RESUMO
Leukemia cells accumulate DNA damage, but altered DNA repair mechanisms protect them from apoptosis. We showed here that formaldehyde generated by serine/1-carbon cycle metabolism contributed to the accumulation of toxic DNA-protein crosslinks (DPCs) in leukemia cells, especially in driver clones harboring oncogenic tyrosine kinases (OTKs: FLT3(internal tandem duplication [ITD]), JAK2(V617F), BCR-ABL1). To counteract this effect, OTKs enhanced the expression of DNA polymerase theta (POLθ) via ERK1/2 serine/threonine kinase-dependent inhibition of c-CBL E3 ligase-mediated ubiquitination of POLθ and its proteasomal degradation. Overexpression of POLθ in OTK-positive cells resulted in the efficient repair of DPC-containing DNA double-strand breaks by POLθ-mediated end-joining. The transforming activities of OTKs and other leukemia-inducing oncogenes, especially of those causing the inhibition of BRCA1/2-mediated homologous recombination with and without concomitant inhibition of DNA-PK-dependent nonhomologous end-joining, was abrogated in Polq-/- murine bone marrow cells. Genetic and pharmacological targeting of POLθ polymerase and helicase activities revealed that both activities are promising targets in leukemia cells. Moreover, OTK inhibitors or DPC-inducing drug etoposide enhanced the antileukemia effect of POLθ inhibitor in vitro and in vivo. In conclusion, we demonstrated that POLθ plays an essential role in protecting leukemia cells from metabolically induced toxic DNA lesions triggered by formaldehyde, and it can be targeted to achieve a therapeutic effect.
Assuntos
Proteína BRCA1 , Dano ao DNA , Leucemia , Animais , Camundongos , Proteína BRCA2 , DNA/metabolismo , Leucemia/enzimologia , Leucemia/genética , DNA Polimerase tetaRESUMO
Growth arrest and DNA damage 45 (Gadd45) family genes, Gadd45A, Gadd45B, and GADD45 G are implicated as stress sensors that are rapidly induced upon genotoxic/physiological stress. They are involved in regulation of various cellular functions such as DNA repair, senescence, and cell cycle control. Gadd45 family of genes serve as tumor suppressors in response to different stimuli and defects in Gadd45 pathway can give rise to oncogenesis. More recently, Gadd45 has been shown to promote gene activation by demethylation and this function is important for transcriptional regulation and differentiation during development. Gadd45 serves as an adaptor for DNA repair factors to promote removal of 5-methylcytosine from DNA at gene specific loci. Therefore, Gadd45 serves as a powerful link between DNA repair and epigenetic gene regulation.
Assuntos
Proteínas de Ciclo Celular , Desmetilação do DNA , Pontos de Checagem do Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Dano ao DNA/genética , Reparo do DNA/genéticaRESUMO
Replication fork stalling can promote genomic instability, predisposing to cancer and other diseases. Stalled replication forks may be processed by sister chromatid recombination (SCR), generating error-free or error-prone homologous recombination (HR) outcomes. In mammalian cells, a long-standing hypothesis proposes that the major hereditary breast/ovarian cancer predisposition gene products, BRCA1 and BRCA2, control HR/SCR at stalled replication forks. Although BRCA1 and BRCA2 affect replication fork processing, direct evidence that BRCA gene products regulate homologous recombination at stalled chromosomal replication forks is lacking, due to a dearth of tools for studying this process. Here we report that the Escherichia coli Tus/Ter complex can be engineered to induce site-specific replication fork stalling and chromosomal HR/SCR in mouse cells. Tus/Ter-induced homologous recombination entails processing of bidirectionally arrested forks. We find that the Brca1 carboxy (C)-terminal tandem BRCT repeat and regions of Brca1 encoded by exon 11-two Brca1 elements implicated in tumour suppression-control Tus/Ter-induced homologous recombination. Inactivation of either Brca1 or Brca2 increases the absolute frequency of 'long-tract' gene conversions at Tus/Ter-stalled forks, an outcome not observed in response to a site-specific endonuclease-mediated chromosomal double-strand break. Therefore, homologous recombination at stalled forks is regulated differently from homologous recombination at double-strand breaks arising independently of a replication fork. We propose that aberrant long-tract homologous recombination at stalled replication forks contributes to genomic instability and breast/ovarian cancer predisposition in BRCA mutant cells.
Assuntos
Proteína BRCA1/metabolismo , Replicação do DNA , Proteínas de Escherichia coli/metabolismo , Recombinação Homóloga , Animais , Proteína BRCA1/química , Proteína BRCA1/genética , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Quebras de DNA de Cadeia Dupla , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Éxons/genética , Conversão Gênica/genética , Instabilidade Genômica/genética , Síndrome Hereditária de Câncer de Mama e Ovário/genética , CamundongosRESUMO
Ataxia telangiectasia mutated (ATM) is activated upon DNA double strand breaks (DSBs) and phosphorylates numerous DSB response proteins, including histone H2AX on serine 139 (Ser-139) to form γ-H2AX. Through interaction with MDC1, γ-H2AX promotes DSB repair by homologous recombination (HR). H2AX Ser-139 can also be phosphorylated by DNA-dependent protein kinase catalytic subunit and ataxia telangiectasia- and Rad3-related kinase. Thus, we tested whether ATM functions in HR, particularly that controlled by γ-H2AX, by comparing HR occurring at the euchromatic ROSA26 locus between mouse embryonic stem cells lacking either ATM, H2AX, or both. We show here that loss of ATM does not impair HR, including H2AX-dependent HR, but confers sensitivity to inhibition of poly(ADP-ribose) polymerases. Loss of ATM or H2AX has independent contributions to cellular sensitivity to ionizing radiation. The ATM-independent HR function of H2AX requires both Ser-139 phosphorylation and γ-H2AX/MDC1 interaction. Our data suggest that ATM is dispensable for HR, including that controlled by H2AX, in the context of euchromatin, excluding the implication of such an HR function in genomic instability, hypersensitivity to DNA damage, and poly(ADP-ribose) polymerase inhibition associated with ATM deficiency.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Recombinação Homóloga , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Proteínas Mutadas de Ataxia Telangiectasia , Western Blotting , Proteínas de Ciclo Celular/genética , Células Cultivadas , Cromonas/farmacologia , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Proteína Quinase Ativada por DNA/antagonistas & inibidores , Proteína Quinase Ativada por DNA/metabolismo , Proteínas de Ligação a DNA/genética , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/efeitos da radiação , Endodesoxirribonucleases/metabolismo , Histonas/genética , Histonas/metabolismo , Camundongos , Morfolinas/farmacologia , Fosforilação/efeitos dos fármacos , Fosforilação/efeitos da radiação , Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases , Poli(ADP-Ribose) Polimerases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas/genética , Proteínas/metabolismo , RNA não Traduzido , Radiação Ionizante , Serina/metabolismo , Proteínas Supressoras de Tumor/genéticaRESUMO
Epithelial tissue morphogenesis is accompanied by the formation of a polarity axis--a feature of tissue architecture that is initiated by the binding of integrins to the basement membrane. Polarity plays a crucial role in tissue homeostasis, preserving differentiation, cell survival and resistance to chemotherapeutic drugs among others. An important aspect in the maintenance of tissue homeostasis is genome integrity. As normal tissues frequently experience DNA double-strand breaks (DSBs), we asked how tissue architecture might participate in the DNA damage response. Using 3D culture models that mimic mammary glandular morphogenesis and tumor formation, we show that DSB repair activity is higher in basally polarized tissues, regardless of the malignant status of cells, and is controlled by hemidesmosomal integrin signaling. In the absence of glandular morphogenesis, in 2D flat monolayer cultures, basal polarity does not affect DNA repair activity but enhances H2AX phosphorylation, an early chromatin response to DNA damage. The nuclear mitotic apparatus protein 1 (NuMA), which controls breast glandular morphogenesis by acting on the organization of chromatin, displays a polarity-dependent pattern and redistributes in the cell nucleus of basally polarized cells upon the induction of DSBs. This is shown using high-content analysis of nuclear morphometric descriptors. Furthermore, silencing NuMA impairs H2AX phosphorylation--thus, tissue polarity and NuMA cooperate to maintain genome integrity.
Assuntos
Antígenos Nucleares/fisiologia , Reparo do DNA , Morfogênese , Proteínas Associadas à Matriz Nuclear/fisiologia , Células Acinares/metabolismo , Antígenos Nucleares/metabolismo , Membrana Basal/metabolismo , Mama/citologia , Técnicas de Cultura de Células , Proteínas de Ciclo Celular , Linhagem Celular , Linhagem Celular Tumoral , Polaridade Celular , Quebras de DNA de Cadeia Dupla , Células Epiteliais , Epitélio/crescimento & desenvolvimento , Feminino , Histonas/metabolismo , Humanos , Proteínas Associadas à Matriz Nuclear/metabolismoRESUMO
DNA polymerase theta (Polθ)-mediated end-joining (TMEJ) repairs DNA double-strand breaks and confers resistance to genotoxic agents. How Polθ is regulated at the molecular level to exert TMEJ remains poorly characterized. We find that Polθ interacts with and is PARylated by PARP1 in a HPF1-independent manner. PARP1 recruits Polθ to the vicinity of DNA damage via PARylation dependent liquid demixing, however, PARylated Polθ cannot perform TMEJ due to its inability to bind DNA. PARG-mediated de-PARylation of Polθ reactivates its DNA binding and end-joining activities. Consistent with this, PARG is essential for TMEJ and the temporal recruitment of PARG to DNA damage corresponds with TMEJ activation and dissipation of PARP1 and PAR. In conclusion, we show a two-step spatiotemporal mechanism of TMEJ regulation. First, PARP1 PARylates Polθ and facilitates its recruitment to DNA damage sites in an inactivated state. PARG subsequently activates TMEJ by removing repressive PAR marks on Polθ.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , DNA Polimerase teta , DNA Polimerase Dirigida por DNA , Poli(ADP-Ribose) Polimerase-1 , Humanos , Poli(ADP-Ribose) Polimerase-1/metabolismo , Poli(ADP-Ribose) Polimerase-1/genética , DNA Polimerase Dirigida por DNA/metabolismo , Poli Adenosina Difosfato Ribose/metabolismo , Dano ao DNA , Animais , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , DNA/metabolismo , DNA/genética , Células HEK293 , Poli ADP Ribosilação , Poli(ADP-Ribose) Polimerases/metabolismo , Poli(ADP-Ribose) Polimerases/genética , Proteínas de Transporte , Glicosídeo Hidrolases , Proteínas NuclearesRESUMO
DNA polymerase theta (Polθ) is a DNA helicase-polymerase protein that facilitates DNA repair and is synthetic lethal with homology-directed repair (HDR) factors. Thus, Polθ is a promising precision oncology drug-target in HDR-deficient cancers. Here, we characterize the binding and mechanism of action of a Polθ helicase (Polθ-hel) small-molecule inhibitor (AB25583) using cryo-EM. AB25583 exhibits 6 nM IC50 against Polθ-hel, selectively kills BRCA1/2-deficient cells, and acts synergistically with olaparib in cancer cells harboring pathogenic BRCA1/2 mutations. Cryo-EM uncovers predominantly dimeric Polθ-hel:AB25583 complex structures at 3.0-3.2 Å. The structures reveal a binding-pocket deep inside the helicase central-channel, which underscores the high specificity and potency of AB25583. The cryo-EM structures in conjunction with biochemical data indicate that AB25583 inhibits the ATPase activity of Polθ-hel helicase via an allosteric mechanism. These detailed structural data and insights about AB25583 inhibition pave the way for accelerating drug development targeting Polθ-hel in HDR-deficient cancers.
Assuntos
Microscopia Crioeletrônica , DNA Helicases , DNA Polimerase teta , DNA Polimerase Dirigida por DNA , Humanos , DNA Helicases/metabolismo , DNA Helicases/química , DNA Helicases/genética , DNA Helicases/antagonistas & inibidores , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/genética , Proteína BRCA2/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/química , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/química , Piperazinas/farmacologia , Piperazinas/química , Linhagem Celular Tumoral , Ftalazinas/farmacologia , Ftalazinas/química , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Modelos Moleculares , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/antagonistas & inibidores , Ligação ProteicaRESUMO
The DNA damage response (DDR) protein DNA Polymerase θ (Polθ) is synthetic lethal with homologous recombination (HR) factors and is therefore a promising drug target in BRCA1/2 mutant cancers. We discover an allosteric Polθ inhibitor (Polθi) class with 4-6 nM IC50 that selectively kills HR-deficient cells and acts synergistically with PARP inhibitors (PARPi) in multiple genetic backgrounds. X-ray crystallography and biochemistry reveal that Polθi selectively inhibits Polθ polymerase (Polθ-pol) in the closed conformation on B-form DNA/DNA via an induced fit mechanism. In contrast, Polθi fails to inhibit Polθ-pol catalytic activity on A-form DNA/RNA in which the enzyme binds in the open configuration. Remarkably, Polθi binding to the Polθ-pol:DNA/DNA closed complex traps the polymerase on DNA for more than forty minutes which elucidates the inhibitory mechanism of action. These data reveal a unique small-molecule DNA polymerase:DNA trapping mechanism that induces synthetic lethality in HR-deficient cells and potentiates the activity of PARPi.
Assuntos
Proteína BRCA1 , Inibidores de Poli(ADP-Ribose) Polimerases , Proteína BRCA1/genética , Proteína BRCA2/genética , DNA/metabolismo , Reparo do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Recombinação Homóloga , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , HumanosRESUMO
The double-strand break (DSB) repair pathway called microhomology-mediated end-joining (MMEJ) is thought to be dependent on DNA polymerase theta (Polθ) and occur independently of nonhomologous end-joining (NHEJ) factors. An unresolved question is whether MMEJ is facilitated by a single Polθ-mediated end-joining pathway or consists of additional undiscovered pathways. We find that human X-family Polλ, which functions in NHEJ, additionally exhibits robust MMEJ activity like Polθ. Polλ promotes MMEJ in mammalian cells independently of essential NHEJ factors LIG4/XRCC4 and Polθ, which reveals a distinct Polλ-dependent MMEJ mechanism. X-ray crystallography employing in situ photo-induced DSB formation captured Polλ in the act of stabilizing a microhomology-mediated DNA synapse with incoming nucleotide at 2.0 Å resolution and reveals how Polλ performs replication across a DNA synapse joined by minimal base-pairing. Last, we find that Polλ is semisynthetic lethal with BRCA1 and BRCA2. Together, these studies indicate Polλ MMEJ as a distinct DSB repair mechanism.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Animais , Humanos , Reparo do DNA por Junção de Extremidades , DNA , MamíferosRESUMO
Anticancer nucleosides are effective against solid tumors and hematological malignancies, but typically are prone to nucleoside metabolism resistance mechanisms. Using a nucleoside-specific multiplexed high-throughput screening approach, we discovered 4'-ethynyl-2'-deoxycytidine (EdC) as a third-generation anticancer nucleoside prodrug with preferential activity against diffuse large B-cell lymphoma (DLBCL) and acute lymphoblastic leukemia (ALL). EdC requires deoxycytidine kinase (DCK) phosphorylation for its activity and induced replication fork arrest and accumulation of cells in S-phase, indicating it acts as a chain terminator. A 2.1Å co-crystal structure of DCK bound to EdC and UDP reveals how the rigid 4'-alkyne of EdC fits within the active site of DCK. Remarkably, EdC was resistant to cytidine deamination and SAMHD1 metabolism mechanisms and exhibited higher potency against ALL compared to FDA approved nelarabine. Finally, EdC was highly effective against DLBCL tumors and B-ALL in vivo. These data characterize EdC as a pre-clinical nucleoside prodrug candidate for DLBCL and ALL.
RESUMO
The hereditary breast and ovarian cancer predisposition genes BRCA1 and BRCA2 account for the lion's share of heritable breast cancer risk in the human population. Loss of function of either gene results in defective homologous recombination (HR) and triggers genomic instability, accelerating breast tumorigenesis. A long-standing hypothesis proposes that BRCA1 and BRCA2 mediate HR following attempted replication across damaged DNA, ensuring error-free processing of the stalled replication fork. A recent paper describes a new replication fork protective function of BRCA2, which appears to collaborate with its HR function to suppress genomic instability.
Assuntos
Proteína BRCA2/fisiologia , Neoplasias da Mama/genética , Replicação do DNA , Recombinação Homóloga , Proteína BRCA1/metabolismo , Replicação do DNA/efeitos dos fármacos , Feminino , Instabilidade Genômica , Humanos , Hidroxiureia/farmacologia , MutaçãoRESUMO
DNA polymerase θ (Polθ) confers resistance to chemotherapy agents that cause DNA-protein crosslinks (DPCs) at double-strand breaks (DSBs), such as topoisomerase inhibitors. This suggests Polθ might facilitate DPC repair by microhomology-mediated end-joining (MMEJ). Here, we investigate Polθ repair of DSBs carrying DPCs by monitoring MMEJ in Xenopus egg extracts. MMEJ in extracts is dependent on Polθ, exhibits the MMEJ repair signature, and efficiently repairs 5' terminal DPCs independently of non-homologous end-joining and the replisome. We demonstrate that Polθ promotes the repair of 5' terminal DPCs in mammalian cells by using an MMEJ reporter and find that Polθ confers resistance to formaldehyde in addition to topoisomerase inhibitors. Dual deficiency in Polθ and tyrosyl-DNA phosphodiesterase 2 (TDP2) causes severe cellular sensitivity to etoposide, which demonstrates MMEJ as an independent DPC repair pathway. These studies recapitulate MMEJ in vitro and elucidate how Polθ confers resistance to etoposide.
Assuntos
Reagentes de Ligações Cruzadas/farmacologia , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/metabolismo , Animais , Linhagem Celular , DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/deficiência , DNA Polimerase Dirigida por DNA/genética , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Formaldeído/farmacologia , Humanos , Camundongos , Óvulo/metabolismo , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , RNA Guia de Cinetoplastídeos/metabolismo , Xenopus/crescimento & desenvolvimento , Xenopus/metabolismo , DNA Polimerase tetaRESUMO
Genome-embedded ribonucleotides arrest replicative DNA polymerases (Pols) and cause DNA breaks. Whether mammalian DNA repair Pols efficiently use template ribonucleotides and promote RNA-templated DNA repair synthesis remains unknown. We find that human Polθ reverse transcribes RNA, similar to retroviral reverse transcriptases (RTs). Polθ exhibits a significantly higher velocity and fidelity of deoxyribonucleotide incorporation on RNA versus DNA. The 3.2-Šcrystal structure of Polθ on a DNA/RNA primer-template with bound deoxyribonucleotide reveals that the enzyme undergoes a major structural transformation within the thumb subdomain to accommodate A-form DNA/RNA and forms multiple hydrogen bonds with template ribose 2'-hydroxyl groups like retroviral RTs. Last, we find that Polθ promotes RNA-templated DNA repair in mammalian cells. These findings suggest that Polθ was selected to accommodate template ribonucleotides during DNA repair.
Assuntos
DNA Polimerase Dirigida por DNA , RNA , Animais , DNA/química , Reparo do DNA , DNA Polimerase Dirigida por DNA/química , Desoxirribonucleotídeos , Humanos , Mamíferos/genética , RibonucleotídeosRESUMO
PARP inhibitor monotherapies are effective to treat patients with breast, ovary, prostate, and pancreatic cancer with BRCA1 mutations, but not to the much more frequent BRCA wild-type cancers. Searching for strategies that would extend the use of PARP inhibitors to BRCA1-proficient tumors, we found that the stability of BRCA1 protein following ionizing radiation (IR) is maintained by postphosphorylational prolyl-isomerization adjacent to Ser1191 of BRCA1, catalyzed by prolyl-isomerase Pin1. Extinction of Pin1 decreased homologous recombination (HR) to the level of BRCA1-deficient cells. Pin1 stabilizes BRCA1 by preventing ubiquitination of Lys1037 of BRCA1. Loss of Pin1, or introduction of a BRCA1-mutant refractory to Pin1 binding, decreased the ability of BRCA1 to localize to repair foci and augmented IR-induced DNA damage. In vitro growth of HR-proficient breast, prostate, and pancreatic cancer cells were modestly repressed by olaparib or Pin1 inhibition using all-trans retinoic acid (ATRA), while combination treatment resulted in near-complete block of cell proliferation. In MDA-MB-231 xenografts and triple-negative breast cancer patient-derived xenografts, either loss of Pin1 or ATRA treatment reduced BRCA1 expression and sensitized breast tumors to olaparib. Together, our study reveals that Pin1 inhibition, with clinical widely used ATRA, acts as an effective HR disrupter that sensitizes BRCA1-proficient tumors to PARP inhibition. SIGNIFICANCE: PARP inhibitors have been limited to treat homologous recombination-deficient tumors. All-trans retinoic acid, by inhibiting Pin1 and destabilizing BRCA1, extends benefit of PARP inhibitors to patients with homologous recombination-proficient tumors.See related commentary by Cai, p. 2977.
Assuntos
Inibidores de Poli(ADP-Ribose) Polimerases , Neoplasias de Mama Triplo Negativas , Proteína BRCA1/genética , Linhagem Celular Tumoral , Feminino , Humanos , Masculino , Peptidilprolil Isomerase de Interação com NIMA/genética , Peptidilprolil Isomerase , Ftalazinas/farmacologia , Ftalazinas/uso terapêutico , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genéticaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
DNA polymerase θ (Polθ) is a unique polymerase-helicase fusion protein that promotes microhomology-mediated end-joining (MMEJ) of DNA double-strand breaks (DSBs). How full-length human Polθ performs MMEJ at the molecular level remains unknown. Using a biochemical approach, we find that the helicase is essential for Polθ MMEJ of long ssDNA overhangs which model resected DSBs. Remarkably, Polθ MMEJ of ssDNA overhangs requires polymerase-helicase attachment, but not the disordered central domain, and occurs independently of helicase ATPase activity. Using single-particle microscopy and biophysical methods, we find that polymerase-helicase attachment promotes multimeric gel-like Polθ complexes that facilitate DNA accumulation, DNA synapsis, and MMEJ. We further find that the central domain regulates Polθ multimerization and governs its DNA substrate requirements for MMEJ. These studies identify unexpected functions for the helicase and central domain and demonstrate the importance of polymerase-helicase tethering in MMEJ and the structural organization of Polθ.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/fisiologia , DNA Helicases/metabolismo , DNA de Cadeia Simples/metabolismo , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Domínio Catalítico , Quebras de DNA , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , Humanos , Modelos Moleculares , Mutagênese Sítio-Dirigida , DNA Polimerase tetaRESUMO
Genetic studies in yeast indicate that RNA transcripts facilitate homology-directed DNA repair in a manner that is dependent on RAD52. The molecular basis for so-called RNA-DNA repair, however, remains unknown. Using reconstitution assays, we demonstrate that RAD52 directly cooperates with RNA as a sequence-directed ribonucleoprotein complex to promote two related modes of RNA-DNA repair. In a RNA-bridging mechanism, RAD52 assembles recombinant RNA-DNA hybrids that coordinate synapsis and ligation of homologous DNA breaks. In an RNA-templated mechanism, RAD52-mediated RNA-DNA hybrids enable reverse transcription-dependent RNA-to-DNA sequence transfer at DNA breaks that licenses subsequent DNA recombination. Notably, we show that both mechanisms of RNA-DNA repair are promoted by transcription of a homologous DNA template in trans. In summary, these data elucidate how RNA transcripts cooperate with RAD52 to coordinate homology-directed DNA recombination and repair in the absence of a DNA donor, and demonstrate a direct role for transcription in RNA-DNA repair.
Assuntos
Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , Reparo do DNA/fisiologia , RNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Reparo de DNA por Recombinação/genética , Reparo de DNA por Recombinação/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Microhomology-mediated end-joining (MMEJ) is an error-prone alternative double-strand break-repair pathway that uses sequence microhomology to recombine broken DNA. Although MMEJ has been implicated in cancer development, the mechanism of this pathway is unknown. We demonstrate that purified human DNA polymerase θ (Polθ) performs MMEJ of DNA containing 3' single-strand DNA overhangs with ≥2 bp of homology, including DNA modeled after telomeres, and show that MMEJ is dependent on Polθ in human cells. Our data support a mechanism whereby Polθ facilitates end-joining and microhomology annealing, then uses the opposing overhang as a template in trans to stabilize the DNA synapse. Polθ exhibits a preference for DNA containing a 5'-terminal phosphate, similarly to polymerases involved in nonhomologous end-joining. Finally, we identify a conserved loop domain that is essential for MMEJ and higher-order structures of Polθ that probably promote DNA synapse formation.
Assuntos
Reparo do DNA por Junção de Extremidades/fisiologia , DNA Polimerase Dirigida por DNA/fisiologia , Modelos Genéticos , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , DNA Polimerase Dirigida por DNA/química , Humanos , Modelos Moleculares , DNA Polimerase tetaRESUMO
Suppression of RAD52 causes synthetic lethality in BRCA-deficient cells. Yet pharmacological inhibition of RAD52, which binds single-strand DNA (ssDNA) and lacks enzymatic activity, has not been demonstrated. Here, we identify the small molecule 6-hydroxy-DL-dopa (6-OH-dopa) as a major allosteric inhibitor of the RAD52 ssDNA binding domain. For example, we find that multiple small molecules bind to and completely transform RAD52 undecamer rings into dimers, which abolishes the ssDNA binding channel observed in crystal structures. 6-OH-Dopa also disrupts RAD52 heptamer and undecamer ring superstructures, and suppresses RAD52 recruitment and recombination activity in cells with negligible effects on other double-strand break repair pathways. Importantly, we show that 6-OH-dopa selectively inhibits the proliferation of BRCA-deficient cancer cells, including those obtained from leukemia patients. Taken together, these data demonstrate small-molecule disruption of RAD52 rings as a promising mechanism for precision medicine in BRCA-deficient cancers.