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1.
Nat Commun ; 15(1): 2862, 2024 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-38580648

RESUMO

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 , Humanos
2.
Mol Cell ; 83(13): 2258-2275.e11, 2023 07 06.
Artigo em Inglês | MEDLINE | ID: mdl-37369199

RESUMO

The pre-mRNA life cycle requires intron processing; yet, how intron-processing defects influence splicing and gene expression is unclear. Here, we find that TTDN1/MPLKIP, which is encoded by a gene implicated in non-photosensitive trichothiodystrophy (NP-TTD), functionally links intron lariat processing to spliceosomal function. The conserved TTDN1 C-terminal region directly binds lariat debranching enzyme DBR1, whereas its N-terminal intrinsically disordered region (IDR) binds the intron-binding complex (IBC). TTDN1 loss, or a mutated IDR, causes significant intron lariat accumulation, as well as splicing and gene expression defects, mirroring phenotypes observed in NP-TTD patient cells. A Ttdn1-deficient mouse model recapitulates intron-processing defects and certain neurodevelopmental phenotypes seen in NP-TTD. Fusing DBR1 to the TTDN1 IDR is sufficient to recruit DBR1 to the IBC and circumvents the functional requirement for TTDN1. Collectively, our findings link RNA lariat processing with splicing outcomes by revealing the molecular function of TTDN1.


Assuntos
Síndromes de Tricotiodistrofia , Animais , Camundongos , Íntrons/genética , Síndromes de Tricotiodistrofia/genética , RNA Nucleotidiltransferases/genética , Splicing de RNA
3.
J Biol Chem ; 298(2): 101545, 2022 02.
Artigo em Inglês | MEDLINE | ID: mdl-34971705

RESUMO

Alkylation of DNA and RNA is a potentially toxic lesion that can result in mutations and even cell death. In response to alkylation damage, K63-linked polyubiquitin chains are assembled that localize the Alpha-ketoglutarate-dependent dioxygenase alkB homolog 3-Activating Signal Cointegrator 1 Complex Subunit (ASCC) repair complex to damage sites in the nucleus. The protein ASCC2, a subunit of the ASCC complex, selectively binds K63-linked polyubiquitin chains via its coupling of ubiquitin conjugation to ER degradation (CUE) domain. The basis for polyubiquitin-binding specificity was unclear, because CUE domains in other proteins typically bind a single ubiquitin and do not discriminate among different polyubiquitin linkage types. We report here that the ASCC2 CUE domain selectively binds K63-linked diubiquitin by contacting both the distal and proximal ubiquitin. The ASCC2 CUE domain binds the distal ubiquitin in a manner similar to that reported for other CUE domains bound to a single ubiquitin, whereas the contacts with the proximal ubiquitin are unique to ASCC2. Residues in the N-terminal portion of the ASCC2 α1 helix contribute to the binding interaction with the proximal ubiquitin of K63-linked diubiquitin. Mutation of residues within the N-terminal portion of the ASCC2 α1 helix decreases ASCC2 recruitment in response to DNA alkylation, supporting the functional significance of these interactions during the alkylation damage response. Our study reveals the versatility of CUE domains in ubiquitin recognition.


Assuntos
Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato , Reparo do DNA , Proteínas Nucleares , Poliubiquitina , Ubiquitina , Ubiquitinas , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/genética , Homólogo AlkB 3 da Dioxigenase Dependente de alfa-Cetoglutarato/metabolismo , DNA/metabolismo , Modelos Moleculares , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Poliubiquitina/genética , Poliubiquitina/metabolismo , Ligação Proteica , Ubiquitina/genética , Ubiquitina/metabolismo , Ubiquitinas/genética , Ubiquitinas/metabolismo
4.
Sci Adv ; 7(24)2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34117057

RESUMO

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 , Ribonucleotídeos
5.
Cell Rep ; 34(10): 108820, 2021 03 09.
Artigo em Inglês | MEDLINE | ID: mdl-33691100

RESUMO

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 teta
7.
Nat Commun ; 10(1): 4423, 2019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31562312

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 teta
8.
Nucleic Acids Res ; 47(7): 3272-3283, 2019 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-30818397

RESUMO

Site-specific modification of synthetic and cellular RNA such as with specific nucleobases, fluorophores and attachment chemistries is important for a variety of basic and applied research applications. However, simple and efficient methods to modify RNA such as at the 3' terminus with specific nucleobases or nucleotide analogs conjugated to various chemical moieties are lacking. Here, we develop and characterize a one-step enzymatic method to modify RNA 3' termini using recombinant human polymerase theta (Polθ). We demonstrate that Polθ efficiently adds 30-50 2'-deoxyribonucleotides to the 3' terminus of RNA molecules of various lengths and sequences, and extends RNA 3' termini with an assortment of 2'-deoxy and 2',3'-dideoxy ribonucleotide analogs containing functional chemistries, such as high affinity attachment moieties and fluorophores. In contrast to Polθ, terminal deoxynucleotidyl transferase (TdT) is unable to use RNA as a substrate altogether. Overall, Polθ shows a strong preference for adding deoxyribonucleotides to RNA, but can also add ribonucleotides with relatively high efficiency in particular sequence contexts. We anticipate that this unique activity of Polθ will become invaluable for applications requiring 3' terminal modification of RNA and potentially enzymatic synthesis of RNA.


Assuntos
DNA Polimerase Dirigida por DNA/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Regiões 3' não Traduzidas/genética , DNA Nucleotidilexotransferase/química , DNA Nucleotidilexotransferase/metabolismo , DNA Polimerase Dirigida por DNA/química , Humanos , RNA Mensageiro/genética , DNA Polimerase teta
9.
Sci Rep ; 8(1): 17384, 2018 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-30478404

RESUMO

The spread of Zika virus (ZIKV) has caused an international health emergency due to its ability to cause microcephaly in infants. Yet, our knowledge of how ZIKV replicates at the molecular level is limited. For example, how the non-structural protein 5 (NS5) performs replication, and in particular whether the N-terminal methytransferase (MTase) domain is essential for the function of the C-terminal RNA-dependent RNA polymerase (RdRp) remains unclear. In contrast to previous reports, we find that MTase is absolutely essential for all activities of RdRp in vitro. For instance, the MTase domain confers stability onto the RdRp elongation complex (EC) and and is required for de novo RNA synthesis and nucleotide incorporation by RdRp. Finally, structure function analyses identify key conserved residues at the MTase-RdRp interface that specifically activate RdRp elongation and are essential for ZIKV replication in Huh-7.5 cells. These data demonstrate the requirement for the MTase-RdRp interface in ZIKV replication and identify a specific site within this region as a potential site for therapeutic development.


Assuntos
Metiltransferases/genética , RNA Polimerase Dependente de RNA/genética , Replicação Viral/genética , Zika virus/genética , Sequência de Aminoácidos , Linhagem Celular Tumoral , Humanos , Proteínas não Estruturais Virais/genética , Infecção por Zika virus/virologia
10.
Nat Commun ; 9(1): 1091, 2018 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-29545568

RESUMO

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/metabolismo
11.
J Biol Chem ; 293(14): 5259-5269, 2018 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-29444826

RESUMO

POLQ is a unique multifunctional replication and repair gene that encodes for a N-terminal superfamily 2 helicase and a C-terminal A-family polymerase. Although the function of the polymerase domain has been investigated, little is understood regarding the helicase domain. Multiple studies have reported that polymerase θ-helicase (Polθ-helicase) is unable to unwind DNA. However, it exhibits ATPase activity that is stimulated by single-stranded DNA, which presents a biochemical conundrum. In contrast to previous reports, we demonstrate that Polθ-helicase (residues 1-894) efficiently unwinds DNA with 3'-5' polarity, including DNA with 3' or 5' overhangs, blunt-ended DNA, and replication forks. Polθ-helicase also efficiently unwinds RNA-DNA hybrids and exhibits a preference for unwinding the lagging strand at replication forks, similar to related HELQ helicase. Finally, we find that Polθ-helicase can facilitate strand displacement synthesis by Polθ-polymerase, suggesting a plausible function for the helicase domain. Taken together, these findings indicate nucleic acid unwinding as a relevant activity for Polθ in replication repair.


Assuntos
DNA Helicases/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Sequência de Aminoácidos , Cristalografia por Raios X , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades/fisiologia , Replicação do DNA/fisiologia , DNA de Cadeia Simples/metabolismo , DNA Polimerase Dirigida por DNA/fisiologia , Humanos , Hibridização de Ácido Nucleico , Ligação Proteica , Recombinação Genética/genética , DNA Polimerase teta
12.
Nucleic Acids Res ; 44(19): 9381-9392, 2016 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-27591252

RESUMO

DNA polymerase θ (Polθ) is a unique A-family polymerase that is essential for alternative end-joining (alt-EJ) of double-strand breaks (DSBs) and performs translesion synthesis. Because Polθ is highly expressed in cancer cells, confers resistance to ionizing radiation and chemotherapy agents, and promotes the survival of homologous recombination (HR) deficient cells, it represents a promising new cancer drug target. As a result, identifying substrates that are selective for this enzyme is a priority. Here, we demonstrate that Polθ efficiently and selectively incorporates into DNA large benzo-expanded nucleotide analogs (dxAMP, dxGMP, dxTMP, dxAMP) which exhibit canonical base-pairing and enhanced base stacking. In contrast, functionally related Y-family translesion polymerases exhibit a severely reduced ability to incorporate dxNMPs, and all other human polymerases tested from the X, B and A families fail to incorporate them under the same conditions as Polθ. We further find that Polθ is inhibited after multiple dxGMP incorporation events, and that Polθ efficiency for dxGMP incorporation approaches that of native dGMP. These data demonstrate a unique function for Polθ in incorporating synthetic large-sized nucleotides and suggest the future possibility of the use of dxG nucleoside or related prodrug analogs as selective inhibitors of Polθ activity.


Assuntos
Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA/genética , DNA/metabolismo , Humanos , Nucleotídeos/metabolismo , Ligação Proteica , DNA Polimerase teta
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