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1.
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
2.
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
3.
Nat Struct Mol Biol ; 24(12): 1116-1123, 2017 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-29058711

RESUMO

Mammalian polymerase theta (Polθ) is a multifunctional enzyme that promotes error-prone DNA repair by alternative nonhomologous end joining (alt-NHEJ). Here we present structure-function analyses that reveal that, in addition to the polymerase domain, Polθ-helicase activity plays a central role during double-strand break (DSB) repair. Our results show that the helicase domain promotes chromosomal translocations by alt-NHEJ in mouse embryonic stem cells and also suppresses CRISPR-Cas9- mediated gene targeting by homologous recombination (HR). In vitro assays demonstrate that Polθ-helicase activity facilitates the removal of RPA from resected DSBs to allow their annealing and subsequent joining by alt-NHEJ. Consistent with an antagonistic role for RPA during alt-NHEJ, inhibition of RPA1 enhances end joining and suppresses recombination. Taken together, our results reveal that the balance between HR and alt-NHEJ is controlled by opposing activities of Polθ and RPA, providing further insight into the regulation of repair-pathway choice in mammalian cells.


Assuntos
Domínio Catalítico/genética , Reparo do DNA por Junção de Extremidades/genética , DNA Polimerase Dirigida por DNA/genética , Células-Tronco Embrionárias/citologia , Proteína de Replicação A/antagonistas & inibidores , Animais , Sistemas CRISPR-Cas/genética , Linhagem Celular , Quebras de DNA de Cadeia Dupla , Recombinação Homóloga/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína de Replicação A/genética , Relação Estrutura-Atividade , Translocação Genética/genética , DNA Polimerase teta
4.
Virology ; 507: 220-230, 2017 07.
Artigo em Inglês | MEDLINE | ID: mdl-28456021

RESUMO

The Epstein Barr virus (EBV) genome persists in infected host cells as a chromatinized episome and is subject to chromatin-mediated regulation. Binding of the host insulator protein CTCF to the EBV genome has an established role in maintaining viral latency type, and in other herpesviruses, loss of CTCF binding at specific regions correlates with viral reactivation. Here, we demonstrate that binding of PARP1, an important cofactor of CTCF, at the BZLF1 lytic switch promoter restricts EBV reactivation. Knockdown of PARP1 in the Akata-EBV cell line significantly increases viral copy number and lytic protein expression. Interestingly, CTCF knockdown has no effect on viral reactivation, and CTCF binding across the EBV genome is largely unchanged following reactivation. Moreover, EBV reactivation attenuates PARP activity, and Zta expression alone is sufficient to decrease PARP activity. Here we demonstrate a restrictive function of PARP1 in EBV lytic reactivation.


Assuntos
Infecções por Vírus Epstein-Barr/enzimologia , Infecções por Vírus Epstein-Barr/virologia , Herpesvirus Humano 4/fisiologia , Poli(ADP-Ribose) Polimerase-1/metabolismo , Regiões Promotoras Genéticas , Transativadores/genética , Ativação Viral , Linhagem Celular , Infecções por Vírus Epstein-Barr/genética , Regulação Viral da Expressão Gênica , Herpesvirus Humano 4/genética , Interações Hospedeiro-Patógeno , Humanos , Poli(ADP-Ribose) Polimerase-1/genética , Ligação Proteica , Transativadores/metabolismo , Latência Viral
5.
Chem Biol ; 22(11): 1491-1504, 2015 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-26548611

RESUMO

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.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Proteína Rad52 de Recombinação e Reparo de DNA/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/química , Regulação Alostérica , Apoptose/efeitos dos fármacos , Proteína BRCA1/deficiência , Proteína BRCA2/deficiência , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , Di-Hidroxifenilalanina/análogos & derivados , Di-Hidroxifenilalanina/química , Di-Hidroxifenilalanina/metabolismo , Di-Hidroxifenilalanina/toxicidade , Ensaio de Desvio de Mobilidade Eletroforética , Humanos , Concentração Inibidora 50 , Microscopia de Fluorescência , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Ligação Proteica , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Bibliotecas de Moléculas Pequenas/metabolismo , Bibliotecas de Moléculas Pequenas/toxicidade
6.
Nat Struct Mol Biol ; 22(3): 230-7, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25643323

RESUMO

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 teta
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