RESUMO
James German's work to establish the natural history and cancer risk associated with Bloom syndrome (BS) has had a strong influence on the generation of scientists and clinicians working to understand other RECQ deficiencies and heritable cancer predisposition syndromes. I summarize work by us and others below, inspired by James German's precedents with BS, to understand and compare BS with the other heritable RECQ deficiency syndromes with a focus on Werner syndrome (WS). What we know, unanswered questions and new opportunities are discussed, as are potential ways to treat or modify WS-associated disease mechanisms and pathways.
Assuntos
Síndrome de Bloom , RecQ Helicases , Síndrome de Werner , Humanos , RecQ Helicases/genética , RecQ Helicases/metabolismo , RecQ Helicases/deficiência , Síndrome de Bloom/genética , Síndrome de Werner/genética , História do Século XXRESUMO
Metazoan genomes are copied bidirectionally from thousands of replication origins. Replication initiation entails the assembly and activation of two CMG helicases (Cdc45â Mcm2-7â GINS) at each origin. This requires several replication firing factors (including TopBP1, RecQL4, and DONSON) whose exact roles are still under debate. How two helicases are correctly assembled and activated at each origin is a long-standing question. By visualizing the recruitment of GINS, Cdc45, TopBP1, RecQL4, and DONSON in real time, we uncovered that replication initiation is surprisingly dynamic. First, TopBP1 transiently binds to the origin and dissociates before the start of DNA synthesis. Second, two Cdc45 are recruited together, even though Cdc45 alone cannot dimerize. Next, two copies of DONSON and two GINS simultaneously arrive at the origin, completing the assembly of two CMG helicases. Finally, RecQL4 is recruited to the CMGâ DONSONâ DONSONâ CMG complex and promotes DONSON dissociation and CMG activation via its ATPase activity.
Assuntos
Proteínas de Ciclo Celular , Replicação do DNA , Imagem Individual de Molécula , Humanos , Proteínas de Ciclo Celular/metabolismo , Origem de Replicação , Animais , DNA Helicases/metabolismo , RecQ Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismoRESUMO
Generation of O6-methylguanine (O6-meG) by DNA-alkylating agents such as N-methyl N-nitrosourea (MNU) activates the multiprotein mismatch repair (MMR) complex and the checkpoint response involving ATR/CHK1 and ATM/CHK2 kinases, which may in turn trigger cell cycle arrest and apoptosis. The Bloom syndrome DNA helicase BLM interacts with the MMR complex, suggesting functional relevance to repair and checkpoint responses. We observed a strong interaction of BLM with MMR proteins in HeLa cells upon treatment with MNU as evidenced by co-immunoprecipitation as well as colocalization in the nucleus as revealed by dual immunofluorescence staining. Knockout of BLM sensitized HeLa MR cells to MNU-induced cell cycle disruption and enhanced expression of the apoptosis markers cleaved caspase-9 and PARP1. MNU-treated BLM-deficient cells also exhibited a greater number of 53BP1 foci and greater phosphorylation levels of H2AX at S139 and RPA32 at S8, indicating the accumulation of DNA double-strand breaks. These findings suggest that BLM prevents double-strand DNA breaks during the MMR-dependent DNA damage response and mitigates O6-meG-induced apoptosis.
Assuntos
Apoptose , Reparo de Erro de Pareamento de DNA , RecQ Helicases , Humanos , RecQ Helicases/metabolismo , RecQ Helicases/genética , Células HeLa , Quebras de DNA de Cadeia Dupla , Metilnitrosoureia/toxicidade , Síndrome de Bloom/genética , Síndrome de Bloom/metabolismo , Síndrome de Bloom/patologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli(ADP-Ribose) Polimerase-1/genéticaRESUMO
Homology-dependent targeted DNA integration, generally referred to as gene targeting, provides a powerful tool for precise genome modification; however, its fundamental mechanisms remain poorly understood in human cells. Here we reveal a noncanonical gene targeting mechanism that does not rely on the homologous recombination (HR) protein Rad51. This mechanism is suppressed by Rad52 inhibition, suggesting the involvement of single-strand annealing (SSA). The SSA-mediated gene targeting becomes prominent when DSB repair by HR or end-joining pathways is defective and does not require isogenic DNA, permitting 5% sequence divergence. Intriguingly, loss of Msh2, loss of BLM, and induction of a target-site DNA break all significantly and synergistically enhance SSA-mediated targeted integration. Most notably, SSA-mediated integration is cell cycle-independent, occurring in the G1 phase as well. Our findings provide unequivocal evidence for Rad51-independent targeted integration and unveil multiple mechanisms to regulate SSA-mediated targeted as well as random integration.
Assuntos
Ciclo Celular , Marcação de Genes , Proteína 2 Homóloga a MutS , Rad51 Recombinase , Proteína Rad52 de Recombinação e Reparo de DNA , Humanos , Rad51 Recombinase/metabolismo , Rad51 Recombinase/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Ciclo Celular/genética , Proteína 2 Homóloga a MutS/metabolismo , Proteína 2 Homóloga a MutS/genética , RecQ Helicases/metabolismo , RecQ Helicases/genética , Recombinação Homóloga , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Reparo do DNA por Junção de Extremidades , Fase G1/genéticaRESUMO
BACKGROUND: Telomeropathies are a group of inherited disorders caused by germline pathogenic variants in genes involved in telomere maintenance, resulting in excessive telomere attrition that affects several tissues, including hematopoiesis. RecQ and RTEL1 helicases contribute to telomere maintenance by unwinding telomeric structures such as G-quadruplexes (G4), preventing replication defects. Germline RTEL1 variants also are etiologic in telomeropathies. METHODS AND RESULTS: Here we investigated the expression of RecQ (RECQL1, BLM, WRN, RECQL4, and RECQL5) and RTEL1 helicase genes in peripheral blood mononuclear cells (PBMCs) from human telomeropathy patients. The mRNA expression levels of all RecQ helicases, but not RTEL1, were significantly downregulated in patients' primary cells. Reduced RecQ expression was not attributable to cell proliferative exhaustion, as RecQ helicases were not attenuated in T cells exhausted in vitro. An additional fifteen genes involved in DNA damage repair and RecQ functional partners also were downregulated in the telomeropathy cells. CONCLUSION: These findings indicate that the expression of RecQ helicases and functional partners involved in DNA repair is downregulated in PBMCs of telomeropathy patients.
Assuntos
Leucócitos Mononucleares , RecQ Helicases , Adulto , Feminino , Humanos , Masculino , DNA Helicases/genética , DNA Helicases/metabolismo , Reparo do DNA/genética , Leucócitos Mononucleares/metabolismo , RecQ Helicases/genética , RecQ Helicases/metabolismo , Telômero/metabolismo , Telômero/genética , Homeostase do Telômero/genéticaRESUMO
Homologous recombination (HR) is a key process for repairing DNA double strand breaks and for promoting genetic diversity. However, HR occurs unevenly across the genome, and certain genomic features can influence its activity. One such feature is the presence of guanine quadruplexes (G4s), stable secondary structures widely distributed throughout the genome. These G4s play essential roles in gene transcription and genome stability regulation. Especially, elevated G4 levels in cells deficient in the Bloom syndrome helicase (BLM) significantly enhance HR at G4 sites, potentially threatening genome stability. Here, we investigated the role of G4-binding protein Yin Yang-1 (YY1) in modulating HR at G4 sites in human cells. Our results show that YY1's binding to G4 structures suppresses sister chromatid exchange after BLM knockdown, and YY1's chromatin occupancy negatively correlates with the overall HR rate observed across the genome. By limiting RAD51 homolog 1 (RAD51) access, YY1 preferentially binds to essential genomic regions, shielding them from excessive HR. Our findings unveil a novel role of YY1-G4 interaction, revealing novel insights into cellular mechanisms involved in HR regulation.
Assuntos
Quadruplex G , Recombinação Homóloga , Rad51 Recombinase , Troca de Cromátide Irmã , Fator de Transcrição YY1 , Humanos , Fator de Transcrição YY1/metabolismo , Fator de Transcrição YY1/genética , Recombinação Homóloga/genética , Rad51 Recombinase/metabolismo , Rad51 Recombinase/genética , Troca de Cromátide Irmã/genética , RecQ Helicases/metabolismo , RecQ Helicases/genética , Cromatina/metabolismo , Cromatina/genética , Quebras de DNA de Cadeia Dupla , Instabilidade Genômica/genéticaRESUMO
The Mec1/ATR kinase is crucial for genome stability, yet the mechanism by which it prevents gross chromosomal rearrangements (GCRs) remains unknown. Here we find that in cells with deficient Mec1 signaling, GCRs accumulate due to the deregulation of multiple steps in homologous recombination (HR). Mec1 primarily suppresses GCRs through its role in activating the canonical checkpoint kinase Rad53, which ensures the proper control of DNA end resection. Upon loss of Rad53 signaling and resection control, Mec1 becomes hyperactivated and triggers a salvage pathway in which the Sgs1 helicase is recruited to sites of DNA lesions via the 911-Dpb11 scaffolds and phosphorylated by Mec1 to favor heteroduplex rejection and limit HR-driven GCR accumulation. Fusing an ssDNA recognition domain to Sgs1 bypasses the requirement of Mec1 signaling for GCR suppression and nearly eliminates D-loop formation, thus preventing non-allelic recombination events. We propose that Mec1 regulates multiple steps of HR to prevent GCRs while ensuring balanced HR usage when needed for promoting tolerance to replication stress.
Assuntos
Recombinação Homóloga , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Serina-Treonina Quinases , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Quinase do Ponto de Checagem 2/metabolismo , Quinase do Ponto de Checagem 2/genética , RecQ Helicases/metabolismo , RecQ Helicases/genética , Transdução de Sinais , Fosforilação , Aberrações Cromossômicas , Rearranjo GênicoRESUMO
To identify new molecular components of the Brh2-governed homologous recombination (HR)-network in the highly radiation-resistant fungus Ustilago maydis, we undertook a genetic screen for suppressors of blm-KR hydroxyurea (HU)-sensitivity. Twenty DNA-damage sensitive mutants were obtained, three of which showing slow-growth phenotypes. Focusing on the "normally" growing candidates we identified five mutations, two in previously well-defined genes (Rec2 and Rad51) and the remaining three in completely uncharacterized genes (named Rec3, Bls9 and Zdr1). A common feature among these novel factors is their prominent role in DNA repair. Rec3 contains the P-loop NTPase domain which is most similar to that found in U. maydis Rec2 protein, and like Rec2, Rec3 plays critical roles in induced allelic recombination, is crucial for completion of meiosis, and with regard to DNA repair Δrec3 and Δrec2 are epistatic to one another. Importantly, overexpression of Brh2 in Δrec3 can effectively restore DNA-damage resistance, indicating a close functional connection between Brh2 and Rec3. The Bls9 does not seem to have any convincing domains that would give a clue as to its function. Nevertheless, we present evidence that, besides being involved in DNA-repair, Bls9 is also necessary for HR between chromosome homologs. Moreover, Δbls9 showed epistasis with Δbrh2 with respect to killing by DNA-damaging agents. Both, Rec3 and Bls9, play an important role in protecting the genome from mutations. Zdr1 is Cys2-His2 zinc finger (C2H2-ZF) protein, whose loss does not cause a detectable change in HR. Also, the functions of both Bls9 and Zdr1 genes are dispensable in meiosis and sporulation. However, Zdr1 appears to have overlapping activities with Blm and Mus81 in protecting the organism from methyl methanesulfonate- and diepoxybutane-induced DNA-damage. Finally, while deletion of Rec3 and Zdr1 can suppress HU-sensitivity of blm-KR, Δgen1, and Δmus81 mutants, interestingly loss of Bls9 does not rescue HU-sensitivity of Δgen1.
Assuntos
Reparo do DNA , Proteínas Fúngicas , RecQ Helicases , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/genética , RecQ Helicases/metabolismo , RecQ Helicases/genética , Hidroxiureia/farmacologia , Dano ao DNA , Mutação , Recombinação Homóloga , Meiose , BasidiomycotaRESUMO
RECQL5 is a member of the conserved RecQ family of DNA helicases involved in the maintenance of genome stability that is specifically found in higher eukaryotes and associates with the elongating RNA polymerase II. To expand our understanding of its function we expressed human RECQL5 in the yeast Saccharomyces cerevisiae, which does not have a RECQL5 ortholog. We found that RECQL5 expression leads to cell growth inhibition, increased genotoxic sensitivity and transcription-associated hyperrecombination. Chromatin immunoprecipitation and transcriptomic analysis of yeast cells expressing human RECQL5 shows that this is recruited to transcribed genes and although it causes only a weak impact on gene expression, in particular at G + C-rich genes, it leads to a transcription termination defect detected as readthrough transcription. The data indicate that the interaction between RNAPII and RECQL5 is conserved from yeast to humans. Unexpectedly, however, the RECQL5-ID mutant, previously shown to have reduced the association with RNAPII in vitro, associates with the transcribing polymerase in cells. As a result, expression of RECQL5-ID leads to similar although weaker phenotypes than wild-type RECQL5 that could be transcription-mediated. Altogether, the data suggests that RECQL5 has the intrinsic ability to function in transcription-dependent and independent genome dynamics in S. cerevisiae.
Assuntos
Instabilidade Genômica , RecQ Helicases , Saccharomyces cerevisiae , Transcrição Gênica , Saccharomyces cerevisiae/genética , Instabilidade Genômica/genética , RecQ Helicases/genética , RecQ Helicases/metabolismo , Humanos , Transcrição Gênica/genética , RNA Polimerase II/genética , RNA Polimerase II/metabolismoRESUMO
Human RECQL4, a member of the RecQ helicase family, plays a role in maintaining genomic stability, but its precise function remains unclear. The N-terminus of RECQL4 has similarity to Sld2, a protein required for the firing of DNA replication origins in budding yeast. Consistent with this sequence similarity, the Xenopus laevis homolog of RECQL4 has been implicated in initiating DNA replication in egg extracts. To determine whether human RECQL4 is required for firing of DNA replication origins, we generated cells in which both RECQL4 alleles were targeted, resulting in either lack of protein expression (knock-out; KO) or expression of a full-length, mutant protein lacking helicase activity (helicase-dead; HD). Interestingly, both the RECQL4 KO and HD cells were viable and exhibited essentially identical origin firing profiles as the parental cells. Analysis of the rate of fork progression revealed increased rates in the RECQL4 KO cells, which might be indicative of decreased origin firing efficiency. Our results are consistent with human RECQL4 having a less critical role in firing of DNA replication origins, than its budding yeast homolog Sld2.
Assuntos
RecQ Helicases , Origem de Replicação , Animais , Humanos , RecQ Helicases/genética , RecQ Helicases/metabolismo , Replicação do DNA , Xenopus laevis/metabolismo , DNA/metabolismoRESUMO
Complex chromosomal rearrangements (CCRs) are often observed in clinical samples from patients with cancer and congenital diseases but are difficult to induce experimentally. Here, we report the first success in establishing animal models for CCRs. Mutation in Recql5, a crucial member of the DNA helicase RecQ family involved in DNA replication, transcription, and repair, enabled CRISPR/Cas9-mediated CCRs, establishing a mouse model containing triple fusion genes and megabase-sized inversions. Some of these structural features of individual chromosomal rearrangements use template switching and microhomology-mediated break-induced replication mechanisms and are reminiscent of the newly described phenomenon "chromoanasynthesis." These data show that Recql5 mutant mice could be a powerful tool to analyze the pathogenesis of CCRs (particularly chromoanasynthesis) whose underlying mechanisms are poorly understood. The Recql5 mutants generated in this study are to be deposited at key animal research facilities, thereby making them accessible for future research on CCRs.
Assuntos
Sistemas CRISPR-Cas , RecQ Helicases , Zigoto , Animais , RecQ Helicases/genética , RecQ Helicases/metabolismo , Camundongos , Zigoto/metabolismo , MutaçãoRESUMO
The Bloom syndrome (BLM) helicase is critical for alternative lengthening of telomeres (ALT), a homology-directed repair (HDR)-mediated telomere maintenance mechanism that is prevalent in cancers of mesenchymal origin. The DNA substrates that BLM engages to direct telomere recombination during ALT remain unknown. Here, we determine that BLM helicase acts on lagging strand telomere intermediates that occur specifically in ALT-positive cells to assemble a replication-associated DNA damage response. Loss of ATRX was permissive for BLM localization to ALT telomeres in S and G2, commensurate with the appearance of telomere C-strand-specific single-stranded DNA (ssDNA). DNA2 nuclease deficiency increased 5'-flap formation in a BLM-dependent manner, while telomere C-strand, but not G-strand, nicks promoted ALT. These findings define the seminal events in the ALT DNA damage response, linking aberrant telomeric lagging strand DNA replication with a BLM-directed HDR mechanism that sustains telomere length in a subset of human cancers.
Assuntos
Dano ao DNA , Replicação do DNA , RecQ Helicases , Homeostase do Telômero , Telômero , RecQ Helicases/metabolismo , RecQ Helicases/genética , Humanos , Telômero/metabolismo , Telômero/genética , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , Proteína Nuclear Ligada ao X/genética , Proteína Nuclear Ligada ao X/metabolismo , DNA Helicases/metabolismo , DNA Helicases/genética , Síndrome de Bloom/genética , Síndrome de Bloom/metabolismo , Síndrome de Bloom/enzimologia , Síndrome de Bloom/patologia , Linhagem Celular TumoralRESUMO
Replication fork reversal, a critical protective mechanism against replication stress in higher eukaryotic cells, is orchestrated via a series of coordinated enzymatic reactions. The Bloom syndrome gene product, BLM, a member of the highly conserved RecQ helicase family, is implicated in this process, yet its precise regulation and role remain poorly understood. In this study, we demonstrate that the GCFC domain-containing protein TFIP11 forms a complex with the BLM helicase. TFIP11 exhibits a preference for binding to DNA substrates that mimic the structure generated at stalled replication forks. Loss of either TFIP11 or BLM leads to the accumulation of the other protein at stalled forks. This abnormal accumulation, in turn, impairs RAD51-mediated fork reversal and slowing, sensitizes cells to replication stress-inducing agents, and enhances chromosomal instability. These findings reveal a previously unidentified regulatory mechanism that modulates the activities of BLM and RAD51 at stalled forks, thereby impacting genome integrity.
Assuntos
Tolerância ao Dano no DNA , Replicação do DNA , Humanos , RecQ Helicases/genética , RecQ Helicases/metabolismo , DNA/genética , DNA/metabolismo , Proteínas/metabolismo , Instabilidade Genômica , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Fatores de Processamento de RNA/metabolismoRESUMO
We recently demonstrated that 1,6-hexanediol inhibits the formation of assemblysomes. These membraneless cell organelles have important roles in co-translational protein complex assembly and also store halfway translated DNA damage response proteins for a timely stress response. Recognizing the therapeutic potential of 1,6-hexanediol in dismantling assemblysomes likely to be involved in chemo- or radiotherapy resistance of tumor cells, we initiated an investigation into the properties of 1,6-hexanediol. Our particular interest was to determine if this compound induces DNA double-strand breaks by releasing the BLM helicase. Its yeast ortholog Sgs1 was confirmed to be a component of assemblysomes. The BLM helicase induces DNA damage when overexpressed due to the DNA double-strand breaks it generates during its normal function to repair DNA damage sites. It is evident that storing Sgs1 helicase in assemblysomes is crucial to express the full-length functional protein only in the event of DNA damage. Alternatively, if we dissolve assemblysomes using 1,6-hexanediol, ribosome-nascent chain complexes might become targets of ribosome quality control. We explored these possibilities and found, through the Drosophila wing-spot test assay, that 1,6-hexanediol induces DNA double-strand breaks. Lethality connected to recombination events following 1,6-hexanediol treatment can be mitigated by inducing DNA double-strand breaks with X-ray. Additionally, we confirmed that SMC5 recruits DmBLM to DNA damage sites, as knocking it down abolishes the rescue effect of DNA double-strand breaks on 1,6-hexanediol-induced lethality in Drosophila melanogaster.
Assuntos
DNA Helicases , Proteínas de Drosophila , Drosophila melanogaster , Glicóis , Animais , DNA/metabolismo , DNA Helicases/metabolismo , Reparo do DNA , Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Recombinação Homóloga , RecQ Helicases/genética , RecQ Helicases/metabolismoRESUMO
Many patients with hepatocellular carcinoma (HCC) respond poorly to radiotherapy despite remarkable advances in treatment. A deeper insight into the mechanism of sensitivity of HCC to this therapy is urgently required. It is demonstrated that RECQL4 is upregulated in the malignant cells of patients with HCC. Elevated RECQL4 levels reduce the sensitivity of HCC to radiotherapy by repairing radiation-induced double-stranded DNA (dsDNA) fragments. Mechanistically, the inhibitory effect of RECQL4 on radiotherapy is due to the reduced recruitment of dendritic cells and CD8+ T cells in the tumor microenvironment (TME). RECQL4 disrupts the radiation-induced transformation of the TME into a tumoricidal niche by inhibiting the cGAS-STING pathway in dendritic cells. Knocking out STING in dendritic cells can block the impact of RECQL4 on HCC radiosensitivity. Notably, high RECQL4 expressions in HCC is significantly associated with poor prognosis in multiple independent cohorts. In conclusion, this study highlights how HCC-derived RECQL4 disrupts cGAS-STING pathway activation in dendritic cells through DNA repair, thus reducing the radiosensitivity of HCC. These findings provide new perspectives on the clinical treatment of HCC.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Proteínas de Membrana , Nucleotidiltransferases , RecQ Helicases , Transdução de Sinais , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/imunologia , Carcinoma Hepatocelular/radioterapia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/radioterapia , Neoplasias Hepáticas/imunologia , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Animais , RecQ Helicases/genética , RecQ Helicases/metabolismo , Microambiente Tumoral/imunologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Modelos Animais de Doenças , Tolerância a Radiação/genética , Linhagem Celular TumoralRESUMO
The upregulation of RecQ helicases has been associated with cancer cell survival and resistance to chemotherapy, making them appealing targets for therapeutic intervention. In this study, twenty-nine novel quinazolinone derivatives were designed and synthesized. The anti-proliferative activity of all compounds was evaluated against 60 cancer cell lines at the National Cancer Institute Developmental Therapeutic Program, with six compounds (11f, 11g, 11k, 11n, 11p, and 11q) being promoted to a five-dose screen. Compound 11g demonstrated high cytotoxic activity against all examined cell lines. The compounds were further assayed for Bloom syndrome (BLM) helicase inhibition, where 11g, 11q, and 11u showed moderate activity. These compounds were counter-screened against WRN and RECQ1 helicases, where 11g moderately inhibited both enzymes. An ATP competition assay confirmed that the compounds bound to the ATP site of RecQ helicases, and molecular docking simulations were used to study the binding mode within the active site of BLM, WRN, and RECQ1 helicases. Compound 11g induced apoptosis in both HCT-116 and MDA-MB-231 cell lines, but also caused an G2/M phase cell cycle arrest in HCT-116 cells. This data revealed the potential of 11g as a modulator of cell cycle dynamics and supports its interaction with RecQ helicases. In addition, compound 11g displayed non-significant toxicity against FCH normal colon cells at doses up to 100 µM, which confirming its high safety margin and selectivity on cancer cells. Overall, these findings suggest compound 11g as a potential pan RecQ helicase inhibitor with high anticancer potency and a favorable safety margin and selectivity.
Assuntos
Antineoplásicos , RecQ Helicases , Simulação de Acoplamento Molecular , RecQ Helicases/metabolismo , Quinazolinonas/farmacologia , Antineoplásicos/farmacologia , Trifosfato de AdenosinaRESUMO
The Bloom syndrome helicase BLM interacts with topoisomerase IIIα (TOP3A), RMI1, and RMI2 to form the BTR complex, which dissolves double Holliday junctions and DNA replication intermediates to promote sister chromatid disjunction before cell division. In its absence, structure-specific nucleases like the SMX complex (comprising SLX1-SLX4, MUS81-EME1, and XPF-ERCC1) can cleave joint DNA molecules instead, but cells deficient in both BTR and SMX are not viable. Here, we identify a negative genetic interaction between BLM loss and deficiency in the BRCA1-BARD1 tumor suppressor complex. We show that this is due to a previously overlooked role for BARD1 in recruiting SLX4 to resolve DNA intermediates left unprocessed by BLM in the preceding interphase. Consequently, cells with defective BLM and BRCA1-BARD1 accumulate catastrophic levels of chromosome breakage and micronucleation, leading to cell death. Thus, we reveal mechanistic insights into SLX4 recruitment to DNA lesions, with potential clinical implications for treating BRCA1-deficient tumors.
Assuntos
Proteínas de Ligação a DNA , Recombinases , Humanos , DNA/genética , Reparo do DNA , Replicação do DNA , DNA Cruciforme , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Recombinases/genética , RecQ Helicases/genética , RecQ Helicases/metabolismoRESUMO
Leukemia is a type of malignant hematological disease that is generally resistant to chemotherapy and has poor therapeutic outcomes. Werner (WRN) DNA helicase, an important member of the RecQ family of helicases, plays an important role in DNA repair and telomere stability maintenance. WRN gene dysfunction leads to premature aging and predisposes humans to various types of cancers. However, the biological function of WRN in cancer remains unknown. In this study, the expression of this RecQ family helicase was investigated in different types of leukemia cells, and the leukemia cell line K562 with high WRN expression was selected to construct a WRN knockdown cell line. The results showed that WRN knockdown inhibited leukemia occurrence and development by regulating the proliferation, cell cycle, differentiation, and aging of cells and other biological processes. The results of transcriptome sequencing revealed that WRN promoted the sensitivity of leukemia cells to the DNA damage inducer Etoposide by regulating cell cycle-related proteins, such as CDC2, cyclin B1, p16, and p21, as well as key proteins in DNA damage repair pathways, such as p53, RAD50, RAD51, and MER11. Our findings show that WRN helicase is a promising potential target for leukemia treatment, providing new ideas for the development of targeted drugs against leukemia.
Assuntos
Exodesoxirribonucleases , Leucemia , Humanos , Helicase da Síndrome de Werner/genética , Helicase da Síndrome de Werner/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , RecQ Helicases/genética , RecQ Helicases/metabolismo , Ciclo Celular/genética , Reparo do DNA , Dano ao DNA , Leucemia/genéticaRESUMO
Addiction to the WRN helicase is a unique vulnerability of human cancers with high levels of microsatellite instability (MSI-H). However, while prolonged loss of WRN ultimately leads to cell death, little is known about how MSI-H cancers initially respond to acute loss of WRN-knowledge that would be helpful for informing clinical development of WRN targeting therapy, predicting possible resistance mechanisms, and identifying useful biomarkers of successful WRN inhibition. Here, we report the construction of an inducible ligand-mediated degradation system in which the stability of endogenous WRN protein can be rapidly and specifically tuned, enabling us to track the complete sequence of cellular events elicited by acute loss of WRN function. We found that WRN degradation leads to immediate accrual of DNA damage in a replication-dependent manner that curiously did not robustly engage checkpoint mechanisms to halt DNA synthesis. As a result, WRN-degraded MSI-H cancer cells accumulate DNA damage across multiple replicative cycles and undergo successive rounds of increasingly aberrant mitoses, ultimately triggering cell death. Of potential therapeutic importance, we found no evidence of any generalized mechanism by which MSI-H cancers could adapt to near-complete loss of WRN. However, under conditions of partial WRN degradation, addition of low-dose ATR inhibitor significantly increased their combined efficacy to levels approaching full inactivation of WRN. Overall, our results provide the first comprehensive view of molecular events linking upstream inhibition of WRN to subsequent cell death and suggest that dual targeting of WRN and ATR might be a useful strategy for treating MSI-H cancers.
Assuntos
Replicação do DNA , Neoplasias , Humanos , Replicação do DNA/genética , DNA Helicases/metabolismo , Repetições de Microssatélites , Dano ao DNA , Neoplasias/tratamento farmacológico , Neoplasias/genética , RecQ Helicases/genética , RecQ Helicases/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Helicase da Síndrome de Werner/genética , Helicase da Síndrome de Werner/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismoRESUMO
The high expression of BLM (Bloom syndrome) DNA helicase in tumors involves its strong association with cell expansion. Bisbenzylisoquinoline alkaloids own an antitumor property and have developed as candidates for anticancer drugs. This paper aimed to study the antitumor effect of fangchinoline derivative HY-2 by targeting BLM642-1290 DNA helicase, and then explore its inhibitory mechanism on proliferation of MDA-MB-435 breast cancer cells. We confirmed that the mRNA and protein levels of BLM DNA helicase in breast cancer were higher than those in normal tissues. HY-2 could inhibit the DNA binding, ATPase and DNA unwinding of BLM642-1290 DNA helicase with enzymatic assay. HY-2 could also inhibit the DNA unwinding of DNA helicase in cells. In addition, HY-2 showed an inhibiting the MDA-MB-435, MDA-MB-231, MDA-MB-436 breast cancer cells expansion. The mRNA and protein levels of BLM DNA helicase in MDA-MB-435 cells increased after HY-2 treatment, which might contribute to HY-2 inhibiting the DNA binding, ATPase and DNA unwinding of BLM DNA helicase. The mechanism of HY-2 inhibition on BLM DNA helicase was further confirmed with the effect of HY-2 on the ultraviolet spectrogram of BLM642-1290 DNA helicase and Molecular dynamics simulation of the interacting between HY-2 and BLM640-1291 DNA helicase. Our study provided some valuable clues for the exploration of HY-2 in the living body and developing it as an anticancer drug.