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Methods in Molecular Biology (Clifton,... 2023The Gibson Assembly is a popular method for molecular cloning which has been developed specifically to join several fragments together in a specific order, without the...
The Gibson Assembly is a popular method for molecular cloning which has been developed specifically to join several fragments together in a specific order, without the constraint of restriction enzyme sites. This method is based on the assembly of overlapping fragments, generally produced by PCR, and then combining them using three enzymes: a 5' exonuclease, a DNA polymerase, and a DNA ligase, in an isothermal reaction. Here, we describe this method, including the design of primers for the generation of the overlapping fragments and the assembly; to this end, we provide an example involving joining two fragments in a single plasmid.
Topics: Cloning, Molecular; DNA Ligase ATP; DNA Ligases; DNA Primers; Nucleotidyltransferases
PubMed: 36853455
DOI: 10.1007/978-1-0716-3004-4_4 -
Methods in Molecular Biology (Clifton,... 2023Traditional molecular cloning involves a series of linked experimental steps performed with the overall goal of isolating ("cloning") a specific DNA sequence-often a...
Traditional molecular cloning involves a series of linked experimental steps performed with the overall goal of isolating ("cloning") a specific DNA sequence-often a gene. The main purpose of cloning is to study either that DNA sequence or the RNA or protein product it encodes. Building on key enzymatic discoveries in the late 1960s, gene cloning was pioneered in the early 1970s. Since then, DNA cloning and manipulation have been used in every area of biological and biomedical research, from molecular genetics, structural biology, and developmental biology to neurobiology, ancient DNA studies, and immunology. It is a versatile technique that can be applied to a variety of starting DNA types and lengths, including cDNAs, genes, gene fragments, chromosomal regions, or shorter fragments such as PCR products and functional control regions such as enhancers or promoters. The starting DNA can originate from any cell, tissue, or organism. In this chapter we will cover traditional ("classic") molecular cloning strategy. This comprises six linked stages in which (1) PCR is used to amplify a DNA region of interest that is then (2) digested with restriction enzymes, alongside a selected vector, to produce complementary ends crucial for the two molecules to be (3) ligated by an ATP-dependent DNA ligase, creating a recombinant DNA molecule. The recombinant DNA is then (4) introduced into competent bacterial cells by transformation and (5) grown on a selective agar media, followed by (6) colony-PCR for screening purposes. We provide a worked example to demonstrate the cloning of an average-size gene (in this case the 2 kb DNA ligase A gene) from E. coli into a common plasmid expression vector. We have included six color figures and two tables to depict the key stages of a classical molecular cloning protocol. If you are cloning a segment of DNA or a gene, remember that each DNA cloning experiment is unique in terms of sequence, length, and experimental purpose. However, the principles of traditional cloning covered in this chapter are the same for any DNA sequence; we have included a detailed notes section, so you should easily be able to transfer them to your own work. Some of the following chapters in this volume will cover other, more recently developed, cloning protocols.
Topics: DNA, Recombinant; Escherichia coli; Cloning, Molecular; Polymerase Chain Reaction; Genetic Vectors; DNA Ligase ATP
PubMed: 36853452
DOI: 10.1007/978-1-0716-3004-4_1 -
Nature Nov 2021Extrachromosomal circular DNA elements (eccDNAs) have been described in the literature for several decades, and are known for their broad existence across different...
Extrachromosomal circular DNA elements (eccDNAs) have been described in the literature for several decades, and are known for their broad existence across different species. However, their biogenesis and functions are largely unknown. By developing a new circular DNA enrichment method, here we purified and sequenced full-length eccDNAs with Nanopore sequencing. We found that eccDNAs map across the entire genome in a close to random manner, suggesting a biogenesis mechanism of random ligation of genomic DNA fragments. Consistent with this idea, we found that apoptosis inducers can increase eccDNA generation, which is dependent on apoptotic DNA fragmentation followed by ligation by DNA ligase 3. Importantly, we demonstrated that eccDNAs can function as potent innate immunostimulants in a manner that is independent of eccDNA sequence but dependent on eccDNA circularity and the cytosolic DNA sensor Sting. Collectively, our study not only revealed the origin, biogenesis and immunostimulant function of eccDNAs but also uncovered their sensing pathway and potential clinical implications in immune response.
Topics: Animals; Apoptosis; Cells, Cultured; Chromosome Mapping; DNA Fragmentation; DNA Ligase ATP; DNA, Circular; Endodeoxyribonucleases; Gene Expression Regulation; Genome; Immunity, Innate; Male; Membrane Proteins; Mice; Poly-ADP-Ribose Binding Proteins
PubMed: 34671165
DOI: 10.1038/s41586-021-04009-w -
Nature Aug 2021The BRCA1-BARD1 tumour suppressor is an E3 ubiquitin ligase necessary for the repair of DNA double-strand breaks by homologous recombination. The BRCA1-BARD1 complex...
The BRCA1-BARD1 tumour suppressor is an E3 ubiquitin ligase necessary for the repair of DNA double-strand breaks by homologous recombination. The BRCA1-BARD1 complex localizes to damaged chromatin after DNA replication and catalyses the ubiquitylation of histone H2A and other cellular targets. The molecular bases for the recruitment to double-strand breaks and target recognition of BRCA1-BARD1 remain unknown. Here we use cryo-electron microscopy to show that the ankyrin repeat and tandem BRCT domains in BARD1 adopt a compact fold and bind to nucleosomal histones, DNA and monoubiquitin attached to H2A amino-terminal K13 or K15, two signals known to be specific for double-strand breaks. We further show that RING domains in BRCA1-BARD1 orient an E2 ubiquitin-conjugating enzyme atop the nucleosome in a dynamic conformation, primed for ubiquitin transfer to the flexible carboxy-terminal tails of H2A and variant H2AX. Our work reveals a regulatory crosstalk in which recognition of monoubiquitin by BRCA1-BARD1 at the N terminus of H2A blocks the formation of polyubiquitin chains and cooperatively promotes ubiquitylation at the C terminus of H2A. These findings elucidate the mechanisms of BRCA1-BARD1 chromatin recruitment and ubiquitylation specificity, highlight key functions of BARD1 in both processes and explain how BRCA1-BARD1 promotes homologous recombination by opposing the DNA repair protein 53BP1 in post-replicative chromatin. These data provide a structural framework to evaluate BARD1 variants and help to identify mutations that drive the development of cancer.
Topics: BRCA1 Protein; Cryoelectron Microscopy; DNA Repair; Histones; Homologous Recombination; Humans; Models, Molecular; Mutation; Neoplasms; Nucleosomes; Protein Domains; Tumor Suppressor Proteins; Tumor Suppressor p53-Binding Protein 1; Ubiquitin; Ubiquitin-Conjugating Enzymes; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 34321665
DOI: 10.1038/s41586-021-03716-8 -
Cancer Research Sep 2023The DNA damage response (DDR) is essential for the maintenance of genomic stability. Protein posttranslational modifications play pivotal roles in regulating the DDR...
UNLABELLED
The DNA damage response (DDR) is essential for the maintenance of genomic stability. Protein posttranslational modifications play pivotal roles in regulating the DDR process. Here, we found that SUMOylated RNF168 undergoes liquid-liquid phase separation (LLPS), which restricts the recruitment of RNF168 to DNA damage sites, reduces RNF168-catalyzed H2A ubiquitination, restrains 53BP1 in nuclear condensates, and ultimately impairs nonhomologous DNA end joining repair efficiency. Sentrin/SUMO-specific protease 1 (SENP1) was identified as a specific deSUMOylase of RNF168, and it was highly expressed in colorectal adenocarcinoma. In response to DNA damage, SENP1 decreased RNF168 SUMOylation and prevented RNF168 from forming nuclear condensates, thus promoting damage repair efficiency and cancer cell resistance to DNA damaging agents. Moreover, high SENP1 expression correlated with poor prognosis in patients with cancer, and SENP1 depletion sensitized cancer cells to chemotherapy. In summary, these findings reveal DDR is suppressed by SUMOylation-induced LLPS of RNF168 and suggest that SENP1 is a potential target for cancer therapy.
SIGNIFICANCE
Sentrin/SUMO-specific protease 1 decreases RNF168 SUMOylation and liquid-liquid phase separation to promote DNA damage repair, safeguarding genomic integrity and driving chemotherapy resistance.
Topics: Humans; Ubiquitin-Protein Ligases; Peptide Hydrolases; DNA Repair; Ubiquitination; DNA Damage; Endopeptidases; Colonic Neoplasms; Drug Resistance; Small Ubiquitin-Related Modifier Proteins; Cysteine Endopeptidases
PubMed: 37350666
DOI: 10.1158/0008-5472.CAN-22-4017 -
Cell Feb 2021Mutations in DNA damage response (DDR) genes endanger genome integrity and predispose to cancer and genetic disorders. Here, using CRISPR-dependent cytosine base editing...
Mutations in DNA damage response (DDR) genes endanger genome integrity and predispose to cancer and genetic disorders. Here, using CRISPR-dependent cytosine base editing screens, we identify > 2,000 sgRNAs that generate nucleotide variants in 86 DDR genes, resulting in altered cellular fitness upon DNA damage. Among those variants, we discover loss- and gain-of-function mutants in the Tudor domain of the DDR regulator 53BP1 that define a non-canonical surface required for binding the deubiquitinase USP28. Moreover, we characterize variants of the TRAIP ubiquitin ligase that define a domain, whose loss renders cells resistant to topoisomerase I inhibition. Finally, we identify mutations in the ATM kinase with opposing genome stability phenotypes and loss-of-function mutations in the CHK2 kinase previously categorized as variants of uncertain significance for breast cancer. We anticipate that this resource will enable the discovery of additional DDR gene functions and expedite studies of DDR variants in human disease.
Topics: Amino Acid Sequence; Ataxia Telangiectasia Mutated Proteins; Base Sequence; CRISPR-Cas Systems; Camptothecin; Cell Line; DNA Damage; DNA Repair; Female; Gene Editing; Genetic Testing; Humans; Mutation; Phenotype; Protein Binding; Protein Domains; RNA, Guide, CRISPR-Cas Systems; Topoisomerase Inhibitors; Tumor Suppressor p53-Binding Protein 1; Ubiquitin Thiolesterase; Ubiquitin-Protein Ligases
PubMed: 33606978
DOI: 10.1016/j.cell.2021.01.041 -
Molecular Cell Jul 2021Mammalian DNA base excision repair (BER) is accelerated by poly(ADP-ribose) polymerases (PARPs) and the scaffold protein XRCC1. PARPs are sensors that detect...
Mammalian DNA base excision repair (BER) is accelerated by poly(ADP-ribose) polymerases (PARPs) and the scaffold protein XRCC1. PARPs are sensors that detect single-strand break intermediates, but the critical role of XRCC1 during BER is unknown. Here, we show that protein complexes containing DNA polymerase β and DNA ligase III that are assembled by XRCC1 prevent excessive engagement and activity of PARP1 during BER. As a result, PARP1 becomes "trapped" on BER intermediates in XRCC1-deficient cells in a manner similar to that induced by PARP inhibitors, including in patient fibroblasts from XRCC1-mutated disease. This excessive PARP1 engagement and trapping renders BER intermediates inaccessible to enzymes such as DNA polymerase β and impedes their repair. Consequently, PARP1 deletion rescues BER and resistance to base damage in XRCC1 cells. These data reveal excessive PARP1 engagement during BER as a threat to genome integrity and identify XRCC1 as an "anti-trapper" that prevents toxic PARP1 activity.
Topics: Animals; Cell Line; DNA; DNA Breaks, Single-Stranded; DNA Damage; DNA Ligase ATP; DNA Polymerase beta; DNA Repair; DNA-Binding Proteins; Fibroblasts; Humans; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Protein Binding; X-ray Repair Cross Complementing Protein 1
PubMed: 34102106
DOI: 10.1016/j.molcel.2021.05.009 -
American Journal of Physiology.... Jul 2020Mitochondrial injury in granulosa cells is associated with the pathogenesis of polycystic ovary syndrome (PCOS). However, the protective effects of melatonin against...
Mitochondrial injury in granulosa cells is associated with the pathogenesis of polycystic ovary syndrome (PCOS). However, the protective effects of melatonin against mitochondrial injury in the granulosa cells of PCOS remain unclear. In this study, decreased mitochondrial membrane potential and mtDNA content, increased number of autophagosomes were found in the granulosa cells of PCOS patients and the dihydrotestosterone (DHT)-treated KGN cells, with decreased protein level of the autophagy substrate p62 and increased levels of the cellular autophagy markers Beclin 1 and LC3B-II, while the protein levels of PTEN-induced kinase-1 (PINK1) and Parkin were increased and the level of sirtuin 1 (SIRT1) was decreased. DHT-induced PCOS-like mice also showed enhanced mitophagy and decreased mRNA expression. Melatonin treatment significantly increased the protein level of SIRT1 and decreased the levels of PINK1/Parkin, whereas it ameliorated the mitochondrial dysfunction and PCOS phenotype in vitro and in vivo. However, when the KGN cells were treated with siRNA to knock down SIRT1 expression, melatonin treatment failed to repress the excessive mitophagy. In conclusion, melatonin protects against mitochondrial injury in granulosa cells of PCOS by enhancing SIRT1 expression to inhibit excessive PINK1/Parkin-mediated mitophagy.
Topics: Adult; Animals; Antioxidants; Autophagosomes; Autophagy; Beclin-1; Case-Control Studies; Cell Line; DNA, Mitochondrial; Dihydrotestosterone; Female; Granulosa Cells; Humans; Melatonin; Membrane Potential, Mitochondrial; Mice; Microtubule-Associated Proteins; Mitophagy; Polycystic Ovary Syndrome; Protein Kinases; Sirtuin 1; Ubiquitin-Protein Ligases
PubMed: 32343612
DOI: 10.1152/ajpendo.00006.2020 -
Molecular Cell Oct 2021PRIMPOL repriming allows DNA replication to skip DNA lesions, leading to ssDNA gaps. These gaps must be filled to preserve genome stability. Using a DNA fiber approach...
PRIMPOL repriming allows DNA replication to skip DNA lesions, leading to ssDNA gaps. These gaps must be filled to preserve genome stability. Using a DNA fiber approach to directly monitor gap filling, we studied the post-replicative mechanisms that fill the ssDNA gaps generated in cisplatin-treated cells upon increased PRIMPOL expression or when replication fork reversal is defective because of SMARCAL1 inactivation or PARP inhibition. We found that a mechanism dependent on the E3 ubiquitin ligase RAD18, PCNA monoubiquitination, and the REV1 and POLζ translesion synthesis polymerases promotes gap filling in G2. The E2-conjugating enzyme UBC13, the RAD51 recombinase, and REV1-POLζ are instead responsible for gap filling in S, suggesting that temporally distinct pathways of gap filling operate throughout the cell cycle. Furthermore, we found that BRCA1 and BRCA2 promote gap filling by limiting MRE11 activity and that simultaneously targeting fork reversal and gap filling enhances chemosensitivity in BRCA-deficient cells.
Topics: Antineoplastic Agents; BRCA1 Protein; BRCA2 Protein; Cell Line, Tumor; DNA Breaks, Single-Stranded; DNA Helicases; DNA Primase; DNA Repair; DNA Replication; DNA, Neoplasm; DNA-Binding Proteins; DNA-Directed DNA Polymerase; G2 Phase; Genomic Instability; HEK293 Cells; Humans; MRE11 Homologue Protein; Multifunctional Enzymes; Neoplasms; Nucleotidyltransferases; Proliferating Cell Nuclear Antigen; S Phase; Time Factors; Ubiquitin-Conjugating Enzymes; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 34624216
DOI: 10.1016/j.molcel.2021.09.013 -
International Journal of Molecular... Dec 2023Mitochondrial dysregulation, such as mitochondrial complex I deficiency, increased oxidative stress, perturbation of mitochondrial dynamics and mitophagy, has long been... (Review)
Review
Mitochondrial dysregulation, such as mitochondrial complex I deficiency, increased oxidative stress, perturbation of mitochondrial dynamics and mitophagy, has long been implicated in the pathogenesis of PD. Initiating from the observation that mitochondrial toxins cause PD-like symptoms and mitochondrial DNA mutations are associated with increased risk of PD, many mutated genes linked to familial forms of PD, including , , and , have also been found to affect the mitochondrial features. Recent research has uncovered a much more complex involvement of mitochondria in PD. Disruption of mitochondrial quality control coupled with abnormal secretion of mitochondrial contents to dispose damaged organelles may play a role in the pathogenesis of PD. Furthermore, due to its bacterial ancestry, circulating mitochondrial DNAs can function as damage-associated molecular patterns eliciting inflammatory response. In this review, we summarize and discuss the connection between mitochondrial dysfunction and PD, highlighting the molecular triggers of the disease process, the intra- and extracellular roles of mitochondria in PD as well as the therapeutic potential of mitochondrial transplantation.
Topics: Humans; Parkinson Disease; Ubiquitin-Protein Ligases; Mitochondria; DNA, Mitochondrial; Mitophagy
PubMed: 38069350
DOI: 10.3390/ijms242317027