RÉSUMÉ
DNA-protein crosslinks (DPCs) are toxic lesions that inhibit DNA related processes. Post-translational modifications (PTMs), including SUMOylation and ubiquitylation, play a central role in DPC resolution, but whether other PTMs are also involved remains elusive. Here, we identify a DPC repair pathway orchestrated by poly-ADP-ribosylation (PARylation). Using Xenopus egg extracts, we show that DPCs on single-stranded DNA gaps can be targeted for degradation via a replication-independent mechanism. During this process, DPCs are initially PARylated by PARP1 and subsequently ubiquitylated and degraded by the proteasome. Notably, PARP1-mediated DPC resolution is required for resolving topoisomerase 1-DNA cleavage complexes (TOP1ccs) induced by camptothecin. Using the Flp-nick system, we further reveal that in the absence of PARP1 activity, the TOP1cc-like lesion persists and induces replisome disassembly when encountered by a DNA replication fork. In summary, our work uncovers a PARP1-mediated DPC repair pathway that may underlie the synergistic toxicity between TOP1 poisons and PARP inhibitors.
Sujet(s)
Réparation de l'ADN , Réplication de l'ADN , ADN topoisomérases de type I , Poly (ADP-Ribose) polymerase-1 , Poly(ADP-ribosylation) , Animaux , Poly (ADP-Ribose) polymerase-1/métabolisme , Poly (ADP-Ribose) polymerase-1/génétique , ADN topoisomérases de type I/métabolisme , Xenopus laevis , Ubiquitination , Humains , ADN/métabolisme , Altération de l'ADN , Camptothécine/pharmacologie , Maturation post-traductionnelle des protéines , ADN simple brin/métabolisme , Protéines de Xénope/métabolismeRÉSUMÉ
Despite the potential of small molecules and recombinant proteins to enhance the efficiency of homology-directed repair (HDR), single-stranded DNA (ssDNA) donors, as currently designed and chemically modified, remain suboptimal for precise gene editing. Here, we screen the biased ssDNA binding sequences of DNA repair-related proteins and engineer RAD51-preferred sequences into HDR-boosting modules for ssDNA donors. Donors with these modules exhibit an augmented affinity for RAD51, thereby enhancing HDR efficiency across various genomic loci and cell types when cooperated with Cas9, nCas9, and Cas12a. By combining with an inhibitor of non-homologous end joining (NHEJ) or the HDRobust strategy, these modular ssDNA donors achieve up to 90.03% (median 74.81%) HDR efficiency. The HDR-boosting modules targeting an endogenous protein enable a chemical modification-free strategy to improve the efficacy of ssDNA donors for precise gene editing.
Sujet(s)
ADN simple brin , Édition de gène , Rad51 Recombinase , Réparation de l'ADN par recombinaison , ADN simple brin/métabolisme , ADN simple brin/génétique , Humains , Édition de gène/méthodes , Rad51 Recombinase/métabolisme , Rad51 Recombinase/génétique , Systèmes CRISPR-Cas , Cellules HEK293 , Protéines associées aux CRISPR/métabolisme , Protéines associées aux CRISPR/génétique , Protéine-9 associée à CRISPR/métabolisme , Protéine-9 associée à CRISPR/génétique , Protéines bactériennes/métabolisme , Protéines bactériennes/génétique , Endodeoxyribonucleases/métabolisme , Endodeoxyribonucleases/génétique , Réparation de l'ADN par jonction d'extrémitésRÉSUMÉ
Cis-regulatory elements (CREs) are pivotal in orchestrating gene expression throughout diverse biological systems. Accurate identification and in-depth characterization of functional CREs are crucial for decoding gene regulation networks during cellular processes. In this study, we develop Kethoxal-Assisted Single-stranded DNA Assay for Transposase-Accessible Chromatin with Sequencing (KAS-ATAC-seq) to quantitatively analyze the transcriptional activity of CREs. A main advantage of KAS-ATAC-seq lies in its precise measurement of ssDNA levels within both proximal and distal ATAC-seq peaks, enabling the identification of transcriptional regulatory sequences. This feature is particularly adept at defining Single-Stranded Transcribing Enhancers (SSTEs). SSTEs are highly enriched with nascent RNAs and specific transcription factors (TFs) binding sites that define cellular identity. Moreover, KAS-ATAC-seq provides a detailed characterization and functional implications of various SSTE subtypes. Our analysis of CREs during mouse neural differentiation demonstrates that KAS-ATAC-seq can effectively identify immediate-early activated CREs in response to retinoic acid (RA) treatment. Our findings indicate that KAS-ATAC-seq provides more precise annotation of functional CREs in transcription. Future applications of KAS-ATAC-seq would help elucidate the intricate dynamics of gene regulation in diverse biological processes.
Sujet(s)
Facteurs de transcription , Animaux , Souris , Facteurs de transcription/métabolisme , Facteurs de transcription/génétique , Transcription génétique , Éléments activateurs (génétique)/génétique , Chromatine/métabolisme , Chromatine/génétique , Sites de fixation , Humains , ADN simple brin/génétique , ADN simple brin/métabolisme , Séquençage après immunoprécipitation de la chromatine/méthodes , Transposases/métabolisme , Transposases/génétique , Éléments de régulation transcriptionnelle , Trétinoïne/pharmacologie , Trétinoïne/métabolisme , Régulation de l'expression des gènes , Différenciation cellulaire/génétique , Analyse de séquence d'ADN/méthodes , Séquences d'acides nucléiques régulatrices/génétiqueRÉSUMÉ
Homologous recombination (HR) is essential for the maintenance of genome stability. During HR, Replication Protein A (RPA) rapidly coats the 3'-tailed single-strand DNA (ssDNA) generated by end resection. Then, the ssDNA-bound RPA must be timely replaced by Rad51 recombinase to form Rad51 nucleoprotein filaments that drive homology search and HR repair. How cells regulate Rad51 assembly dynamics and coordinate RPA and Rad51 actions to ensure proper HR remains poorly understood. Here, we identified that Rtt105, a Ty1 transposon regulator, acts to stimulate Rad51 assembly and orchestrate RPA and Rad51 actions during HR. We found that Rtt105 interacts with Rad51 in vitro and in vivo and restrains the adenosine 5' triphosphate (ATP) hydrolysis activity of Rad51. We showed that Rtt105 directly stimulates dynamic Rad51-ssDNA assembly, strand exchange, and D-loop formation in vitro. Notably, we found that Rtt105 physically regulates the binding of Rad51 and RPA to ssDNA via different motifs and that both regulations are necessary and epistatic in promoting Rad51 nucleation, strand exchange, and HR repair. Consequently, disrupting either of the interactions impaired HR and conferred DNA damage sensitivity, underscoring the importance of Rtt105 in orchestrating the actions of Rad51 and RPA. Our work reveals additional layers of mechanisms regulating Rad51 filament dynamics and the coordination of HR.
Sujet(s)
ADN simple brin , Rad51 Recombinase , Réparation de l'ADN par recombinaison , Protéine A de réplication , Protéines de Saccharomyces cerevisiae , Rad51 Recombinase/métabolisme , Protéine A de réplication/métabolisme , Protéine A de réplication/génétique , ADN simple brin/métabolisme , ADN simple brin/génétique , Protéines de Saccharomyces cerevisiae/métabolisme , Protéines de Saccharomyces cerevisiae/génétique , Saccharomyces cerevisiae/métabolisme , Saccharomyces cerevisiae/génétique , Liaison aux protéinesRÉSUMÉ
Poly(ADP-ribose) polymerase inhibitors (PARPis) exhibit remarkable anticancer activity in tumors with homologous recombination (HR) gene mutations. However, the role of other DNA repair proteins in PARPi-induced lethality remains elusive. Here, we reveal that FANCM promotes PARPi resistance independent of the core Fanconi anemia (FA) complex. FANCM-depleted cells retain HR proficiency, acting independently of BRCA1 in response to PARPis. FANCM depletion leads to increased DNA damage in the second S phase after PARPi exposure, driven by elevated single-strand DNA (ssDNA) gap formation behind replication forks in the first S phase. These gaps arise from both 53BP1- and primase and DNA directed polymerase (PRIMPOL)-dependent mechanisms. Notably, FANCM-depleted cells also exhibit reduced resection of collapsed forks, while 53BP1 deletion restores resection and mitigates PARPi sensitivity. Our results suggest that FANCM counteracts 53BP1 to repair PARPi-induced DNA damage. Furthermore, FANCM depletion leads to increased chromatin bridges and micronuclei formation after PARPi treatment, elucidating the mechanism underlying extensive cell death in FANCM-depleted cells.
Sujet(s)
ADN simple brin , Inhibiteurs de poly(ADP-ribose) polymérases , Protéine-1 liant le suppresseur de tumeur p53 , Inhibiteurs de poly(ADP-ribose) polymérases/pharmacologie , Humains , ADN simple brin/métabolisme , Protéine-1 liant le suppresseur de tumeur p53/métabolisme , Protéine-1 liant le suppresseur de tumeur p53/génétique , Helicase/métabolisme , Helicase/génétique , Altération de l'ADN , Réparation de l'ADN/effets des médicaments et des substances chimiques , Recombinaison homologue/effets des médicaments et des substances chimiques , Protéine BRCA1/métabolisme , Protéine BRCA1/génétique , Résistance aux médicaments antinéoplasiques/effets des médicaments et des substances chimiques , Résistance aux médicaments antinéoplasiques/génétique , Lignée cellulaire tumoraleRÉSUMÉ
Bacterial exonuclease III (ExoIII), widely acknowledged for specifically targeting double-stranded DNA (dsDNA), has been documented as a DNA repair-associated nuclease with apurinic/apyrimidinic (AP)-endonuclease and 3'â5' exonuclease activities. Due to these enzymatic properties, ExoIII has been broadly applied in molecular biosensors. Here, we demonstrate that ExoIII (Escherichia coli) possesses highly active enzymatic activities on ssDNA. By using a range of ssDNA fluorescence-quenching reporters and fluorophore-labeled probes coupled with mass spectrometry analysis, we found ExoIII cleaved the ssDNA at 5'-bond of phosphodiester from 3' to 5' end by both exonuclease and endonuclease activities. Additional point mutation analysis identified the critical residues for the ssDNase action of ExoIII and suggested the activity shared the same active center with the dsDNA-targeted activities of ExoIII. Notably, ExoIII could also digest the dsDNA structures containing 3'-end ssDNA. Considering most ExoIII-assisted molecular biosensors require the involvement of single-stranded DNA (ssDNA) or nucleic acid aptamer containing ssDNA, the activity will lead to low efficiency or false positive outcome. Our study revealed the multi-enzymatic activity and the underlying molecular mechanism of ExoIII on ssDNA, illuminating novel insights for understanding its biological roles in DNA repair and the rational design of ExoIII-ssDNA involved diagnostics.
Sujet(s)
ADN simple brin , Escherichia coli , Exodeoxyribonucleases , Exodeoxyribonucleases/métabolisme , Exodeoxyribonucleases/génétique , ADN simple brin/métabolisme , ADN simple brin/génétique , Escherichia coli/génétique , Escherichia coli/métabolisme , Escherichia coli/enzymologie , Protéines Escherichia coli/métabolisme , Protéines Escherichia coli/génétiqueRÉSUMÉ
G-quadruplexes (G4s) formed by guanine-rich nucleic acids induce genome instability through impeding DNA replication fork progression. G4s are stable DNA structures, the unfolding of which require the functions of DNA helicases. Pif1 helicase binds preferentially to G4 DNA and plays multiple roles in maintaining genome stability, but the mechanism by which Pif1 unfolds G4s is poorly understood. Here we report the co-crystal structure of Saccharomyces cerevisiae Pif1 (ScPif1) bound to a G4 DNA with a 5' single-stranded DNA (ssDNA) segment. Unlike the Thermus oshimai Pif1-G4 structure, in which the 1B and 2B domains confer G4 recognition, ScPif1 recognizes G4 mainly through the wedge region in the 1A domain that contacts the 5' most G-tetrad directly. A conserved Arg residue in the wedge is required for Okazaki fragment processing but not for mitochondrial function or for suppression of gross chromosomal rearrangements. Multiple substitutions at this position have similar effects on resolution of DNA duplexes and G4s, suggesting that ScPif1 may use the same wedge to unwind G4 and dsDNA. Our results reveal the mechanism governing dsDNA unwinding and G4 unfolding by ScPif1 helicase that can potentially be generalized to other eukaryotic Pif1 helicases and beyond.
Sujet(s)
Helicase , G-quadruplexes , Protéines de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Helicase/métabolisme , Helicase/composition chimique , Helicase/génétique , Protéines de Saccharomyces cerevisiae/métabolisme , Protéines de Saccharomyces cerevisiae/composition chimique , Protéines de Saccharomyces cerevisiae/génétique , Saccharomyces cerevisiae/génétique , Saccharomyces cerevisiae/métabolisme , ADN/métabolisme , ADN/composition chimique , ADN/génétique , ADN simple brin/métabolisme , ADN simple brin/composition chimique , Cristallographie aux rayons X , Modèles moléculaires , Liaison aux protéines , Réplication de l'ADN , Instabilité du génomeRÉSUMÉ
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.
Sujet(s)
Réparation de l'ADN , Réplication de l'ADN , ADN simple brin , Protéines de liaison à l'ADN , Poly(ADP-ribose) polymerases , Antigène nucléaire de prolifération cellulaire , Ubiquitin-protein ligases , Ubiquitination , Humains , Antigène nucléaire de prolifération cellulaire/métabolisme , Ubiquitin-protein ligases/métabolisme , Ubiquitin-protein ligases/génétique , Protéines de liaison à l'ADN/métabolisme , Protéines de liaison à l'ADN/génétique , ADN simple brin/métabolisme , ADN simple brin/génétique , Poly(ADP-ribose) polymerases/métabolisme , Poly(ADP-ribose) polymerases/génétique , Altération de l'ADN , Protéine BRCA2/métabolisme , Protéine BRCA2/génétique , Nucleotidyltransferases/métabolisme , Nucleotidyltransferases/génétique , Protéine BRCA1/métabolisme , Protéine BRCA1/génétique , Lignée cellulaire tumorale , Cellules HEK293 , , DNA-directed DNA polymerase , Protéines proto-oncogènesRÉSUMÉ
The fundamental binding of single-stranded (ssDNA) and double-stranded DNA (dsDNA) with graphene oxide-Ag nanocomposites (GO-AgNCPs) has been systematically investigated by multi spectroscopic methods, i.e. ultraviolet-visible (UV-vis) absorption, fluorescence spectroscopy, and circular dichroism (CD). The experimental and theoretical results demonstrate that both ssDNA and dsDNA can be adsorbed onto the GO-AgNCPs surface. All of the evidence indicated that there were relatively strong binding of ssDNA/dsDNA with GO-AgNCPs. The article compares the differences in binding between the two types of DNA and the nanomaterials using spectroscopic and thermodynamic data. UV-vis absorption spectroscopy experiments indicate that the characteristic absorbance intensity of both ss DNA and ds DNA increases, but the rate of change in absorbance is different. The fluorescence results revealed that ss/dsDNA could interact with the GO-AgNCPs surface, in spite of the different binding affinities. The Ka value of ssDNA binding with GO-AgNCPs is greater than that of dsDNA at each constant temperature, indicating that the affinity of dsDNA toward GO-AgNCPs is comparatively weak. Molecular docking studies have corroborated the mentioned experimental results. The calculated thermodynamic parameters showed that the binding process was thermodynamically spontaneous, van der Waals force and hydrogen bonding played predominant roles in the binding process. The mechanism of ss/ds DNA binding with GO-AgNCPs was also investigated, and the results indicated that GO-AgNCPs directly binds to the minor groove of ss/ds DNA by replacing minor groove binders.
Sujet(s)
ADN simple brin , ADN , Graphite , Nanocomposites , Argent , Thermodynamique , Graphite/composition chimique , Argent/composition chimique , Nanocomposites/composition chimique , ADN simple brin/composition chimique , ADN simple brin/métabolisme , ADN/composition chimique , ADN/métabolisme , Simulation de docking moléculaire , Spectrométrie de fluorescence , Analyse spectrale , Dichroïsme circulaireRÉSUMÉ
The preferential response to PARP inhibitors (PARPis) in BRCA-deficient and Schlafen 11 (SLFN11)-expressing ovarian cancers has been documented, yet the underlying molecular mechanisms remain unclear. As the accumulation of single-strand DNA (ssDNA) gaps behind replication forks is key for the lethality effect of PARPis, we investigated the combined effects of SLFN11 expression and BRCA deficiency on PARPi sensitivity and ssDNA gap formation in human cancer cells. PARPis increased chromatin-bound RPA2 and ssDNA gaps in SLFN11-expressing cells and even more in cells with BRCA1 or BRCA2 deficiency. SLFN11 was co-localized with chromatin-bound RPA2 under PARPis treatment, with enhanced recruitment in BRCA2-deficient cells. Notably, the chromatin-bound SLFN11 under PARPis did not block replication, contrary to its function under replication stress. SLFN11 recruitment was attenuated by the inactivation of MRE11. Hence, under PARPi treatment, MRE11 expression and BRCA deficiency lead to ssDNA gaps behind replication forks, where SLFN11 binds and increases their accumulation. As ovarian cancer patients who responded (progression-free survival >2 years) to olaparib maintenance therapy had a significantly higher SLFN11-positivity than short-responders (<6 months), our findings provide a mechanistic understanding of the favorable responses to PARPis in SLFN11-expressing and BRCA-deficient tumors. It highlight the clinical implications of SLFN11.
Sujet(s)
Protéine BRCA1 , Protéine BRCA2 , Réplication de l'ADN , ADN simple brin , Protéine homologue de MRE11 , Tumeurs de l'ovaire , Inhibiteurs de poly(ADP-ribose) polymérases , Humains , Inhibiteurs de poly(ADP-ribose) polymérases/pharmacologie , Réplication de l'ADN/effets des médicaments et des substances chimiques , ADN simple brin/génétique , ADN simple brin/métabolisme , Femelle , Tumeurs de l'ovaire/traitement médicamenteux , Tumeurs de l'ovaire/génétique , Tumeurs de l'ovaire/anatomopathologie , Tumeurs de l'ovaire/métabolisme , Protéine BRCA2/génétique , Protéine BRCA2/métabolisme , Protéine homologue de MRE11/métabolisme , Protéine homologue de MRE11/génétique , Protéine BRCA1/génétique , Protéine BRCA1/métabolisme , Lignée cellulaire tumorale , Protéines nucléaires/métabolisme , Protéines nucléaires/génétique , Protéine A de réplication/métabolisme , Protéine A de réplication/génétique , Chromatine/métabolisme , Phtalazines/pharmacologieRÉSUMÉ
The influence of environmental factors on the interactions between phages and bacteria, particularly single-stranded DNA (ssDNA) phages, has been largely unexplored. In this study, we used Finnlakevirus FLiP, the first known ssDNA phage species with a lipid membrane, as our model phage. We examined the infectivity of FLiP with three Flavobacterium host strains, B330, B167 and B114. We discovered that FLiP infection is contingent on the host strain and conditions such as temperature and bacterial growth phase. FLiP can infect its hosts across a wide temperature range, but optimal phage replication varies with each host. We uncovered some unique aspects of phage infectivity: FLiP has limited infectivity in liquid-suspended cells, but it improves when cells are surface-attached. Moreover, FLiP infects stationary phase B167 and B114 cells more rapidly and efficiently than exponentially growing cells, a pattern not observed with the B330 host. We also present the first experimental evidence of endolysin function in ssDNA phages. The activity of FLiP's lytic enzymes was found to be condition-dependent. Our findings underscore the importance of studying phage ecology in contexts that are relevant to the environment, as both the host and the surrounding conditions can significantly alter the outcome of phage-host interactions.
Sujet(s)
Bactériophages , ADN simple brin , Flavobacterium , ADN simple brin/métabolisme , ADN simple brin/génétique , Bactériophages/génétique , Bactériophages/physiologie , Flavobacterium/virologie , Flavobacterium/croissance et développement , Flavobacterium/génétique , Interactions hôte-microbes , Endopeptidases/métabolisme , Endopeptidases/génétique , Réplication virale , TempératureRÉSUMÉ
DNA glycosylases are a group of enzymes that play a crucial role in the DNA repair process by recognizing and removing damaged or incorrect bases from DNA molecules, which maintains the integrity of the genetic information. The abnormal expression of uracil-DNA glycosylase (UDG), one of significant DNA glycosylases in the base-excision repair pathway, is linked to numerous diseases. Here, we proposed a simple UDG activity detection method based on toehold region triggered CRISPR/Cas12a trans-cleavage. The toehold region on hairpin DNA probe (HP) produced by UDG could induce the trans-cleavage of ssDNA with fluorophore and quencher, generating an obvious fluorescence signal. This protospacer adjacent motif (PAM)-free approach achieves remarkable sensitivity and specificity in detecting UDG, with a detection limit as low as 0.000368 U mL-1. Moreover, this method is able to screen inhibitors and measure UDG in complex biological samples. These advantages render it highly promising for applications in clinical diagnosis and drug discovery.
Sujet(s)
Systèmes CRISPR-Cas , Uracil-DNA glycosidase , Uracil-DNA glycosidase/métabolisme , Uracil-DNA glycosidase/génétique , Systèmes CRISPR-Cas/génétique , Humains , Protéines associées aux CRISPR/métabolisme , Protéines associées aux CRISPR/génétique , Protéines bactériennes/génétique , Protéines bactériennes/métabolisme , ADN simple brin/métabolisme , ADN simple brin/génétiqueRÉSUMÉ
Significant attention has been shifted toward the use and development of biodegradable polymeric materials to mitigate environmental accumulation and potential health impacts. One such material, poly(aspartic acid) (PAA), is a biodegradable alternative to superabsorbent poly(carboxylates), like poly(acrylate). Three enzymes are known to hydrolyze PAA: PahZ1KT-1 and PahZ2KT-1 from Sphingomonas sp. KT-1 and PahZ1KP-2 from Pedobacter sp. KP-2. We previously reported the X-ray crystal structure for PahZ1KT-1, which revealed a homodimer complex with a strongly cationic surface spanning one side of each monomer. Here, we report the first characterization of any polymer hydrolase binding to DNA, where modeling data predict binding of the polyanionic DNA near the cationic substrate binding surface. Our data reveal that PahZ1 homologues from Sphingomonas sp. KT-1 and Pedobacter sp. KP-2 bind ssDNA and dsDNA with nanomolar binding affinities. PahZ1KT-1 binds ssDNA and dsDNA with an apparent dissociation constant, KD,app = 81 ± 14 and 19 ± 1 nM, respectively, and these estimates are similar to the same behaviors exhibited by PahZ1KP-2. Gel permeation chromatography data reveal that dsDNA binding promotes inhibition of PahZ1-catalyzed PAA biodegradation for each homologue. We propose a working model wherein binding of PahZ1 to extracellular biofilm DNA aids in the localization of the hydrolase to the environment in which PAA would first be encountered, thereby providing a mechanism to degrade extracellular PAA and potentially harvest aspartic acid for nutritional uptake.
Sujet(s)
Sphingomonas , Sphingomonas/enzymologie , Pedobacter/enzymologie , ADN/métabolisme , Hydrolases/métabolisme , Hydrolases/composition chimique , Protéines bactériennes/métabolisme , Protéines bactériennes/composition chimique , Protéines bactériennes/génétique , Peptides/métabolisme , Peptides/composition chimique , ADN simple brin/métabolisme , Modèles moléculaires , Liaison aux protéines , Acide aspartique/métabolisme , Acide aspartique/composition chimiqueRÉSUMÉ
Theta-mediated end joining (TMEJ) is critical for survival of cancer cells when other DNA double-stranded break repair pathways are impaired. Human DNA polymerase theta (Pol θ) can extend ssDNA oligonucleotides, but little is known about preferred substrates and mechanism. We show that Pol θ can extend both ssDNA and RNA substrates by unimolecular stem-loop synthesis initiated by only two 3' terminal base pairs. Given sufficient time, Pol θ uses alternative pairing configurations that greatly expand the repertoire of sequence outcomes. Further primer-template adjustments yield low-fidelity outcomes when the nucleotide pool is imbalanced. Unimolecular stem-loop synthesis competes with bimolecular end joining, even when a longer terminal microhomology for end joining is available. Both reactions are partially suppressed by the ssDNA-binding protein replication protein A. Protein-primer grasp residues that are specific to Pol θ are needed for rapid stem-loop synthesis. The ability to perform stem-loop synthesis from a minimally paired primer is rare among human DNA polymerases, but we show that human DNA polymerases Pol η and Pol λ can catalyze related reactions. Using purified human Pol θ, we reconstituted in vitro TMEJ incorporating an insertion arising from a stem-loop extension. These activities may help explain TMEJ repair events that include inverted repeat sequences.
Sujet(s)
, DNA-directed DNA polymerase , Humains , DNA-directed DNA polymerase/métabolisme , DNA-directed DNA polymerase/génétique , ADN simple brin/métabolisme , ADN simple brin/génétique , ADN simple brin/composition chimique , Réparation de l'ADN par jonction d'extrémités , Réparation de l'ADN , Protéine A de réplication/métabolisme , Protéine A de réplication/génétique , DNA polymerase beta/métabolisme , DNA polymerase beta/génétique , DNA polymerase beta/composition chimiqueRÉSUMÉ
The abnormal expression of protein tyrosine phosphatase 1B (PTP1B) is highly related to several serious human diseases. Therefore, an accurate PTP1B activity assay is beneficial to the diagnosis and treatment of these diseases. In this study, a dual-mode biosensing platform that enabled the sensitive and accurate assay of PTP1B activity was constructed based on the high-frequency (100 MHz) quartz crystal microbalance (QCM) and dual-signaling electrochemical (EC) ratiometric strategy. Covalent-organic framework@gold nanoparticles@ferrocene@single-strand DNA (COF@Au@Fc-S0) was introduced onto the QCM Au chip via the chelation between Zr4+ and phosphate groups (phosphate group of the phosphopeptide (P-peptide) on the QCM Au chip and the phosphate group of thiol-labeled single-stranded DNA (S0) on COF@Au@Fc-S0) and used as a signal reporter. When PTP1B was present, the dephosphorylation of the P-peptide led to the release of COF@Au@Fc-S0 from the QCM Au chip, resulting in an increase in the frequency of the QCM. Meanwhile, the released COF@Au@Fc-S0 hybridized with thiol/methylene blue (MB)-labeled hairpin DNA (S1-MB) on the Au NPs-modified indium-tin oxide (ITO) electrode. This caused MB to be far away from the electrode surface and Fc to be close to the electrode, leading to a decrease in the oxidation peak current of MB and an increase in the oxidation peak current of Fc. Thus, PTP1B-induced dephosphorylation of the P-peptide was monitored in real time by QCM, and PTP1B activity was detected sensitively and reliably using this innovative QCM-EC dual-mode sensing platform with an ultralow detection limit. This platform is anticipated to serve as a robust tool for the analysis of protein phosphatase activity and the discovery of drugs targeting protein phosphatase.
Sujet(s)
Techniques électrochimiques , Composés du fer II , Or , Réseaux organométalliques , Métallocènes , Protein Tyrosine Phosphatase, Non-Receptor Type 1 , Techniques de microbalance à cristal de quartz , Protein Tyrosine Phosphatase, Non-Receptor Type 1/métabolisme , Protein Tyrosine Phosphatase, Non-Receptor Type 1/analyse , Or/composition chimique , Humains , Réseaux organométalliques/composition chimique , Composés du fer II/composition chimique , Métallocènes/composition chimique , ADN simple brin/composition chimique , ADN simple brin/métabolisme , Nanoparticules métalliques/composition chimique , Techniques de biocapteur/méthodes , Zirconium/composition chimique , Dosages enzymatiques/méthodesRÉSUMÉ
i-Motifs (iMs) are non-canonical, four-stranded secondary structures formed by stacking of hemi-protonated CH+·C base pairs in cytosine-rich DNA sequences, predominantly at pH < 7. The presence of iM structures in cells was a matter of debate until the recent development of iM-specific antibody, iMab, which was instrumental for several studies that suggested the existence of iMs in live cells and their putative biological roles. We assessed the interaction of iMab with cytosine-rich oligonucleotides by biolayer interferometry (BLI), pull-down assay and bulk-FRET experiments. Our results suggest that binding of iMab to DNA oligonucleotides is governed by the presence of runs of at least two consecutive cytosines and is generally increased in acidic conditions, irrespectively of the capacity of the sequence to adopt, or not, an iM structure. Moreover, the results of the bulk-FRET assay indicate that interaction with iMab results in unfolding of iM structures even in acidic conditions, similarly to what has been observed with hnRNP K, well-studied single-stranded DNA binding protein. Taken together, our results strongly suggest that iMab actually binds to blocks of 2-3 cytosines in single-stranded DNA, and call for more careful interpretation of results obtained with this antibody.
Sujet(s)
Cytosine , ADN simple brin , Motifs nucléotidiques , Cytosine/composition chimique , Cytosine/métabolisme , ADN simple brin/composition chimique , ADN simple brin/métabolisme , Conformation d'acide nucléique , Liaison aux protéines , Anticorps/composition chimique , Anticorps/métabolisme , ADN/composition chimique , ADN/métabolisme , Séquence nucléotidiqueRÉSUMÉ
Replication repriming by the specialized primase-polymerase PRIMPOL ensures the continuity of DNA synthesis during replication stress. PRIMPOL activity generates residual post-replicative single-stranded nascent DNA gaps, which are linked with mutagenesis and chemosensitivity in BRCA1/2-deficient models, and which are suppressed by replication fork reversal mediated by the DNA translocases SMARCAL1 and ZRANB3. Here, we report that the MRE11 regulator MRNIP limits the prevalence of PRIMPOL and MRE11-dependent ssDNA gaps in cells in which fork reversal is perturbed either by treatment with the PARP inhibitor Olaparib, or by depletion of SMARCAL1 or ZRANB3. MRNIP-deficient cells are sensitive to PARP inhibition and accumulate PRIMPOL-dependent DNA damage, supportive of a pro-survival role for MRNIP linked to the regulation of gap prevalence. In MRNIP-deficient cells, post-replicative gap filling is driven in S-phase by UBC13-mediated template switching involving REV1 and the TLS polymerase Pol-ζ. Our findings represent the first report of modulation of post-replicative ssDNA gap dynamics by a direct MRE11 regulator.
Sujet(s)
Helicase , DNA primase , Réplication de l'ADN , ADN simple brin , DNA-directed DNA polymerase , Protéine homologue de MRE11 , ADN simple brin/métabolisme , ADN simple brin/génétique , Protéine homologue de MRE11/métabolisme , Protéine homologue de MRE11/génétique , Humains , DNA-directed DNA polymerase/métabolisme , DNA-directed DNA polymerase/génétique , DNA primase/métabolisme , DNA primase/génétique , Helicase/métabolisme , Helicase/génétique , Protéines de liaison à l'ADN/métabolisme , Protéines de liaison à l'ADN/génétique , Altération de l'ADN , Phtalazines/pharmacologie , Pipérazines/pharmacologie , Enzymes multifonctionnelles/génétique , Enzymes multifonctionnelles/métabolisme , Inhibiteurs de poly(ADP-ribose) polymérases/pharmacologie , Protéines de transport/métabolisme , Protéines de transport/génétiqueRÉSUMÉ
TRIP4 is a conserved transcriptional coactivator that is involved in the regulation of the expression of multiple genes. It consists of a classical N-terminal C2HC5-like zinc-finger domain and a conserved C-terminal ASCH domain. Here, we characterized the DNA-binding properties of the human TRIP4 ASCH domain. Our biochemical data show that TRIP4-ASCH has comparable binding affinities toward ssDNA and dsDNA of different lengths, sequences, and structures. The crystal structures reveal that TRIP4-ASCH binds to DNA substrates in a sequence-independent manner through two adjacent positively charged surface patches: one binds to the 5'-end of DNA, and the other binds to the 3'-end of DNA. Further mutagenesis experiments and binding assays confirm the functional roles of key residues involved in DNA binding. In summary, our data demonstrate that TRIP4-ASCH binds to the 5' and 3'-ends of DNA in a sequence-independent manner, which will facilitate further studies of the biological function of TRIP4.
Sujet(s)
Protéines de liaison à l'ADN , ADN , Modèles moléculaires , Liaison aux protéines , Facteurs de transcription , Humains , Protéines de liaison à l'ADN/métabolisme , Protéines de liaison à l'ADN/composition chimique , Protéines de liaison à l'ADN/génétique , Facteurs de transcription/métabolisme , Facteurs de transcription/composition chimique , Cristallographie aux rayons X , Sites de fixation , ADN/métabolisme , ADN/composition chimique , ADN simple brin/métabolisme , ADN simple brin/composition chimique , Domaines protéiques , Séquence d'acides aminés , Doigts de zinc , Protéines de répressionRÉSUMÉ
Bacteriophage RNA polymerases, in particular T7 RNA polymerase (RNAP), are well-characterized and popular enzymes for many RNA applications in biotechnology both in vitro and in cellular settings. These monomeric polymerases are relatively inexpensive and have high transcription rates and processivity to quickly produce large quantities of RNA. T7 RNAP also has high promoter-specificity on double-stranded DNA (dsDNA) such that it only initiates transcription downstream of its 17-base promoter site on dsDNA templates. However, there are many promoter-independent T7 RNAP transcription reactions involving transcription initiation in regions of single-stranded DNA (ssDNA) that have been reported and characterized. These promoter-independent transcription reactions are important to consider when using T7 RNAP transcriptional systems for DNA nanotechnology and DNA computing applications, in which ssDNA domains often stabilize, organize, and functionalize DNA nanostructures and facilitate strand displacement reactions. Here we review the existing literature on promoter-independent transcription by bacteriophage RNA polymerases with a specific focus on T7 RNAP, and provide examples of how promoter-independent reactions can disrupt the functionality of DNA strand displacement circuit components and alter the stability and functionality of DNA-based materials. We then highlight design strategies for DNA nanotechnology applications that can mitigate the effects of promoter-independent T7 RNAP transcription. The design strategies we present should have an immediate impact by increasing the rate of success of using T7 RNAP for applications in DNA nanotechnology and DNA computing.
Sujet(s)
DNA-directed RNA polymerases , ADN , Nanostructures , Régions promotrices (génétique) , Transcription génétique , Protéines virales , DNA-directed RNA polymerases/métabolisme , DNA-directed RNA polymerases/génétique , Protéines virales/métabolisme , Protéines virales/génétique , Nanostructures/composition chimique , ADN/métabolisme , ADN/génétique , ADN/composition chimique , ADN simple brin/génétique , ADN simple brin/métabolisme , ADN simple brin/composition chimique , Nanotechnologie/méthodes , Bactériophage T7/génétiqueRÉSUMÉ
Despite extensive studies on DNA replication, the exchange mechanisms of DNA polymerase during replication remain unclear. Existing models propose that this exchange is facilitated by protein partners like helicase. Here we present data, employing a combination of mechanical DNA manipulation and single fluorescent protein observation, that reveal DNA polymerase undergoing rapid and autonomous exchange during replication not coordinated by other proteins. The DNA polymerase shows fast unbinding and rebinding dynamics, displaying a preference for either exonuclease or polymerase activity, or pausing events, during each brief binding event. We also observed a 'memory effect' in DNA polymerase rebinding, i.e., the enzyme tends to preserve its prior activity upon reassociation. This effect, potentially linked to the ssDNA/dsDNA junction's conformation, might play a role in regulating binding preference enabling high processivity amidst rapid protein exchange. Taken together, our findings support an autonomous replication model that includes rapid protein exchange, burst of activity, and a 'memory effect' while moving processively forward.