigated the previously poorly described role of chromatin-associated PALB2 in undamaged cells. We found that PALB2 associates with active genes through its major binding partner, MRG15, which recognizes histone H3 trimethylated at lysine 36 (H3K36me3) by the SETD2 methyltransferase. Missense mutations that ablate PALB2 binding to MRG15 confer elevated sensitivity to the topoisomerase inhibitor camptothecin (CPT) and increased levels of aberrant metaphase chromosomes and DNA stress in gene bodies, which were suppressed by preventing DNA replication. Remarkably, the level of PALB2 at genic regions was frequently decreased, rather than increased, upon CPT treatment. We propose that the steady-state presence of PALB2 at active genes, mediated through the SETD2/H3K36me3/MRG15 axis, ensures an immediate response to DNA stress and therefore effective protection of these regions during DNA replication. This study provides a conceptual advance in demonstrating that the constitutive chromatin association of repair factors plays a key role in the maintenance of genome stability and furthers our understanding of why PALB2 defects lead to human genome instability syndromes.
Assuntos
Cromatina/ultraestrutura , Dano ao DNA , Proteína do Grupo de Complementação N da Anemia de Fanconi/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Fatores de Transcrição/metabolismo , Proteína BRCA2/genética , Linhagem Celular Tumoral , Cromossomos/ultraestrutura , Reparo do DNA , Replicação do DNA , Genoma Humano , Células HEK293 , Células HeLa , Humanos , Concentração Inibidora 50 , Mutação , Ligação Proteica , Proteômica , Transcrição Gênica , Proteínas Supressoras de Tumor/metabolismoRESUMO
Inhibition of type 1 IGF receptor (IGF-1R) sensitizes to DNA-damaging cancer treatments, and delays repair of DNA double strand breaks (DSBs) by non-homologous end-joining and homologous recombination (HR). In a recent screen for mediators of resistance to IGF-1R inhibitor AZ12253801, we identified RAD51, required for the strand invasion step of HR. These findings prompted us to test the hypothesis that IGF-1R-inhibited cells accumulate DSBs formed at endogenous DNA lesions, and depend on residual HR for their repair. Indeed, initial experiments showed time-dependent accumulation of γH2AX foci in IGF-1R -inhibited or -depleted prostate cancer cells. We then tested effects of suppressing HR, and found that RAD51 depletion enhanced AZ12253801 sensitivity in PTEN wild-type prostate cancer cells but not in cells lacking functional PTEN. Similar sensitization was induced in prostate cancer cells by depletion of BRCA2, required for RAD51 loading onto DNA, and in BRCA2(-/-) colorectal cancer cells, compared with isogenic BRCA2(+/-) cells. We also assessed chemical HR inhibitors, finding that RAD51 inhibitor BO2 blocked RAD51 focus formation and sensitized to AZ12253801. Finally, we tested CDK1 inhibitor RO-3306, which impairs HR by inhibiting CDK1-mediated BRCA1 phosphorylation. R0-3306 suppressed RAD51 focus formation consistent with HR attenuation, and sensitized prostate cancer cells to IGF-1R inhibition, with 2.4-fold reduction in AZ12253801 GI50 and 13-fold reduction in GI80. These data suggest that responses to IGF-1R inhibition are enhanced by genetic and chemical approaches to suppress HR, defining a population of cancers (PTEN wild-type, BRCA mutant) that may be intrinsically sensitive to IGF-1R inhibitory drugs.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Recombinação Homóloga/genética , Receptor IGF Tipo 1/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Western Blotting , Compostos de Boro/farmacologia , Proteína Quinase CDC2/antagonistas & inibidores , Proteína Quinase CDC2/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Histonas/metabolismo , Recombinação Homóloga/efeitos dos fármacos , Humanos , Isoxazóis/farmacologia , Masculino , Microscopia de Fluorescência , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Fosforilação/efeitos dos fármacos , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Pirimidinas/farmacologia , Quinolinas/farmacologia , Interferência de RNA , Rad51 Recombinase/antagonistas & inibidores , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Receptor IGF Tipo 1/antagonistas & inibidores , Tiazóis/farmacologiaRESUMO
The partner and localizer of breast cancer 2 susceptibility protein (PALB2) is crucial for the repair of DNA damage by homologous recombination. Here, we report that chromatin-association motif (ChAM), an evolutionarily conserved motif in PALB2, is necessary and sufficient to mediate its chromatin association in both unperturbed and damaged cells. ChAM is distinct from the previously described PALB2 DNA-binding regions. Deletion of ChAM decreases PALB2 and Rad51 accumulation at DNA damage sites and confers cellular hypersensitivity to the genotoxic drug mitomycin C. These results suggest that PALB2 chromatin association via ChAM facilitates PALB2 function in the cellular resistance to DNA damage.
Assuntos
Cromatina/metabolismo , Reparo do DNA , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Supressoras de Tumor/química , Proteínas Supressoras de Tumor/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Linhagem Celular , Sequência Conservada/genética , Dano ao DNA , Evolução Molecular , Proteína do Grupo de Complementação N da Anemia de Fanconi , Recombinação Homóloga/genética , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Nucleossomos/metabolismo , Ligação Proteica , Deleção de Sequência , Relação Estrutura-AtividadeRESUMO
p21 is a well-established regulator of cell cycle progression. The role of p21 in DNA repair, however, remains poorly characterized. Here, we describe a critical role of p21 in a replication-coupled DNA double-strand break (DSB) repair that is mechanistically distinct from its cell cycle checkpoint function. We demonstrate that p21-deficient cells exhibit elevated chromatid-type aberrations, including gaps and breaks, dicentrics and radial formations, following exposure to several DSB-inducing agents. p21-/- cells also exhibit an increased DNA damage-inducible DNA-PKCS S2056 phosphorylation, indicative of elevated non-homologous DNA end joining. Concomitantly, p21-/- cells are defective in replication-coupled homologous recombination (HR), exhibiting decreased sister chromatid exchanges and HR-dependent repair as determined using a crosslinked GFP reporter assay. Importantly, we establish that the DSB hypersensitivity of p21-/- cells is associated with increased cyclin-dependent kinase (CDK)-dependent BRCA2 S3291 phosphorylation and MRE11 nuclear foci formation and can be rescued by inhibition of CDK or MRE11 nuclease activity. Collectively, our results uncover a novel mechanism by which p21 regulates the fidelity of replication-coupled DSB repair and the maintenance of chromosome stability distinct from its role in the G1-S phase checkpoint.
Assuntos
Inibidor de Quinase Dependente de Ciclina p21/fisiologia , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Replicação do DNA , Animais , Proteína BRCA2/metabolismo , Camptotecina/toxicidade , Instabilidade Cromossômica , Reagentes de Ligações Cruzadas/toxicidade , Inibidor de Quinase Dependente de Ciclina p21/genética , Reparo do DNA por Junção de Extremidades , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , Etoposídeo/toxicidade , Pontos de Checagem da Fase G1 do Ciclo Celular , Células HCT116 , Células HeLa , Humanos , Proteína Homóloga a MRE11 , Camundongos , Mitomicina/toxicidade , Fosforilação , Reparo de DNA por Recombinação , Inibidores da Topoisomerase/toxicidadeRESUMO
Detecting DNA breaks in defined regions of the genome is critical to advancing our understanding of genome stability maintenance. Here, we present exo-FISH, a protocol to label exposed single-stranded DNA in defined repetitive regions of mammalian genomes by combining in vitro restriction enzyme digestion on fixed cells with fluorescence in situ hybridization (FISH). We describe steps for cell harvesting and fixation, slide treatments, and FISH probe hybridization. We then detail procedures for imaging and analysis. For complete details on the use and execution of this protocol, please refer to Saayman et al. (2023).1.
Assuntos
DNA , Sequências Repetitivas de Ácido Nucleico , Animais , Hibridização in Situ Fluorescente/métodos , DNA/genética , Sequências Repetitivas de Ácido Nucleico/genética , DNA de Cadeia Simples , Quebras de DNA , Mamíferos/genéticaRESUMO
The human breast cancer susceptibility gene BRCA2 is required for the regulation of RAD51-mediated homologous recombinational repair. BRCA2 interacts with RAD51 monomers, as well as nucleoprotein filaments, primarily though the conserved BRC motifs. The unrelated C-terminal region of BRCA2 also interacts with RAD51. Here we show that the BRCA2 C terminus interacts directly with RAD51 filaments, but not monomers, by binding an interface created by two adjacent RAD51 protomers. These interactions stabilize filaments so that they cannot be dissociated by association with BRC repeats. Interaction of the BRCA2 C terminus with the RAD51 filament causes a large movement of the flexible RAD51 N-terminal domain that is important in regulating filament dynamics. We suggest that interactions of the BRCA2 C-terminal region with RAD51 may facilitate efficient nucleation of RAD51 multimers on DNA and thereby stimulate recombination-mediated repair.
Assuntos
Proteína BRCA2/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Nucleoproteínas/metabolismo , Rad51 Recombinase/metabolismo , Proteínas Reguladoras de Apoptose , Proteína BRCA2/genética , Cromatografia em Gel , Reparo do DNA/fisiologia , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Microscopia Eletrônica , Modelos Biológicos , Modelos Moleculares , Nucleoproteínas/ultraestrutura , Ligação Proteica , Rad51 Recombinase/genéticaRESUMO
DNA double-strand breaks (DSBs) can result from both exogenous and endogenous sources and are potentially toxic lesions to the human genome. If improperly repaired, DSBs can threaten genome integrity and contribute to premature ageing, neurodegenerative disorders and carcinogenesis. Through decades of work on genome stability, it has become evident that certain regions of the genome are inherently more prone to breakage than others, known as genome instability hotspots. Recent advancements in sequencing-based technologies now enable the profiling of genome-wide distributions of DSBs, also known as breakomes, to systematically map these instability hotspots. Here, we review the application of these technologies and their implications for our current understanding of the genomic regions most likely to drive genome instability. These breakomes ultimately highlight both new and established breakage hotspots including actively transcribed regions, loop boundaries and early-replicating regions of the genome. Further, these breakomes challenge the paradigm that DNA breakage primarily occurs in hard-to-replicate regions. With these advancements, we begin to gain insights into the biological mechanisms both invoking and protecting against genome instability.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Animais , DNA/genética , Reparo do DNA/genética , Genoma Humano , Instabilidade Genômica , Humanos , Mamíferos/genéticaRESUMO
The tumour suppressor PALB2 stimulates RAD51-mediated homologous recombination (HR) repair of DNA damage, whilst its steady-state association with active genes protects these loci from replication stress. Here, we report that the lysine acetyltransferases 2A and 2B (KAT2A/2B, also called GCN5/PCAF), two well-known transcriptional regulators, acetylate a cluster of seven lysine residues (7K-patch) within the PALB2 chromatin association motif (ChAM) and, in this way, regulate context-dependent PALB2 binding to chromatin. In unperturbed cells, the 7K-patch is targeted for KAT2A/2B-mediated acetylation, which in turn enhances the direct association of PALB2 with nucleosomes. Importantly, DNA damage triggers a rapid deacetylation of ChAM and increases the overall mobility of PALB2. Distinct missense mutations of the 7K-patch render the mode of PALB2 chromatin binding, making it either unstably chromatin-bound (7Q) or randomly bound with a reduced capacity for mobilisation (7R). Significantly, both of these mutations confer a deficiency in RAD51 foci formation and increase DNA damage in S phase, leading to the reduction of overall cell survival. Thus, our study reveals that acetylation of the ChAM 7K-patch acts as a molecular switch to enable dynamic PALB2 shuttling for HR repair while protecting active genes during DNA replication.
Assuntos
Cromatina , Proteínas Supressoras de Tumor , Acetilação , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Reparo do DNA , Dano ao DNA , NucleossomosRESUMO
Inherited mutations in BRCA2 are associated with a predisposition to early-onset breast cancers. The underlying basis of tumorigenesis is thought to be linked to defects in DNA double-strand break repair by homologous recombination. Here we show that the carboxy-terminal region of BRCA2, which interacts directly with the essential recombination protein RAD51, contains a site (serine 3291; S3291) that is phosphorylated by cyclin-dependent kinases. Phosphorylation of S3291 is low in S phase when recombination is active, but increases as cells progress towards mitosis. This modification blocks C-terminal interactions between BRCA2 and RAD51. However, DNA damage overcomes cell cycle regulation by decreasing S3291 phosphorylation and stimulating interactions with RAD51. These results indicate that S3291 phosphorylation might provide a molecular switch to regulate RAD51 recombination activity, providing new insight into why BRCA2 C-terminal deletions lead to radiation sensitivity and cancer predisposition.
Assuntos
Proteína BRCA2/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Reparo do DNA/genética , Recombinação Genética/genética , Sequência de Aminoácidos , Animais , Proteína BRCA2/química , Proteína BRCA2/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular , Dano ao DNA/genética , Proteínas de Ligação a DNA/metabolismo , Humanos , Dados de Sequência Molecular , Sinais de Localização Nuclear , Fosforilação , Ligação Proteica , Rad51 Recombinase , Deleção de Sequência/genéticaRESUMO
The RAD51 recombinase plays critical roles in safeguarding genome integrity, which is fundamentally important for all living cells. While interphase functions of RAD51 in maintaining genome stability are well-characterised, its role in mitosis remains contentious. In this study, we show that RAD51 protects under-replicated DNA in mitotic human cells and, in this way, promotes mitotic DNA synthesis (MiDAS) and successful chromosome segregation. In cells experiencing mild replication stress, MiDAS was detected irrespective of mitotically generated DNA damage. MiDAS broadly required de novo RAD51 recruitment to single-stranded DNA, which was supported by the phosphorylation of RAD51 by the key mitotic regulator Polo-like kinase 1. Importantly, acute inhibition of MiDAS delayed anaphase onset and induced centromere fragility, suggesting a mechanism that prevents the satisfaction of the spindle assembly checkpoint while chromosomal replication remains incomplete. This study hence identifies an unexpected function of RAD51 in promoting genomic stability in mitosis.
Assuntos
Anáfase/genética , Cromatina/metabolismo , Reparo do DNA , Pontos de Checagem da Fase M do Ciclo Celular/genética , Rad51 Recombinase/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Segregação de Cromossomos , DNA/biossíntese , Dano ao DNA , Replicação do DNA , Instabilidade Genômica , Humanos , Microscopia Intravital , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Quinase 1 Polo-LikeRESUMO
Monopolar spindle-one binder (MOBs) proteins are evolutionarily conserved and contribute to various cellular signalling pathways. Recently, we reported that hMOB2 functions in preventing the accumulation of endogenous DNA damage and a subsequent p53/p21-dependent G1/S cell cycle arrest in untransformed cells. However, the question of how hMOB2 protects cells from endogenous DNA damage accumulation remained enigmatic. Here, we uncover hMOB2 as a regulator of double-strand break (DSB) repair by homologous recombination (HR). hMOB2 supports the phosphorylation and accumulation of the RAD51 recombinase on resected single-strand DNA (ssDNA) overhangs. Physiologically, hMOB2 expression supports cancer cell survival in response to DSB-inducing anti-cancer compounds. Specifically, loss of hMOB2 renders ovarian and other cancer cells more vulnerable to FDA-approved PARP inhibitors. Reduced MOB2 expression correlates with increased overall survival in patients suffering from ovarian carcinoma. Taken together, our findings suggest that hMOB2 expression may serve as a candidate stratification biomarker of patients for HR-deficiency targeted cancer therapies, such as PARP inhibitor treatments.
Assuntos
Neoplasias , Inibidores de Poli(ADP-Ribose) Polimerases , Linhagem Celular Tumoral , Dano ao DNA , Reparo do DNA , Recombinação Homóloga , Humanos , Neoplasias/tratamento farmacológico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêuticoRESUMO
The maintenance of genome integrity is essential for the regulation of cell proliferation and differentiation. DNA must be accurately duplicated and segregated to daughter cells at cell division, a process that is primarily regulated by cyclin-dependent kinases (CDKs). During cell growth, however, it is inevitable that DNA breaks will occur due to endogenous and exogenous stresses. Interestingly, there is increasing evidence that the catalytic activities of CDKs play critical roles in the DNA damage response, especially in the case of damage repaired by the homologous recombination (HR) pathway. In this review, we outline current knowledge of CDK regulation and its roles both in the unperturbed cell cycle and in DNA damage responses, and discuss the physiological roles of CDKs in HR repair.
Assuntos
Quinases Ciclina-Dependentes/metabolismo , Reparo do DNA/fisiologia , Animais , Proteína BRCA2/metabolismo , Ciclo Celular , Divisão Celular , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Humanos , Rad51 Recombinase/metabolismo , RecQ Helicases/metabolismo , Recombinação Genética , Proteína de Replicação A/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
Background: Germline mutations in the PALB2 gene are associated with the genetic disorder Fanconi anaemia and increased predisposition to cancer. Disease-associated variants are mainly protein-truncating mutations, whereas a few missense substitutions are reported to perturb its interaction with breast cancer susceptibility proteins BRCA1 and BRCA2, which play essential roles in homology-directed repair (HDR). More recently, PALB2 was shown to associate with active genes independently of BRCA1, and through this mechanism, safeguards these regions from DNA replicative stresses. However, it is unknown whether PALB2 tumour suppressor function requires its chromatin association. Methods: Mining the public database of cancer mutations, we identified four potentially deleterious cancer-associated missense mutations within the PALB2 chromatin association motif (ChAM). To assess the impact of these mutations on PALB2 function, we generated cell lines expressing PALB2 variants harbouring corresponding ChAM mutations, and evaluated PALB2 chromatin association properties and the cellular resistance to camptothecin (CPT). Additionally, we examined the accumulation of γH2A.X and the RAD51 recombinase as readouts of DNA damage signalling and HDR, respectively. Results: We demonstrate that a small-cell lung cancer (SCLC)-associated T413S mutation in PALB2 impairs its chromatin association and confers reduced resistance to CPT, the only FDA-approved drug for relapsed SCLC. Unexpectedly, we found a less efficient γH2A.X nuclear foci formation in PALB2 T413S expressing cells, whereas a near-normal level of RAD51 nuclear foci was visible. Conclusions: These findings support the importance of PALB2 chromatin association in the suppression of tumours, including SCLC, an unusually aggressive type of cancer with poor prognosis. PALB2 T413S has little impact on RAD51 recruitment, likely due to its intact interaction with BRCA1 and BRCA2. However, this mutant shows inefficient DNA stress signalling. This finding sheds new light on the function of PALB2, playing a role in efficient DNA stress signalling through constitutive chromatin association.