RESUMO
INTRODUCTION: Adaptive mutagenesis observed in colorectal cancer (CRC) cells upon exposure to EGFR inhibitors contributes to the development of resistance and recurrence. Multiple investigations have indicated a parallel between cancer cells and bacteria in terms of exhibiting adaptive mutagenesis. This phenomenon entails a transient and coordinated escalation of error-prone translesion synthesis polymerases (TLS polymerases), resulting in mutagenesis of a magnitude sufficient to drive the selection of resistant phenotypes. METHODS: In this study, we conducted a comprehensive pan-transcriptome analysis of the regulatory framework within CRC cells, with the objective of identifying potential transcriptome modules encompassing certain translesion polymerases and the associated transcription factors (TFs) that govern them. Our sampling strategy involved the collection of transcriptomic data from tumors treated with cetuximab, an EGFR inhibitor, untreated CRC tumors, and colorectal-derived cell lines, resulting in a diverse dataset. Subsequently, we identified co-regulated modules using weighted correlation network analysis with a minKMEtostay threshold set at 0.5 to minimize false-positive module identifications and mapped the modules to STRING annotations. Furthermore, we explored the putative TFs influencing these modules using KBoost, a kernel PCA regression model. RESULTS: Our analysis did not reveal a distinct transcriptional profile specific to cetuximab treatment. Moreover, we elucidated co-expression modules housing genes, for example, POLK, POLI, POLQ, REV1, POLN, and POLM. Specifically, POLK, POLI, and POLQ were assigned to the "blue" module, which also encompassed critical DNA damage response enzymes, for example. BRCA1, BRCA2, MSH6, and MSH2. To delineate the transcriptional control of this module, we investigated associated TFs, highlighting the roles of prominent cancer-associated TFs, such as CENPA, HNF1A, and E2F7. CONCLUSION: We found that translesion polymerases are co-regulated with DNA mismatch repair and cell cycle-associated factors. We did not, however, identified any networks specific to cetuximab treatment indicating that the response to EGFR inhibitors relates to a general stress response mechanism.
Assuntos
Cetuximab , Neoplasias Colorretais , Regulação Neoplásica da Expressão Gênica , Cetuximab/farmacologia , Cetuximab/uso terapêutico , Humanos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Linhagem Celular Tumoral , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , Redes Reguladoras de Genes , Perfilação da Expressão Gênica , Receptores ErbB/metabolismo , Receptores ErbB/genética , Proteínas Mad2/genética , Proteínas Mad2/metabolismo , Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêuticoRESUMO
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
An organism's genomic DNA must be accurately duplicated during each cell cycle. DNA synthesis is catalysed by DNA polymerase enzymes, which extend nucleotide polymers in a 5' to 3' direction. This inherent directionality necessitates that one strand is synthesised forwards (leading), while the other is synthesised backwards discontinuously (lagging) to couple synthesis to the unwinding of duplex DNA. Eukaryotic cells possess many diverse polymerases that coordinate to replicate DNA, with the three main replicative polymerases being Pol α, Pol δ and Pol ε. Studies conducted in yeasts and human cells utilising mutant polymerases that incorporate molecular signatures into nascent DNA implicate Pol ε in leading strand synthesis and Pol α and Pol δ in lagging strand replication. Recent structural insights have revealed how the spatial organization of these enzymes around the core helicase facilitates their strand-specific roles. However, various challenging situations during replication require flexibility in the usage of these enzymes, such as during replication initiation or encounters with replication-blocking adducts. This review summarises the roles of the replicative polymerases in bulk DNA replication and explores their flexible and dynamic deployment to complete genome replication. We also examine how polymerase usage patterns can inform our understanding of global replication dynamics by revealing replication fork directionality to identify regions of replication initiation and termination.
Assuntos
Replicação do DNA , Humanos , DNA/metabolismo , DNA/biossíntese , DNA Polimerase Dirigida por DNA/metabolismo , Animais , DNA Polimerase II/metabolismo , Eucariotos/enzimologia , Eucariotos/genética , DNA Polimerase III/metabolismo , Células Eucarióticas/metabolismo , Células Eucarióticas/enzimologia , DNA Polimerase I/metabolismoRESUMO
Chimeric DNA polymerase with notable performance has been generated for wide applications including DNA amplification and molecular diagnostics. This rational design method aims to improve specific enzymatic characteristics or introduce novel functions by fusing amino acid sequences from different proteins with a single DNA polymerase to create a chimeric DNA polymerase. Several strategies prove to be efficient, including swapping homologous domains between polymerases to combine benefits from different species, incorporating additional domains for exonuclease activity or enhanced binding ability to DNA, and integrating functional protein along with specific protein structural pattern to improve thermal stability and tolerance to inhibitors, as many cases in the past decade shown. The conventional protocol to develop a chimeric DNA polymerase with desired traits involves a Design-Build-Test-Learn (DBTL) cycle. This procedure initiates with the selection of a parent polymerase, followed by the identification of relevant domains and devising a strategy for fusion. After recombinant expression and purification of chimeric polymerase, its performance is evaluated. The outcomes of these evaluations are analyzed for further enhancing and optimizing the functionality of the polymerase. This review, centered on microorganisms, briefly outlines typical instances of chimeric DNA polymerases categorized, and presents a general methodology for their creation. KEY POINTS: ⢠Chimeric DNA polymerase is generated by rational design method. ⢠Strategies include domain exchange and addition of proteins, domains, and motifs. ⢠Chimeric DNA polymerase exhibits improved enzymatic properties or novel functions.
Assuntos
DNA Polimerase Dirigida por DNA , Engenharia de Proteínas , Proteínas Recombinantes de Fusão , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Engenharia de Proteínas/métodosRESUMO
PARP inhibitors (PARPi), known for their ability to induce replication gaps and accelerate replication forks, have become potent agents in anticancer therapy. However, the molecular mechanism underlying PARPi-induced fork acceleration has remained elusive. Here, we show that the first PARPi-induced effect on DNA replication is an increased replication fork rate, followed by a secondary reduction in origin activity. Through the systematic knockdown of human DNA polymerases, we identify POLA1 as mediator of PARPi-induced fork acceleration. This acceleration depends on both DNA polymerase α and primase activities. Additionally, the depletion of POLA1 increases the accumulation of replication gaps induced by PARP inhibition, sensitizing cells to PARPi. BRCA1-depleted cells are especially susceptible to the formation of replication gaps under POLA1 inhibition. Accordingly, BRCA1 deficiency sensitizes cells to POLA1 inhibition. Thus, our findings establish the POLA complex as important player in PARPi-induced fork acceleration and provide evidence that lagging strand synthesis represents a targetable vulnerability in BRCA1-deficient cells.
Assuntos
Proteína BRCA1 , DNA Primase , Replicação do DNA , DNA de Cadeia Simples , Inibidores de Poli(ADP-Ribose) Polimerases , Humanos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , DNA Primase/metabolismo , DNA Primase/genética , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Replicação do DNA/efeitos dos fármacos , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , Linhagem Celular Tumoral , DNA Polimerase IRESUMO
Abasic sites are DNA lesions repaired by base excision repair. Cleavage of unrepaired abasic sites in single-stranded DNA (ssDNA) can lead to chromosomal breakage during DNA replication. How rupture of abasic DNA is prevented remains poorly understood. Here, using cryoelectron microscopy (cryo-EM), Xenopus laevis egg extracts, and human cells, we show that RAD51 nucleofilaments specifically recognize and protect abasic sites, which increase RAD51 association rate to DNA. In the absence of BRCA2 or RAD51, abasic sites accumulate as a result of DNA base methylation, oxidation, and deamination, inducing abasic ssDNA gaps that make replicating DNA fibers sensitive to APE1. RAD51 assembled on abasic DNA prevents abasic site cleavage by the MRE11-RAD50 complex, suppressing replication fork breakage triggered by an excess of abasic sites or POLθ polymerase inhibition. Our study highlights the critical role of BRCA2 and RAD51 in safeguarding against unrepaired abasic sites in DNA templates stemming from base alterations, ensuring genomic stability.
Assuntos
Proteína BRCA2 , Dano ao DNA , Reparo do DNA , Replicação do DNA , DNA de Cadeia Simples , Rad51 Recombinase , Xenopus laevis , Humanos , Rad51 Recombinase/metabolismo , Rad51 Recombinase/genética , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Animais , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/genética , Microscopia Crioeletrônica , DNA Polimerase teta , Metilação de DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , Proteína Homóloga a MRE11/metabolismo , Proteína Homóloga a MRE11/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genéticaRESUMO
When replication forks encounter damaged DNA, cells utilize damage tolerance mechanisms to allow replication to proceed. These include translesion synthesis at the fork, postreplication gap filling, and template switching via fork reversal or homologous recombination. The extent to which these different damage tolerance mechanisms are utilized depends on cell, tissue, and developmental context-specific cues, the last two of which are poorly understood. To address this gap, we have investigated damage tolerance responses in Drosophila melanogaster. We report that tolerance of DNA alkylation damage in rapidly dividing larval tissues depends heavily on translesion synthesis. Furthermore, we show that the REV1 protein plays a multi-faceted role in damage tolerance in Drosophila. Larvae lacking REV1 are hypersensitive to methyl methanesulfonate (MMS) and have highly elevated levels of γ-H2Av (Drosophila γ-H2AX) foci and chromosome aberrations in MMS-treated tissues. Loss of the REV1 C-terminal domain (CTD), which recruits multiple translesion polymerases to damage sites, sensitizes flies to MMS. In the absence of the REV1 CTD, DNA polymerases eta and zeta become critical for MMS tolerance. In addition, flies lacking REV3, the catalytic subunit of polymerase zeta, require the deoxycytidyl transferase activity of REV1 to tolerate MMS. Together, our results demonstrate that Drosophila prioritize the use of multiple translesion polymerases to tolerate alkylation damage and highlight the critical role of REV1 in the coordination of this response to prevent genome instability.
Assuntos
Dano ao DNA , Reparo do DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA , Proteínas de Drosophila , Drosophila melanogaster , Metanossulfonato de Metila , Nucleotidiltransferases , Animais , Drosophila melanogaster/genética , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , Metanossulfonato de Metila/farmacologia , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Nucleotidiltransferases/genética , Nucleotidiltransferases/metabolismo , Alquilação , Reparo do DNA/genética , Replicação do DNA/genética , Larva/genética , Histonas/metabolismo , Histonas/genéticaRESUMO
Cycling cells replicate their DNA during the S phase through a defined temporal program known as replication timing. Mutation frequencies, epigenetic chromatin states, and transcriptional activities are different for genomic regions that are replicated early and late in the S phase. Here, we found from ChIP-Seq analysis that DNA polymerase (Pol) κ is enriched in early-replicating genomic regions in HEK293T cells. In addition, by feeding cells with N 2-heptynyl-2'-deoxyguanosine followed by click chemistry-based enrichment and high-throughput sequencing, we observed elevated Pol κ activities in genomic regions that are replicated early in the S phase. On the basis of the established functions of Pol κ in accurate and efficient nucleotide insertion opposite endogenously induced N 2-modified dG lesions, our work suggests that active engagement of Pol κ may contribute to diminished mutation rates observed in early-replicating regions of the human genome, including cancer genomes. Together, our work expands the functions of Pol κ and offered a plausible mechanism underlying replication timing-dependent mutation accrual in the human genome.
Assuntos
Replicação do DNA , DNA Polimerase Dirigida por DNA , Fase S , Humanos , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , Células HEK293 , Genoma Humano , Período de Replicação do DNARESUMO
DNA polymerase κ (Polκ) is a specialized polymerase that has multiple cellular roles such as translesion DNA synthesis, replication of repetitive sequences, and nucleotide excision repair. We have developed a method for capturing DNA synthesized by Polκ utilizing a Polκ-specific substrate, N2-(4-ethynylbenzyl)-2'-deoxyguanosine (EBndG). After shearing of the DNA into 200 to 500 bp lengths, the EBndG-containing DNA was covalently bound to biotin using the Cu(I)-catalyzed alkyne-azide cycloaddition reaction and isolated with streptavidin beads. Isolated DNA was then ligated to adaptors, followed by PCR amplification and next-generation sequencing to generate genome-wide repair maps. We have termed this method polymerase κ sequencing. Here, we present the human genome maps for Polκ activity in an undamaged cell line. We found that Polκ activity was enhanced in GC-rich regions, euchromatin regions, the promoter of genes, and in DNA that is replicated early in the S phase.
Assuntos
DNA Polimerase Dirigida por DNA , Fibroblastos , Genoma Humano , Humanos , DNA Polimerase Dirigida por DNA/metabolismo , Fibroblastos/metabolismo , Reparo do DNA , DNA/metabolismo , DNA/genética , Linhagem Celular , Replicação do DNARESUMO
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
Random mutagenesis, such as error-prone PCR (epPCR), is a technique capable of generating a wide variety of a single gene. However, epPCR can produce a large number of mutated gene variants, posing a challenge in ligating these mutated PCR products into plasmid vectors. Typically, the primers for mutagenic PCRs incorporate artificial restriction enzyme sites compatible with chosen plasmids. Products are cleaved and ligated to linearized plasmids, then recircularized by DNA ligase. However, this cut-and-paste method known as ligation-dependent process cloning (LDCP), has limited efficiency, as the loss of potential mutants is inevitable leading to a significant reduction in the library's breadth. An alternative to LDCP is the circular polymerase extension cloning (CPEC) method. This technique involves a reaction where a high-fidelity DNA polymerase extends the overlapping regions between the insert and vector, forming a circular molecule. In this study, our objective was to compare the traditional cut-and-paste enzymatic method with CPEC in producing a variant library from the gene encoding the red fluorescent protein (DsRed2) obtained by epPCR. Our findings suggest that CPEC can accelerate the cloning process in gene library generation, enabling the acquisition of a greater number of gene variants compared to methods reliant on restriction enzymes.
Assuntos
Clonagem Molecular , Biblioteca Gênica , Mutagênese , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase/métodos , Clonagem Molecular/métodos , Vetores Genéticos/genética , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , Plasmídeos/genéticaRESUMO
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
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
A stable DNA signal amplification sensor was developed on account of rolling circle amplification (RCA). This sensor includes target DNA-controlled rolling circle amplification technology and locking probe DNA replacement technology, which can be used to detect DNA fragments with genetic information, thus constructing a biosensor for universal detection of DNA. This study takes the homologous DNA of human immunodeficiency virus (HIV) and let-7a as examples to describe this biosensor. The padlock probe is first cyclized by T4 DNA ligase in response to the target's reaction with it. Then, rolling cycle amplification is initiated by Phi29 DNA polymerase, resulting in the formation of a lengthy chain with several triggers. These triggers can open the locked probe LP1 with the fluorescence signal turned off, so that it can continue to react with H2 to form a stable H1-H2 double strand. This regulates the distance between B-DNA modified by the quenching group and H1 modified by fluorescent group, and the fluorescence signal is recovered.
Assuntos
Técnicas Biossensoriais , Sondas de DNA , Técnicas de Amplificação de Ácido Nucleico , Técnicas Biossensoriais/métodos , Técnicas de Amplificação de Ácido Nucleico/métodos , Humanos , Sondas de DNA/química , Sondas de DNA/genética , Corantes Fluorescentes/química , DNA Viral/análise , DNA Viral/genética , DNA/química , DNA/genética , Espectrometria de Fluorescência/métodos , Fluorescência , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/química , Limite de Detecção , HIV/genéticaRESUMO
Accurately modeling the protein fitness landscapes holds great importance for protein engineering. Pre-trained protein language models have achieved state-of-the-art performance in predicting protein fitness without wet-lab experimental data, but their accuracy and interpretability remain limited. On the other hand, traditional supervised deep learning models require abundant labeled training examples for performance improvements, posing a practical barrier. In this work, we introduce FSFP, a training strategy that can effectively optimize protein language models under extreme data scarcity for fitness prediction. By combining meta-transfer learning, learning to rank, and parameter-efficient fine-tuning, FSFP can significantly boost the performance of various protein language models using merely tens of labeled single-site mutants from the target protein. In silico benchmarks across 87 deep mutational scanning datasets demonstrate FSFP's superiority over both unsupervised and supervised baselines. Furthermore, we successfully apply FSFP to engineer the Phi29 DNA polymerase through wet-lab experiments, achieving a 25% increase in the positive rate. These results underscore the potential of our approach in aiding AI-guided protein engineering.
Assuntos
Engenharia de Proteínas , Engenharia de Proteínas/métodos , Aprendizado Profundo , Proteínas/genética , Proteínas/metabolismo , Mutação , DNA Polimerase Dirigida por DNA/metabolismo , Simulação por Computador , Modelos Moleculares , AlgoritmosRESUMO
DNA polymerases are essential enzymes that play a key role in living organisms, as they participate in the synthesis and maintenance of the DNA molecule. The intrinsic properties of these enzymes have been widely observed and studied to understand their functions, activities, and behavior, which has allowed their natural power in DNA synthesis to be exploited in modern biotechnology, to the point of making them true pillars of the field. In this context, the laboratory evolution of these enzymes, either by directed evolution or rational design, has led to the generation of a wide range of new DNA polymerases with novel properties, suitable for a variety of biotechnological needs. In this review, we examine DNA polymerases at the molecular level, their biotechnological use, and their evolutionary methods in relation to the novel properties sought, providing a chronological selection of evolved DNA polymerases cited in the literature that we consider to be of great interest. To our knowledge, this work is the first to bring together the molecular, functional and evolutionary aspects of the DNA polymerase enzyme. We believe it will be of great interest to researchers whose aim is to produce new lines of evolved DNA polymerases.
Assuntos
Biotecnologia , DNA Polimerase Dirigida por DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/genética , Biotecnologia/métodos , Evolução Molecular , Evolução Molecular Direcionada/métodos , Humanos , DNA/genética , DNA/metabolismo , DNA/químicaRESUMO
Glioblastoma (GBM) is a highly aggressive brain tumor associated with poor patient survival. The current standard treatment involves invasive surgery, radiotherapy, and chemotherapy employing temozolomide (TMZ). Resistance to TMZ is, however, a major challenge. Previous work from our group has identified candidate genes linked to TMZ resistance, including genes encoding translesion synthesis (TLS) DNA polymerases iota (PolÉ©) and kappa (Polκ). These specialized enzymes are known for bypassing lesions and tolerating DNA damage. Here, we investigated the roles of PolÉ© and Polκ in TMZ resistance, employing MGMT-deficient U251-MG glioblastoma cells, with knockout of either POLI or POLK genes encoding PolÉ© and Polκ, respectively, and assess their viability and genotoxic stress responses upon subsequent TMZ treatment. Cells lacking either of these polymerases exhibited a significant decrease in viability following TMZ treatment compared to parental counterparts. The restoration of the missing polymerase led to a recovery of cell viability. Furthermore, knockout cells displayed increased cell cycle arrest, mainly in late S-phase, and lower levels of genotoxic stress after TMZ treatment, as assessed by a reduction of γH2AX foci and flow cytometry data. This implies that TMZ treatment does not trigger a significant H2AX phosphorylation response in the absence of these proteins. Interestingly, combining TMZ with Mirin (double-strand break repair pathway inhibitor) further reduced the cell viability and increased DNA damage and γH2AX positive cells in TLS KO cells, but not in parental cells. These findings underscore the crucial roles of PolÉ© and Polκ in conferring TMZ resistance and the potential backup role of homologous recombination in the absence of these TLS polymerases. Targeting these TLS enzymes, along with double-strand break DNA repair inhibition, could, therefore, provide a promising strategy to enhance TMZ's effectiveness in treating GBM.
Assuntos
Metilases de Modificação do DNA , DNA Polimerase iota , Enzimas Reparadoras do DNA , DNA Polimerase Dirigida por DNA , Resistencia a Medicamentos Antineoplásicos , Glioblastoma , Temozolomida , Temozolomida/farmacologia , Humanos , Glioblastoma/genética , Glioblastoma/tratamento farmacológico , Glioblastoma/metabolismo , Glioblastoma/patologia , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , Linhagem Celular Tumoral , Metilases de Modificação do DNA/metabolismo , Metilases de Modificação do DNA/genética , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/genética , Proteínas Supressoras de Tumor/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/deficiência , Antineoplásicos Alquilantes/farmacologia , Antineoplásicos Alquilantes/uso terapêutico , Dano ao DNA , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Reparo do DNA , Técnicas de Inativação de GenesRESUMO
Epigenetic cytosine methylation covers most of genomic CpG dinucleotides in human cells. In addition to common deamination-mediated mutagenesis at CpG sites, an alternative deamination-independent pathway associated with DNA polymerase activity was previously described. This mutagenesis is characterized by the TCGâTTG mutational signature and is believed to arise from dAMP misincorporation opposite 5-methylcytosine (mC) or its oxidized derivative 5-hydroxymethylcytosine (hmC) by B-family replicative DNA polymerases with disrupted proofreading 3â5'-exonuclease activity. In addition to being less stable and pro-mutagenic themselves, cytosine modifications also increase the risk of adjacent nucleotides damage, including the formation of 8-oxo-2'-deoxyguanosine (8-oxoG), a well-known mutagenic lesion. The effect of cytosine methylation on error-prone DNA polymerases lacking proofreading activity and involved in repair and DNA translesion synthesis remains unexplored. Here we analyze the efficiency and fidelity of translesion Y-family polymerases (Pol κ, Pol η, Pol ι and REV1) and primase-polymerase PrimPol opposite mC and hmC as well as opposite 8-oxoG adjacent to mC in the TCG context. We demonstrate that epigenetic cytosine modifications suppress Pol ι and REV1 activities and lead to increasing dAMP misincorporation by PrimPol, Pol κ and Pol ι in vitro. Cytosine methylation also increases misincorporation of dAMP opposite the adjacent 8-oxoG by PrimPol, decreases the TLS activity of Pol η opposite the lesion but increases dCMP incorporation opposite 8-oxoG by REV1. Altogether, these data suggest that methylation and hydroxymethylation of cytosine alter activity and fidelity of translesion DNA polymerases.
Assuntos
5-Metilcitosina , Citosina , Metilação de DNA , DNA Polimerase Dirigida por DNA , Humanos , DNA Polimerase Dirigida por DNA/metabolismo , Citosina/metabolismo , Citosina/análogos & derivados , 5-Metilcitosina/metabolismo , 5-Metilcitosina/análogos & derivados , Reparo do DNA , Dano ao DNA , Nucleotidiltransferases/metabolismo , Nucleotidiltransferases/genética , DNA Polimerase iota , DNA/metabolismo , Enzimas Multifuncionais/metabolismo , Replicação do DNA , 8-Hidroxi-2'-Desoxiguanosina/metabolismoRESUMO
Colorectal cancer (CRC) is one of the most common malignancies worldwide. Double-strand break (DSB) is the most severe type of DNA damage. However, few reviews have thoroughly examined the involvement of DSB in CRC. Latest researches demonstrated that DSB repair plays an important role in CRC. For example, DSB-related genes such as BRCA1, Ku-70 and DNA polymerase theta (POLQ) are associated with the occurrence of CRC, and POLQ even showed to affect the prognosis and resistance for radiotherapy in CRC. This review comprehensively summarizes the DSB role in CRC, explores the mechanisms and discusses the association with CRC treatment. Four pathways for DSB have been demonstrated. 1. Nonhomologous end joining (NHEJ) is the major pathway. Its core genes including Ku70 and Ku80 bind to broken ends and recruit repair factors to form a complex that mediates the connection of DNA breaks. 2. Homologous recombination (HR) is another important pathway. Its key genes including BRCA1 and BRCA2 are involved in finding, pairing, and joining broken ends, and ensure the restoration of breaks in a normal double-stranded DNA structure. 3. Single-strand annealing (SSA) pathway, and 4. POLθ-mediated end-joining (alt-EJ) is a backup pathway. This paper elucidates roles of the DSB repair pathways in CRC, which could contribute to the development of potential new treatment approaches and provide new opportunities for CRC treatment and more individualized treatment options based on therapeutic strategies targeting these DNA repair pathways.
Assuntos
Proteína BRCA1 , Neoplasias Colorretais , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Humanos , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Neoplasias Colorretais/terapia , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , DNA Polimerase teta , Autoantígeno Ku/metabolismo , Autoantígeno Ku/genética , Reparo do DNA , DNA Polimerase Dirigida por DNA/metabolismo , DNA Polimerase Dirigida por DNA/genética , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , AnimaisRESUMO
Enzymatic DNA writing technologies based on the template-independent DNA polymerase terminal deoxynucleotidyl transferase (TdT) have the potential to advance DNA information storage. TdT is unique in its ability to synthesize single-stranded DNA de novo but has limitations, including catalytic inhibition by ribonucleotide presence and slower incorporation rates compared to replicative polymerases. We anticipate that protein engineering can improve, modulate, and tailor the enzyme's properties, but there is limited information on TdT sequence-structure-function relationships to facilitate rational approaches. Therefore, we developed an easily modifiable screening assay that can measure the TdT activity in high-throughput to evaluate large TdT mutant libraries. We demonstrated the assay's capabilities by engineering TdT mutants that exhibit both improved catalytic efficiency and improved activity in the presence of an inhibitor. We screened for and identified TdT variants with greater catalytic efficiency in both selectively incorporating deoxyribonucleotides and in the presence of deoxyribonucleotide/ribonucleotide mixes. Using this information from the screening assay, we rationally engineered other TdT homologues with the same properties. The emulsion-based assay we developed is, to the best of our knowledge, the first high-throughput screening assay that can measure TdT activity quantitatively and without the need for protein purification.