RESUMEN

Timely and accurate DNA replication is critical for safeguarding genome integrity and ensuring cell viability. Yet, this process is challenged by DNA damage blocking the progression of the replication machinery. To counteract replication fork stalling, evolutionary conserved DNA damage tolerance (DDT) mechanisms promote DNA damage bypass and fork movement. One of these mechanisms involves "skipping" DNA damage through repriming downstream of the lesion, leaving single-stranded DNA (ssDNA) gaps behind the advancing forks (also known as post-replicative gaps). In vertebrates, repriming in damaged leading templates is proposed to be mainly promoted by the primase and polymerase PRIMPOL. In this review, we discuss recent advances towards our understanding of the physiological and pathological conditions leading to repriming activation in human models, revealing a regulatory network of PRIMPOL activity. Upon repriming by PRIMPOL, post-replicative gaps formed can be filled-in by the DDT mechanisms translesion synthesis and template switching. We discuss novel findings on how these mechanisms are regulated and coordinated in time to promote gap filling. Finally, we discuss how defective gap filling and aberrant gap expansion by nucleases underlie the cytotoxicity associated with post-replicative gap accumulation. Our increasing knowledge of this repriming mechanism - from gap formation to gap filling - is revealing that targeting the last step of this pathway is a promising approach to exploit post-replicative gaps in anti-cancer therapeutic strategies.

Asunto(s)

Daño del ADN , ADN Primasa , Replicación del ADN , ADN Polimerasa Dirigida por ADN , Humanos , ADN Primasa/metabolismo , ADN Polimerasa Dirigida por ADN/metabolismo , Animales , Reparación del ADN , Enzimas Multifuncionales/metabolismo , ADN de Cadena Simple/metabolismo

RESUMEN

DNA repair proteins became the popular targets in research on cancer treatment. In our studies we hypothesized that inhibition of DNA polymerase theta (Polθ) and its combination with Poly (ADP-ribose) polymerase 1 (PARP1) or RAD52 inhibition and the alkylating drug temozolomide (TMZ) has an anticancer effect on glioblastoma cells (GBM21), whereas it has a low impact on normal human astrocytes (NHA). The effect of the compounds was assessed by analysis of cell viability, apoptosis, proliferation, DNA damage and cell cycle distribution, as well as gene expression. The main results show that Polθ inhibition causes a significant decrease in glioblastoma cell viability. It induces apoptosis, which is accompanied by a reduction in cell proliferation and DNA damage. Moreover, the effect was stronger when dual inhibition of Polθ with PARP1 or RAD52 was applied, and it is further enhanced by addition of TMZ. The impact on normal cells is much lower, especially when considering cell viability and DNA damage. In conclusion, we would like to highlight that Polθ inhibition used in combination with PARP1 or RAD52 inhibition has great potential to kill glioblastoma cells, and shows a synthetic lethal effect, while sparing normal astrocytes.

Asunto(s)

Supervivencia Celular , Glioblastoma , Poli(ADP-Ribosa) Polimerasa-1 , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Proteína Recombinante y Reparadora de ADN Rad52 , Temozolomida , Humanos , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Glioblastoma/metabolismo , Glioblastoma/genética , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Proteína Recombinante y Reparadora de ADN Rad52/genética , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Línea Celular Tumoral , Temozolomida/farmacología , Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidores , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Supervivencia Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , ADN Polimerasa theta , Apoptosis/efectos de los fármacos , Daño del ADN/efectos de los fármacos , ADN Polimerasa Dirigida por ADN/metabolismo , ADN Polimerasa Dirigida por ADN/genética , Mutaciones Letales Sintéticas/efectos de los fármacos , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo

RESUMEN

DNA polymerase ζ (Pol ζ) plays an essential role in replicating damaged DNA templates but contributes to mutagenesis due to its low fidelity. Therefore, ensuring tight control of Pol ζ's activity is critical for continuous and accurate DNA replication, yet the specific mechanisms remain unclear. This study reveals a regulation mechanism of Pol ζ activity in human cells. Under normal conditions, an autoinhibition mechanism keeps the catalytic subunit, REV3L, inactive. Upon encountering replication stress, however, ATR-mediated phosphorylation of REV3L's S279 cluster activates REV3L and triggers its degradation via a caspase-mediated pathway. This regulation confines the activity of Pol ζ, balancing its essential role against its mutations causing potential during replication stress. Overall, our findings elucidate a control scheme that fine tunes the low-fidelity polymerase activity of Pol ζ under challenging replication scenarios.

Asunto(s)

Proteínas de la Ataxia Telangiectasia Mutada , Replicación del ADN , ADN Polimerasa Dirigida por ADN , Humanos , ADN Polimerasa Dirigida por ADN/metabolismo , ADN Polimerasa Dirigida por ADN/genética , Fosforilación , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Daño del ADN , Células HEK293 , Estrés Fisiológico

RESUMEN

G-quadruplexes (G4s) form throughout the genome and influence important cellular processes. Their deregulation can challenge DNA replication fork progression and threaten genome stability. Here, we demonstrate an unexpected role for the double-stranded DNA (dsDNA) translocase helicase-like transcription factor (HLTF) in responding to G4s. We show that HLTF, which is enriched at G4s in the human genome, can directly unfold G4s in vitro and uses this ATP-dependent translocase function to suppress G4 accumulation throughout the cell cycle. Additionally, MSH2 (a component of MutS heterodimers that bind G4s) and HLTF act synergistically to suppress G4 accumulation, restrict alternative lengthening of telomeres, and promote resistance to G4-stabilizing drugs. In a discrete but complementary role, HLTF restrains DNA synthesis when G4s are stabilized by suppressing primase-polymerase (PrimPol)-dependent repriming. Together, the distinct roles of HLTF in the G4 response prevent DNA damage and potentially mutagenic replication to safeguard genome stability.

Asunto(s)

ADN Primasa , Replicación del ADN , Proteínas de Unión al ADN , G-Cuádruplex , Inestabilidad Genómica , Proteína 2 Homóloga a MutS , Factores de Transcripción , Humanos , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteína 2 Homóloga a MutS/metabolismo , Proteína 2 Homóloga a MutS/genética , ADN Primasa/metabolismo , ADN Primasa/genética , Homeostasis del Telómero , Daño del ADN , Células HEK293 , Enzimas Multifuncionales/metabolismo , Enzimas Multifuncionales/genética , ADN Polimerasa Dirigida por ADN

RESUMEN

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.

Asunto(s)

Proteína BRCA2 , Daño del ADN , Reparación del ADN , Replicación del ADN , ADN de Cadena Simple , Recombinasa Rad51 , Xenopus laevis , Humanos , Recombinasa Rad51/metabolismo , Recombinasa Rad51/genética , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Animales , ADN de Cadena Simple/metabolismo , ADN de Cadena Simple/genética , Microscopía por Crioelectrón , ADN Polimerasa theta , Metilación de ADN , ADN Polimerasa Dirigida por ADN/metabolismo , ADN Polimerasa Dirigida por ADN/genética , Proteína Homóloga de MRE11/metabolismo , Proteína Homóloga de MRE11/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética

RESUMEN

BACKGROUND: Viruses with double-stranded (ds) DNA genomes in the realm Duplodnaviria share a conserved structural gene module but show a broad range of variation in their repertoires of DNA replication proteins. Some of the duplodnaviruses encode (nearly) complete replication systems whereas others lack (almost) all genes required for replication, relying on the host replication machinery. DNA polymerases (DNAPs) comprise the centerpiece of the DNA replication apparatus. The replicative DNAPs are classified into 4 unrelated or distantly related families (A-D), with the protein structures and sequences within each family being, generally, highly conserved. More than half of the duplodnaviruses encode a DNAP of family A, B or C. We showed previously that multiple pairs of closely related viruses in the order Crassvirales encode DNAPs of different families. METHODS: Groups of phages in which DNAP swapping likely occurred were identified as subtrees of a defined depth in a comprehensive evolutionary tree of tailed bacteriophages that included phages with DNAPs of different families. The DNAP swaps were validated by constrained tree analysis that was performed on phylogenetic tree of large terminase subunits, and the phage genomes encoding swapped DNAPs were aligned using Mauve. The structures of the discovered unusual DNAPs were predicted using AlphaFold2. RESULTS: We identified four additional groups of tailed phages in the class Caudoviricetes in which the DNAPs apparently were swapped on multiple occasions, with replacements occurring both between families A and B, or A and C, or between distinct subfamilies within the same family. The DNAP swapping always occurs "in situ", without changes in the organization of the surrounding genes. In several cases, the DNAP gene is the only region of substantial divergence between closely related phage genomes, whereas in others, the swap apparently involved neighboring genes encoding other proteins involved in phage genome replication. In addition, we identified two previously undetected, highly divergent groups of family A DNAPs that are encoded in some phage genomes along with the main DNAP implicated in genome replication. CONCLUSIONS: Replacement of the DNAP gene by one encoding a DNAP of a different family occurred on many independent occasions during the evolution of different families of tailed phages, in some cases, resulting in very closely related phages encoding unrelated DNAPs. DNAP swapping was likely driven by selection for avoidance of host antiphage mechanisms targeting the phage DNAP that remain to be identified, and/or by selection against replicon incompatibility.

Asunto(s)

ADN Polimerasa Dirigida por ADN , Filogenia , Proteínas Virales , ADN Polimerasa Dirigida por ADN/genética , Proteínas Virales/genética , Proteínas Virales/metabolismo , Evolución Molecular , Genoma Viral , Caudovirales/genética , Caudovirales/clasificación , ADN Viral/genética , Bacteriófagos/genética , Bacteriófagos/enzimología , Bacteriófagos/clasificación , Replicación del ADN

RESUMEN

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.

Asunto(s)

Proteína BRCA1 , ADN Primasa , Replicación del ADN , ADN de Cadena Simple , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Humanos , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , ADN Primasa/metabolismo , ADN Primasa/genética , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Replicación del ADN/efectos de los fármacos , ADN de Cadena Simple/metabolismo , ADN de Cadena Simple/genética , ADN Polimerasa Dirigida por ADN/metabolismo , ADN Polimerasa Dirigida por ADN/genética , Línea Celular Tumoral , ADN Polimerasa I

RESUMEN

During lagging strand chromatin replication, multiple Okazaki fragments (OFs) require processing and nucleosome assembly, but the mechanisms linking these processes remain unclear. Here, using transmission electron microscopy and rapid degradation of DNA ligase Cdc9, we observed flap structures accumulated on lagging strands, controlled by both Pol δ's strand displacement activity and Fen1's nuclease digestion. The distance between neighboring flap structures exhibits a regular pattern, indicative of matured OF length. While fen1Δ or enhanced strand displacement activities by polymerase δ (Pol δ; pol3exo-) minimally affect inter-flap distance, mutants affecting replication-coupled nucleosome assembly, such as cac1Δ and mcm2-3A, do significantly alter it. Deletion of Pol32, a subunit of DNA Pol δ, significantly increases this distance. Mechanistically, Pol32 binds to histone H3-H4 and is critical for nucleosome assembly on the lagging strand. Together, we propose that Pol32 establishes a connection between nucleosome assembly and the processing of OFs on lagging strands.

Asunto(s)

ADN Polimerasa III , ADN , Histonas , Nucleosomas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Nucleosomas/metabolismo , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , ADN Polimerasa III/metabolismo , ADN Polimerasa III/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , ADN/metabolismo , Replicación del ADN , Unión Proteica , ADN Polimerasa Dirigida por ADN

RESUMEN

Various isothermal amplification methods have been developed for point-of-care testing (POCT) of various infectious diseases. Here, we proposed a novel isothermal amplification method, named as 5'-half complementary primers mediated isothermal amplification (HCPA). Because of the similarity of our method to the previous method competitive annealing mediated isothermal amplification (CAMP) in primer design, we also use the name CAMP for our method. We demonstrated that CAMP is mediated by both a linear isothermal amplification pattern and a loop-mediated isothermal amplification pattern. To improve the specificity and enable multiplex detection, we further developed HiFi-CAMP method that uses a small amount of high-fidelity DNA polymerase to cut HFman probe to release fluorescent signal. The HiFi-CAMP method was demonstrated to have a good specificity and sensitivity, and fast amplification speed in detection of three human respiratory viruses, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), respiratory syncytial virus A (RSV-A) and influenza A viruses (IAV). When compared with gold standard RT-qPCR assays, the HiFi-CAMP assays showed sensitivities of 90.0 %, 71.4 % and 78.1 %, specificities of 100 %, 100 % and 95.5 %, and consistencies of 93.0 %, 93.3 % and 88.2 % for SARS-CoV-2, RSV-A and IAV, respectively. Furthermore, a duplex HiFi-CAMP assay was also developed to simultaneously detect RSV-A and SARS-CoV-2. The HiFi-CAMP will provide a promising candidate for POCT diagnosis in resource-limited settings.

Asunto(s)

ADN Polimerasa Dirigida por ADN , Técnicas de Amplificación de Ácido Nucleico , SARS-CoV-2 , Técnicas de Amplificación de Ácido Nucleico/métodos , Humanos , ADN Polimerasa Dirigida por ADN/metabolismo , SARS-CoV-2/genética , SARS-CoV-2/aislamiento & purificación , Virus de la Influenza A/enzimología , Virus de la Influenza A/genética , Virus de la Influenza A/aislamiento & purificación , Virus Sincitiales Respiratorios/genética , Cartilla de ADN , Técnicas de Diagnóstico Molecular

RESUMEN

PrimPol is a human DNA primase involved in DNA damage tolerance pathways by restarting DNA replication downstream of DNA lesions and non-canonical DNA structures. Activity and affinity to DNA relays on the interaction of PrimPol with replication protein A (RPA). In this work, we report that PrimPol has an intrinsic ability to copy DNA hairpins with a stem length of 5-9 base pairs (bp) but shows pronounced pausing of DNA synthesis. RPA greatly stimulates DNA synthesis across inverted DNA repeats by PrimPol. Moreover, deletion of the C-terminal RPA binding motif (RBM) facilitates DNA hairpin bypass and makes it independent of RPA. This work supports the idea that RBM is a negative regulator of PrimPol and its interaction with RPA is required to achieve the fully active state.

Asunto(s)

ADN Primasa , Replicación del ADN , ADN , Humanos , ADN Primasa/metabolismo , ADN Primasa/química , ADN Primasa/genética , ADN/metabolismo , Enzimas Multifuncionales/metabolismo , Enzimas Multifuncionales/genética , Enzimas Multifuncionales/química , Proteína de Replicación A/metabolismo , Conformación de Ácido Nucleico , ADN Polimerasa Dirigida por ADN/metabolismo , Secuencias Invertidas Repetidas , Unión Proteica

RESUMEN

DNA modified with C2'-methoxy (C2'-OMe) greatly enhances its resistance to nucleases, which is beneficial for the half-life of aptamers and DNA nanomaterials. Although the unnatural DNA polymerases capable of incorporating C2'-OMe modified nucleoside monophosphates (C2'-OMe-NMPs) were engineered via directed evolution, the detailed molecular mechanism by which an evolved DNA polymerase recognizes C2'-OMe-NTPs remains poorly understood. Here, we present the crystal structures of the evolved Stoffel fragment of Taq DNA polymerase SFM4-3 processing the C2'-OMe-GTP in different states. Our results reveal the structural basis for recognition of C2'-methoxy by SFM4-3. Based on the analysis of other mutated residues in SFM4-3, a new Stoffel fragment variant with faster catalytic rate and stronger inhibitor-resistance was obtained. In addition, the capture of a novel pre-insertion co-existing with template 5'-overhang stacking conformation provides insight into the catalytic mechanism of Taq DNA polymerase.

Asunto(s)

Modelos Moleculares , Cristalografía por Rayos X , Conformación Proteica , ADN/metabolismo , ADN/química , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Polimerasa Taq/metabolismo , Polimerasa Taq/química

RESUMEN

By replicating damaged nucleotides, error-prone DNA translesion synthesis (TLS) enables the completion of replication, albeit at the expense of fidelity. TLS of helix-distorting DNA lesions, that usually have reduced capacity of basepairing, comprises insertion opposite the lesion followed by extension, the latter in particular by polymerase ζ (Pol ζ). However, little is known about involvement of Pol ζ in TLS of non- or poorly-distorting, but miscoding, lesions such as O6-methyldeoxyguanosine (O6-medG). Using purified Pol ζ we describe that the enzyme can misincorporate thymidine opposite O6-medG and efficiently extend from terminal mismatches, suggesting its involvement in the mutagenicity of O6-medG. Surprisingly, O6-medG lesions induced by the methylating agent N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) appeared more, rather than less, mutagenic in Pol ζ-deficient mouse embryonic fibroblasts (MEFs) than in wild type MEFs. This suggested that in vivo Pol ζ participates in non-mutagenic TLS of O6-medG. However, we found that the Pol ζ-dependent misinsertions at O6-medG lesions are efficiently corrected by DNA mismatch repair (MMR), which masks the error-proneness of Pol ζ. We also found that the MNNG-induced mutational signature is determined by the adduct spectrum, and modulated by MMR. The signature mimicked single base substitution signature 11 in the catalogue of somatic mutations in cancer, associated with treatment with the methylating drug temozolomide. Our results unravel the individual roles of the major contributors to methylating drug-induced mutagenesis. Moreover, these results warrant caution as to the classification of TLS as mutagenic or error-free based on in vitro data or on the analysis of mutations induced in MMR-proficient cells.

Asunto(s)

Reparación de la Incompatibilidad de ADN , ADN Polimerasa Dirigida por ADN , Metilnitronitrosoguanidina , Animales , ADN Polimerasa Dirigida por ADN/metabolismo , ADN Polimerasa Dirigida por ADN/genética , Ratones , Metilnitronitrosoguanidina/toxicidad , Mutagénesis , Guanina/análogos & derivados , Guanina/metabolismo , Daño del ADN , Metilación de ADN , Fibroblastos/metabolismo , Fibroblastos/efectos de los fármacos , Replicación del ADN , ADN/metabolismo , Síntesis Translesional de ADN

RESUMEN

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.

Asunto(s)

ADN Polimerasa Dirigida por ADN , Ingeniería de Proteínas , Proteínas Recombinantes de Fusión , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , ADN Polimerasa Dirigida por ADN/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Ingeniería de Proteínas/métodos

RESUMEN

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.

Asunto(s)

Cetuximab , Neoplasias Colorrectales , Regulación Neoplásica de la Expresión Génica , Cetuximab/farmacología , Cetuximab/uso terapéutico , Humanos , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Línea Celular Tumoral , ADN Polimerasa Dirigida por ADN/metabolismo , ADN Polimerasa Dirigida por ADN/genética , Redes Reguladoras de Genes , Perfilación de la Expresión Génica , Receptores ErbB/metabolismo , Receptores ErbB/genética , Proteínas Mad2/genética , Proteínas Mad2/metabolismo , Antineoplásicos Inmunológicos/farmacología , Antineoplásicos Inmunológicos/uso terapéutico

RESUMEN

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.

Asunto(s)

Replicación del ADN , Humanos , ADN/metabolismo , ADN/biosíntesis , ADN Polimerasa Dirigida por ADN/metabolismo , Animales , ADN Polimerasa II/metabolismo , Eucariontes/enzimología , Eucariontes/genética , ADN Polimerasa III/metabolismo , Células Eucariotas/metabolismo , Células Eucariotas/enzimología , ADN Polimerasa I/metabolismo

RESUMEN

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.

Asunto(s)

Microscopía por Crioelectrón , ADN Helicasas , ADN Polimerasa theta , ADN Polimerasa Dirigida por ADN , Humanos , ADN Helicasas/metabolismo , ADN Helicasas/química , ADN Helicasas/genética , ADN Helicasas/antagonistas & inhibidores , ADN Polimerasa Dirigida por ADN/metabolismo , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/genética , Proteína BRCA2/metabolismo , Proteína BRCA2/genética , Proteína BRCA2/química , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/química , Piperazinas/farmacología , Piperazinas/química , Línea Celular Tumoral , Ftalazinas/farmacología , Ftalazinas/química , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Modelos Moleculares , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/antagonistas & inhibidores , Unión Proteica

RESUMEN

Unintended on-target chromosomal alterations induced by CRISPR/Cas9 in mammalian cells are common, particularly large deletions and chromosomal translocations, and present a safety challenge for genome editing. Thus, there is still an unmet need to develop safer and more efficient editing tools. We screened diverse DNA polymerases of distinct origins and identified a T4 DNA polymerase derived from phage T4 that strongly prevents undesired on-target damage while increasing the proportion of precise 1- to 2-base-pair insertions generated during CRISPR/Cas9 editing (termed CasPlus). CasPlus induced substantially fewer on-target large deletions while increasing the efficiency of correcting common frameshift mutations in DMD and restored higher level of dystrophin expression than Cas9-alone in human cardiomyocytes. Moreover, CasPlus greatly reduced the frequency of on-target large deletions during mouse germline editing. In multiplexed guide RNAs mediating gene editing, CasPlus repressed chromosomal translocations while maintaining gene disruption efficiency that was higher or comparable to Cas9 in primary human T cells. Therefore, CasPlus offers a safer and more efficient gene editing strategy to treat pathogenic variants or to introduce genetic modifications in human applications.

Asunto(s)

Sistemas CRISPR-Cas , Daño del ADN , Edición Génica , Edición Génica/métodos , Humanos , Animales , Ratones , ADN Polimerasa Dirigida por ADN/metabolismo , ADN Polimerasa Dirigida por ADN/genética , Miocitos Cardíacos/metabolismo , Distrofina/genética , Distrofina/metabolismo , ARN Guía de Sistemas CRISPR-Cas/genética , ARN Guía de Sistemas CRISPR-Cas/metabolismo

RESUMEN

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.

Asunto(s)

Daño del ADN , Reparación del ADN , Replicación del ADN , ADN Polimerasa Dirigida por ADN , Proteínas de Drosophila , Drosophila melanogaster , Metilmetanosulfonato , Nucleotidiltransferasas , Animales , Drosophila melanogaster/genética , ADN Polimerasa Dirigida por ADN/metabolismo , ADN Polimerasa Dirigida por ADN/genética , Metilmetanosulfonato/farmacología , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Alquilación , Reparación del ADN/genética , Replicación del ADN/genética , Larva/genética , Histonas/metabolismo , Histonas/genética

RESUMEN

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.

Asunto(s)

Metilasas de Modificación del ADN , ADN Polimerasa iota , Enzimas Reparadoras del ADN , ADN Polimerasa Dirigida por ADN , Resistencia a Antineoplásicos , Glioblastoma , Temozolomida , Temozolomida/farmacología , Humanos , Glioblastoma/genética , Glioblastoma/tratamiento farmacológico , Glioblastoma/metabolismo , Glioblastoma/patología , ADN Polimerasa Dirigida por ADN/metabolismo , ADN Polimerasa Dirigida por ADN/genética , Línea Celular Tumoral , Metilasas de Modificación del ADN/metabolismo , Metilasas de Modificación del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Enzimas Reparadoras del ADN/genética , Proteínas Supresoras de Tumor/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/deficiencia , Antineoplásicos Alquilantes/farmacología , Antineoplásicos Alquilantes/uso terapéutico , Daño del ADN , Supervivencia Celular/efectos de los fármacos , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Reparación del ADN , Técnicas de Inactivación de Genes

RESUMEN

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.

Asunto(s)

Biotecnología , ADN Polimerasa Dirigida por ADN , ADN Polimerasa Dirigida por ADN/metabolismo , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/genética , Biotecnología/métodos , Evolución Molecular , Evolución Molecular Dirigida/métodos , Humanos , ADN/genética , ADN/metabolismo , ADN/química