RESUMO
Translesion synthesis (TLS) is a mechanism of DNA damage tolerance utilized by eukaryotic cells to replicate DNA across lesions that impede the high-fidelity replication machinery. In TLS, a series of specialized DNA polymerases are employed, which recognize specific DNA lesions, insert nucleotides across the damage, and extend the distorted primer-template. This allows cells to preserve genetic integrity at the cost of mutations. In humans, TLS enzymes include the Y-family, inserter polymerases, Polη, Polι, Polκ, Rev1, and the B-family extender polymerase Polζ, while in S. cerevisiae only Polη, Rev1, and Polζ are present. To bypass DNA lesions, TLS polymerases cooperate, assembling into a complex on the eukaryotic sliding clamp, PCNA, termed the TLS mutasome. The mutasome assembly is contingent on protein-protein interactions (PPIs) between the modular domains and subunits of TLS enzymes, and their interactions with PCNA and DNA. While the structural mechanisms of DNA lesion bypass by the TLS polymerases and PPIs of their individual modules are well understood, the mechanisms by which they cooperate in the context of TLS complexes have remained elusive. This review focuses on structural studies of TLS polymerases and describes the case of TLS holoenzyme assemblies in action emerging from recent high-resolution Cryo-EM studies.
Assuntos
Dano ao DNA , Reparo do DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA , Antígeno Nuclear de Célula em Proliferação , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , Humanos , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Replicação do DNA/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , DNA/genética , DNA/metabolismo , Síntese de DNA TranslesãoRESUMO
Replication stress compromises genomic integrity. Fork blocking lesions such as those induced by cisplatin and other chemotherapeutic agents arrest replication forks. Repriming downstream of these lesions represents an important mechanism of replication restart, however the single stranded DNA (ssDNA) gaps left behind, unless efficiently filled, can serve as entry point for nucleases. Nascent strand gaps can be repaired by BRCA-mediated homology repair. Alternatively, gaps can also be filled by translesion synthesis (TLS) polymerases. How these events are regulated is still not clear. Here, we show that PARP10, a poorly-characterized mono-ADP-ribosyltransferase, is recruited to nascent strand gaps to promote their repair. PARP10 interacts with the ubiquitin ligase RAD18 and recruits it to these structures, resulting in the ubiquitination of the replication factor PCNA. PCNA ubiquitination, in turn, recruits the TLS polymerase REV1 for gap filling. We show that PARP10 recruitment to gaps and the subsequent REV1-mediated gap filling requires both the catalytic activity of PARP10, and its ability to interact with PCNA. We moreover show that PARP10 is hyperactive in BRCA-deficient cells, and its inactivation potentiates gap accumulations and cytotoxicity in these cells. Our work uncovers PARP10 as a regulator of ssDNA gap filling, which promotes genomic stability in BRCA-deficient cells.
Assuntos
Reparo do DNA , Replicação do DNA , DNA de Cadeia Simples , Proteínas de Ligação a DNA , Poli(ADP-Ribose) Polimerases , Antígeno Nuclear de Célula em Proliferação , Ubiquitina-Proteína Ligases , Ubiquitinação , Humanos , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , Poli(ADP-Ribose) Polimerases/metabolismo , Poli(ADP-Ribose) Polimerases/genética , Dano ao DNA , Proteína BRCA2/metabolismo , Proteína BRCA2/genética , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Linhagem Celular Tumoral , Células HEK293 , Síntese de DNA Translesão , DNA Polimerase Dirigida por DNA , Proteínas Proto-OncogênicasRESUMO
The drug floxuridine (5-fluorodeoxyuridine, FUdR) is an active metabolite of 5-Fluorouracil (5-FU). It converts to 5-fluorodeoxyuridine monophosphate (FdUMP) and 5-fluorodeoxyuridine triphosphate (FdUTP), which on incorporation into the genome inhibits DNA replication. Additionally, it inhibits thymidylate synthase, causing dTMP shortage while increasing dUMP availability, which induces uracil incorporation into the genome. However, the mechanisms underlying cellular tolerance to FUdR are yet to be fully elucidated. In this study, we explored the mechanisms underlying cellular resistance to FUdR by screening for FUdR hypersensitive mutants from a collection of DT40 mutants deficient in each genomic maintenance system. We identified REV3, which is involved in translesion DNA synthesis (TLS), to be a critical factor in FUdR tolerance. Replication using a FUdR-damaged template was attenuated in REV3-/- cells, indicating that the TLS function of REV3 is required to maintain replication on the FUdR-damaged template. Notably, FUdR-exposed REV3-/- cells exhibited defective cell cycle arrest in the early S phase, suggesting that REV3 is involved in intra-S checkpoint activation. Furthermore, REV3-/- cells showed defects in Chk1 phosphorylation, which is required for checkpoint activation, but the survival of FUdR-exposed REV3-/- cells was further reduced by the inhibition of Chk1 or ATR. These data indicate that REV3 mediates DNA checkpoint activation at least through Chk1 phosphorylation, but this signal acts in parallel with ATR-Chk1 DNA damage checkpoint pathway. Collectively, we reveal a previously unappreciated role of REV3 in FUdR tolerance.
Assuntos
Dano ao DNA , Replicação do DNA , Floxuridina , Floxuridina/farmacologia , Animais , Quinase 1 do Ponto de Checagem/metabolismo , Quinase 1 do Ponto de Checagem/genética , Pontos de Checagem da Fase S do Ciclo Celular/genética , Pontos de Checagem da Fase S do Ciclo Celular/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , Galinhas , Humanos , Reparo do DNA/genética , Fosforilação , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Síntese de DNA Translesão , Desoxiuridina/análogos & derivadosRESUMO
Mutations in DNA damage response (DDR) factors are associated with human infertility, which affects up to 15% of the population. The DDR is required during germ cell development and meiosis. One pathway implicated in human fertility is DNA translesion synthesis (TLS), which allows replication impediments to be bypassed. We find that TLS is essential for pre-meiotic germ cell development in the embryo. Loss of the central TLS component, REV1, significantly inhibits the induction of human PGC-like cells (hPGCLCs). This is recapitulated in mice, where deficiencies in TLS initiation (Rev1-/- or PcnaK164R/K164R) or extension (Rev7 -/-) result in a > 150-fold reduction in the number of primordial germ cells (PGCs) and complete sterility. In contrast, the absence of TLS does not impact the growth, function, or homeostasis of somatic tissues. Surprisingly, we find a complete failure in both activation of the germ cell transcriptional program and in DNA demethylation, a critical step in germline epigenetic reprogramming. Our findings show that for normal fertility, DNA repair is required not only for meiotic recombination but for progression through the earliest stages of germ cell development in mammals.
Assuntos
Desmetilação do DNA , Reparo do DNA , DNA Polimerase Dirigida por DNA , Células Germinativas , Animais , Feminino , Humanos , Masculino , Camundongos , Dano ao DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , Epigênese Genética , Células Germinativas/metabolismo , Meiose/genética , Camundongos Knockout , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Síntese de DNA TranslesãoRESUMO
RAD18, an important ubiquitin E3 ligase, plays a dual role in translesion DNA synthesis (TLS) and homologous recombination (HR) repair. However, whether and how the regulatory mechanism of O-linked N-acetylglucosamine (O-GlcNAc) modification governing RAD18 and its function during these processes remains unknown. Here, we report that human RAD18, can undergo O-GlcNAcylation at Ser130/Ser164/Thr468, which is important for optimal RAD18 accumulation at DNA damage sites. Mechanistically, abrogation of RAD18 O-GlcNAcylation limits CDC7-dependent RAD18 Ser434 phosphorylation, which in turn significantly reduces damage-induced PCNA monoubiquitination, impairs Polη focus formation and enhances UV sensitivity. Moreover, the ubiquitin and RAD51C binding ability of RAD18 at DNA double-strand breaks (DSBs) is O-GlcNAcylation-dependent. O-GlcNAcylated RAD18 promotes the binding of RAD51 to damaged DNA during HR and decreases CPT hypersensitivity. Our findings demonstrate a novel role of RAD18 O-GlcNAcylation in TLS and HR regulation, establishing a new rationale to improve chemotherapeutic treatment.
Assuntos
Acetilglucosamina , Proteínas de Ligação a DNA , Antígeno Nuclear de Célula em Proliferação , Rad51 Recombinase , Reparo de DNA por Recombinação , Ubiquitina-Proteína Ligases , Humanos , Acetilglucosamina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Quebras de DNA de Cadeia Dupla , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Glicosilação , Células HEK293 , Fosforilação , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ligação Proteica , Rad51 Recombinase/metabolismo , Síntese de DNA Translesão , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação , Raios UltravioletaRESUMO
Translesion synthesis (TLS) is a cellular mechanism through which actively replicating cells recruit specialized, low-fidelity DNA polymerases to damaged DNA to allow for replication past these lesions. REV1 is one of these TLS DNA polymerases that functions primarily as a scaffolding protein to organize the TLS heteroprotein complex and ensure replication occurs in the presence of DNA lesions. The C-Terminal domain of REV1 (REV1-CT) forms many protein-protein interactions (PPIs) with other TLS polymerases, making it essential for TLS function and a promising drug target for anti-cancer drug development. We utilized several lead identification strategies to identify various small molecules capable of disrupting the PPI between REV1-CT and the REV1 Interacting Regions (RIR) present in several other TLS polymerases. These lead compounds were profiled in several in vitro potency and PK assays to identify two scaffolds (1 and 6) as the most promising for further development. Both 1 and 6 synergized with cisplatin in a REV1-dependent fashion and demonstrated promising in vivo PK and toxicity profiles.
Assuntos
Nucleotidiltransferases , Bibliotecas de Moléculas Pequenas , Nucleotidiltransferases/antagonistas & inibidores , Nucleotidiltransferases/metabolismo , Humanos , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/síntese química , Animais , Relação Estrutura-Atividade , Ligação Proteica , Estrutura Molecular , Antineoplásicos/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Relação Dose-Resposta a Droga , DNA Polimerase Dirigida por DNA/metabolismo , Camundongos , Síntese de DNA TranslesãoRESUMO
Translesion DNA synthesis pathways are necessary to ensure bacterial replication in the presence of DNA damage. Translesion DNA synthesis carried out by the PolV mutasome is well-studied in Escherichia coli, but ~one third of bacteria use a functionally homologous protein complex, consisting of ImuA, ImuB, and ImuC (also called DnaE2). Numerous in vivo studies have shown that all three proteins are required for translesion DNA synthesis and that ImuC is the error-prone polymerase, but the roles of ImuA and ImuB are unclear. Here we carry out biochemical characterization of ImuA and a truncation of ImuB from Myxococcus xanthus. We find that ImuA is an ATPase, with ATPase activity enhanced in the presence of DNA. The ATPase activity is likely regulated by the C-terminus, as loss of the ImuA C-terminus results in DNA-independent ATP hydrolysis. We also find that ImuA binds a variety of DNA substrates, with DNA binding affinity affected by the addition of ADP or adenylyl-imidodiphosphate. An ImuB truncation also binds DNA, with lower affinity than ImuA. In the absence of DNA, ImuA directly binds ImuB with moderate affinity. Finally, we show that ImuA and ImuB self-interact, but that ImuA is predominantly a monomer, while truncated ImuB is a trimer in vitro. Together, with our findings and the current literature in the field, we suggest a model for translesion DNA synthesis, where a trimeric ImuB would provide sufficient binding sites for DNA, the ß-clamp, ImuC, and ImuA, and where ImuA ATPase activity may regulate assembly and disassembly of the translesion DNA synthesis complex.
Assuntos
Myxococcus xanthus , Myxococcus xanthus/genética , Myxococcus xanthus/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/química , Síntese de DNA Translesão , Escherichia coli/genética , Escherichia coli/metabolismo , DNA/genética , Replicação do DNARESUMO
Purified translesion synthesis (TLS) DNA polymerases (Pols) replicate through DNA lesions with a low fidelity; however, TLS operates in a predominantly error-free manner in normal human cells. To explain this incongruity, here we determine whether Y family Pols, which play an eminent role in replication through a diversity of DNA lesions, are incorporated into a multiprotein ensemble and whether the intrinsically high error rate of the TLS Pol is ameliorated by the components in the ensemble. To this end, we provide evidence for an indispensable role of Werner syndrome protein (WRN) and WRN-interacting protein 1 (WRNIP1) in Rev1-dependent TLS by Y family Polη, Polι, or Polκ and show that WRN, WRNIP1, and Rev1 assemble together with Y family Pols in response to DNA damage. Importantly, we identify a crucial role of WRN's 3' â 5' exonuclease activity in imparting high fidelity on TLS by Y family Pols in human cells, as the Y family Pols that accomplish TLS in an error-free manner manifest high mutagenicity in the absence of WRN's exonuclease function. Thus, by enforcing high fidelity on TLS Pols, TLS mechanisms have been adapted to safeguard against genome instability and tumorigenesis.
Assuntos
DNA Polimerase Dirigida por DNA , Síntese de DNA Translesão , Helicase da Síndrome de Werner , Humanos , Dano ao DNA , Reparo do DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Exonucleases/metabolismo , Síntese de DNA Translesão/genética , Helicase da Síndrome de Werner/genética , Helicase da Síndrome de Werner/metabolismoRESUMO
Almost all Glioblastoma (GBM) are either intrinsically resistant to the chemotherapeutical drug temozolomide (TMZ) or acquire therapy-induced mutations that cause chemoresistance and recurrence. The genome maintenance mechanisms responsible for GBM chemoresistance and hypermutation are unknown. We show that the E3 ubiquitin ligase RAD18 (a proximal regulator of TLS) is activated in a Mismatch repair (MMR)-dependent manner in TMZ-treated GBM cells, promoting post-replicative gap-filling and survival. An unbiased CRISPR screen provides an aerial map of RAD18-interacting DNA damage response (DDR) pathways deployed by GBM to tolerate TMZ genotoxicity. Analysis of mutation signatures from TMZ-treated GBM reveals a role for RAD18 in error-free bypass of O6mG (the most toxic TMZ-induced lesion), and error-prone bypass of other TMZ-induced lesions. Our analyses of recurrent GBM patient samples establishes a correlation between low RAD18 expression and hypermutation. Taken together we define molecular underpinnings for the hallmark tumorigenic phenotypes of TMZ-treated GBM.
Assuntos
Glioblastoma , Humanos , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Síntese de DNA Translesão , Reparo de Erro de Pareamento de DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Temozolomida/farmacologia , Proteínas de Ligação a DNA , Ubiquitina-Proteína Ligases/genéticaRESUMO
Cell-free extracts derived from Xenopus eggs have been widely used to decipher molecular pathways involved in several cellular processes including DNA synthesis, the DNA damage response, and genome integrity maintenance. We set out assays using Xenopus cell-free extracts to study translesion DNA synthesis (TLS), a branch of the DNA damage tolerance pathway that allows replication of damaged DNA. Using this system, we were able to recapitulate TLS activities that occur naturally in vivo during early embryogenesis. This chapter describes protocols to detect chromatin-bound TLS factors by western blotting and immunofluorescence microscopy upon induction of DNA damage by UV irradiation, monitor TLS-dependent mutagenesis, and perform proteomic screening.
Assuntos
Proteômica , Síntese de DNA Translesão , Animais , Xenopus laevis/genética , Replicação do DNA , Dano ao DNA , DNA/genética , DNA/efeitos da radiação , Reparo do DNARESUMO
5-Fluorouracil (5-FU) is the first-line chemotherapeutic agent in colorectal cancer, and resistance to 5-FU easily emerges. One of the mechanisms of drug action and resistance of 5-FU is through DNA incorporation. Our quantitative reverse-transcription PCR data showed that one of the translesion synthesis (TLS) DNA polymerases, DNA polymerase η (polη), was upregulated within 72 h upon 5-FU administration at 1 and 10 µM, indicating that polη is one of the first responding polymerases, and the only TLS polymerase, upon the 5-FU treatment to incorporate 5-FU into DNA. Our kinetic studies revealed that 5-fluoro-2'-deoxyuridine triphosphate (5FdUTP) was incorporated across dA 41 and 28 times more efficiently than across dG and across inosine, respectively, by polη indicating that the mutagenicity of 5-FU incorporation is higher in the presence of inosine and that DNA lesions could lead to more mutagenic incorporation of 5-FU. Our polη crystal structures complexed with DNA and 5FdUTP revealed that dA:5FdUTP base pair is like dA:dTTP in the active site of polη, while 5FdUTP adopted 4-enol tautomer in the base pairs with dG and HX increasing the insertion efficiency compared to dG:dTTP for the incorrect insertions. These studies confirm that polη engages in the DNA incorporation and bypass of 5-FU.