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Analytical Biochemistry Mar 2021The functionalization of 5'-OH group in nucleic acids is of significant value for molecular biology. In the current work we discovered that acid-labile...
The functionalization of 5'-OH group in nucleic acids is of significant value for molecular biology. In the current work we discovered that acid-labile 4,4'-dimethoxytrityl protecting group (DMT) of oligonucleotides (ONs) is stable under PCR conditions and does not interfere with activity of DNA polymerases. So application of 5'-DMT-protected ONs could allow producing both symmetric and asymmetric 5'-DMT-blocked double-stranded DNA (dsDNA) fragments. We demonstrated that the presence of thiol compounds (mercaptoethanol and dithiothreitol) in PCR mixture is undesirable for the stability of DMT-group. DMT-ONs can be successfully used during polymerase chain assembly of synthetic genes. We tested 5'-DMT dsDNA in blunt-end DNA ligation reaction by T4 DNA ligase and found that it could not be ligated with 5'-phosphorylated DNA fragments, namely linearized plasmid vector pJET1.2/blunt. Possible reason for this is steric hindrance created by bulky and rigid DMT-group, that prevents entering enzyme active site. We also demonstrated that 5'-DMT modification of dsDNA does not affect activity of T5 5',3'-exonuclease towards both ssDNA and dsDNA. Further screening of the exonucleases, sensitive to 5'-DMT-modification or search of ways to separate long 5'-DMT-ssDNA and 5'-OH-ssDNA could allow finding application of 5'-DMT-modified oligo- and polynucleotides.
Topics: DNA Ligases; DNA, Single-Stranded; Exodeoxyribonucleases
PubMed: 33508272
DOI: 10.1016/j.ab.2021.114115 -
Methods in Molecular Biology (Clifton,... 2023Endogenous and exogenous genotoxic agents can generate various types of non-ligatable DNA ends at the site of strand break in the mammalian genome. If not repaired, such...
Endogenous and exogenous genotoxic agents can generate various types of non-ligatable DNA ends at the site of strand break in the mammalian genome. If not repaired, such lesions will impede transcription and replication and can lead to various cellular pathologies. Among various "dirty" DNA ends, 3'-phosphate is one of the most abundant lesions generated in the mammalian cells. Polynucleotide kinase 3'-phosphatase (PNKP) is the major DNA end-processing enzyme for resolving 3'-phosphate termini in the mammalian cells, and thus, it is involved in DNA base excision repair (BER), single-strand break repair, and classical nonhomologous end joining (C-NHEJ)-mediated DNA double-strand break (DSB) repair. The 3'-OH ends generated following PNKP-mediated processing of 3'-P are utilized by a DNA polymerase to fill in the gap, and subsequently, the nick is sealed by a DNA ligase to complete the repair process. Here we describe two novel assay systems to detect phosphate release by PNKP's 3'-phosphatase activity and PNKP-mediated in vitro single-strand break repair with minimal repair components (PNKP, DNA polymerase, and DNA ligase) using either purified proteins or cell-free nuclear extracts from mammalian cells/tissues. These assays are highly reproducible and sensitive, and the researchers would be able to detect any significant difference in PNKP's 3'-phosphatase activity as well as PNKP-mediated single-strand break repair activity in diseased mammalian cells/tissues vs normal healthy controls.
Topics: Animals; DNA Repair Enzymes; Polynucleotide 5'-Hydroxyl-Kinase; Radioactivity; DNA Repair; DNA Ligases; DNA-Directed DNA Polymerase; DNA; Phosphates; Phosphoric Monoester Hydrolases; Mammals
PubMed: 37574474
DOI: 10.1007/978-1-0716-3373-1_3 -
MSphere Jun 2022In mammalian cells, DNA double-strand breaks (DSBs) are mainly repaired by nonhomologous end joining (NHEJ) pathway. Ku (a heterodimer formed by Ku70 and Ku80 proteins)...
In mammalian cells, DNA double-strand breaks (DSBs) are mainly repaired by nonhomologous end joining (NHEJ) pathway. Ku (a heterodimer formed by Ku70 and Ku80 proteins) and DNA ligase IV are the core NHEJ factors. Ku could also be involved in other cellular processes, including telomere length regulation, DNA replication, transcription, and translation control. , an early branching eukaryote and the causative agent of leishmaniasis, has no functional NHEJ pathway due to its lack of DNA ligase IV and other NHEJ factors but retains Ku70 and Ku80 proteins. In this study, we generated Leishmania donovani Ku70 disruption mutants and Ku70 and Ku80 double gene (Ku70/80) disruption mutants. We found that Ku is still involved in DSB repair, possibly through its binding to DNA ends to block and slowdown 5' end resections and Ku-Ku or other protein interactions. Depending on location of a DSB between the direct repeat genomic sequences, Ku could have an inhibiting effect, no effect or a promoting effect on the DSB repair mediated by single strand annealing (SSA), the most frequently used DSB repair pathway in . Ku70/80 proteins are also required for the healthy proliferation of cells. Interestingly, unlike in Trypanosoma brucei and L. mexicana, Ku70/80 proteins are dispensable for maintaining the normal lengths of telomeres in L. donovani. We also show it is possible to reconstitute the two components (Ku and Ligase D) NHEJ pathway derived from Mycobacterium marinum in . This improved DSB repair fidelity and efficiency in and sets up an example that the bacterial NHEJ pathway can be successfully reconstructed in an NHEJ-deficient eukaryotic parasite. Nonhomologous end joining (NHEJ) is the most efficient double-stranded DNA break (DSB) repair pathway in mammalian cells. In contrast, the protozoan parasite has no functional NHEJ pathway but retains the core NHEJ factors of Ku70 and Ku80 proteins. In this study, we found that Ku heterodimers are still participating in DSB repair possibly through blocking 5' end resections and Ku-Ku protein interactions. Depending on the DSB location, Ku could have an inhibiting or promoting effect on DSB repair mediated by the single-strand annealing repair pathway. Ku is also required for the normal growth of the parasite but surprisingly dispensable for maintaining the telomere lengths. Further, we show it is possible to introduce Mycobacterium marinum NHEJ pathway into . Understanding DSB repair mechanisms of may improve the CRISPR gene targeting specificity and efficiency and help identify new drug targets for this important human parasite.
Topics: Animals; DNA; DNA End-Joining Repair; DNA Ligase ATP; DNA-Binding Proteins; Humans; Leishmania; Mammals; Mycobacterium marinum
PubMed: 35695492
DOI: 10.1128/msphere.00156-22 -
Angewandte Chemie (International Ed. in... Apr 2023Small, single-stranded DNA (ssDNA) circles have many applications, such as templating rolling circle amplification (RCA), capturing microRNAs, and scaffolding DNA...
Small, single-stranded DNA (ssDNA) circles have many applications, such as templating rolling circle amplification (RCA), capturing microRNAs, and scaffolding DNA nanostructures. However, it is challenging to prepare such ssDNA circles, particularly when the DNA size becomes very small (e.g. a 20 nucleotide (nt) long ssDNA circle). Often, such short ssDNA dominantly form concatemers (either linear or circular) due to intermolecular ligation, instead of forming monomeric ssDNA circles by intramolecular ligation. Herein, a simple method to overcome this problem by designing the complementary linker molecules is reported. It is demonstrated that ssDNA, as short as 16 nts, can be enzymatically ligated (by the commonly used T4 DNA ligase) into monomeric ssDNA circles at high concentration (100 μM) with high yield (97 %). This method does not require any special sequence, thus, it is expected to be generally applicable. The experimental protocol is identical to regular DNA ligation, thus, is expected to be user friendly for general chemists and biologists.
Topics: DNA, Single-Stranded; DNA; Nucleotides; Nanostructures; DNA Ligases; Nucleic Acid Amplification Techniques; DNA, Circular
PubMed: 36652628
DOI: 10.1002/anie.202218443 -
Advances in Experimental Medicine and... 2018Chromosomal translocations are now well understood to not only constitute signature molecular markers for certain human cancers but often also to be causative in the... (Review)
Review
Chromosomal translocations are now well understood to not only constitute signature molecular markers for certain human cancers but often also to be causative in the genesis of that tumor. Despite the obvious importance of such events, the molecular mechanism of chromosomal translocations in human cells remains poorly understood. Part of the explanation for this dearth of knowledge is due to the complexity of the reaction and the need to archaeologically work backwards from the final product (a translocation) to the original unrearranged chromosomes to infer mechanism. Although not definitive, these studies have indicated that the aberrant usage of endogenous DNA repair pathways likely lies at the heart of the problem. An equally obfuscating aspect of this field, however, has also originated from the unfortunate species-specific differences that appear to exist in the relevant model systems that have been utilized to investigate this process. Specifically, yeast and murine systems (which are often used by basic science investigators) rely on different DNA repair pathways to promote chromosomal translocations than human somatic cells. In this chapter, we will review some of the basic concepts of chromosomal translocations and the DNA repair systems thought to be responsible for their genesis with an emphasis on underscoring the differences between other species and human cells. In addition, we will focus on a specific subset of translocations that involve the very end of a chromosome (a telomere). A better understanding of the relationship between DNA repair pathways and chromosomal translocations is guaranteed to lead to improved therapeutic treatments for cancer.
Topics: Animals; DNA Damage; DNA Ligases; DNA Repair; Humans; Mice; Telomere; Translocation, Genetic
PubMed: 29956293
DOI: 10.1007/978-981-13-0593-1_7 -
Archaea (Vancouver, B.C.) 2015DNA ligases are indispensable in all living cells and ubiquitous in all organs. DNA ligases are broadly utilized in molecular biology research fields, such as genetic... (Review)
Review
DNA ligases are indispensable in all living cells and ubiquitous in all organs. DNA ligases are broadly utilized in molecular biology research fields, such as genetic engineering and DNA sequencing technologies. Here we review the utilization of DNA ligases in a variety of in vitro gene manipulations, developed over the past several decades. During this period, fewer protein engineering attempts for DNA ligases have been made, as compared to those for DNA polymerases. We summarize the recent progress in the elucidation of the DNA ligation mechanisms obtained from the tertiary structures solved thus far, in each step of the ligation reaction scheme. We also present some examples of engineered DNA ligases, developed from the viewpoint of their three-dimensional structures.
Topics: DNA Ligases; Models, Biological; Protein Engineering; Structure-Activity Relationship
PubMed: 26508902
DOI: 10.1155/2015/267570 -
Nature Communications Mar 2016Despite the implication of Wnt signalling in radioresistance, the underlying mechanisms are unknown. Here we find that high Wnt signalling is associated with...
Despite the implication of Wnt signalling in radioresistance, the underlying mechanisms are unknown. Here we find that high Wnt signalling is associated with radioresistance in colorectal cancer (CRC) cells and intestinal stem cells (ISCs). We find that LIG4, a DNA ligase in DNA double-strand break repair, is a direct target of β-catenin. Wnt signalling enhances non-homologous end-joining repair in CRC, which is mediated by LIG4 transactivated by β-catenin. During radiation-induced intestinal regeneration, LIG4 mainly expressed in the crypts is conditionally upregulated in ISCs, accompanied by Wnt/β-catenin signalling activation. Importantly, among the DNA repair genes, LIG4 is highly upregulated in human CRC cells, in correlation with β-catenin hyperactivation. Furthermore, blocking LIG4 sensitizes CRC cells to radiation. Our results reveal the molecular mechanism of Wnt signalling-induced radioresistance in CRC and ISCs, and further unveils the unexpected convergence between Wnt signalling and DNA repair pathways in tumorigenesis and tissue regeneration.
Topics: Animals; Animals, Genetically Modified; Cell Line, Tumor; Cell Proliferation; Cell Survival; Colorectal Neoplasms; Computer Simulation; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA Ligase ATP; DNA Ligases; DNA Repair; Gene Expression Profiling; Gene Expression Regulation, Neoplastic; Humans; Immunohistochemistry; Intestinal Mucosa; Intestines; Mice; Radiation Tolerance; Reverse Transcriptase Polymerase Chain Reaction; Stem Cells; Telomerase; Transcriptional Activation; Wnt Signaling Pathway; beta Catenin
PubMed: 27009971
DOI: 10.1038/ncomms10994 -
DNA Repair Sep 2020DNA polymerase μ is a Family X member that participates in repair of DNA double strand breaks (DSBs) by non-homologous end joining. Its role is to fill short gaps... (Review)
Review
DNA polymerase μ is a Family X member that participates in repair of DNA double strand breaks (DSBs) by non-homologous end joining. Its role is to fill short gaps arising as intermediates in the process of V(D)J recombination and during processing of accidental double strand breaks. Pol μ is the only known template-dependent polymerase that can repair non-complementary DSBs with unpaired 3´primer termini. Here we review the unique properties of Pol μ that allow it to productively engage such a highly unstable substrate to generate a nick that can be sealed by DNA Ligase IV.
Topics: DNA; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA Ligase ATP; DNA-Directed DNA Polymerase; Humans
PubMed: 33087269
DOI: 10.1016/j.dnarep.2020.102932 -
Molecular Carcinogenesis Feb 2017The terminal step of ligation of single and/or double-strand breaks during physiological processes such as DNA replication, repair and recombination requires...
The terminal step of ligation of single and/or double-strand breaks during physiological processes such as DNA replication, repair and recombination requires participation of DNA ligases in all mammals. DNA Ligase I has been well characterised to play vital roles during these processes. Considering the indispensable role of DNA Ligase I, a therapeutic strategy to impede proliferation of cancer cells is by using specific small molecule inhibitors against it. In the present study, we have designed and chemically synthesised putative DNA Ligase I inhibitors. Based on various biochemical and biophysical screening approaches, we identify two prospective DNA Ligase I inhibitors, SCR17 and SCR21. Both the inhibitors blocked ligation of nicks on DNA in a concentration-dependent manner, when catalysed by cell-free extracts or purified Ligase I. Docking studies in conjunction with biolayer interferometry and gel shift assays revealed that both SCR17 and SCR21 can bind to Ligase I, particularly to the DNA Binding Domain of Ligase I with KD values in nanomolar range. The inhibitors did not show significant affinity towards DNA Ligase III and DNA Ligase IV. Further, addition of Ligase I could restore the joining, when the inhibitors were treated with testicular cell-free extracts. Ex vivo studies using multiple assays showed that even though cell death was limited in the presence of inhibitors in cancer cells, their proliferation was compromised. Hence, we identify two promising DNA Ligase I inhibitors, which can be used in biochemical and cellular assays, and could be further modified and optimised to target cancer cells. © 2016 Wiley Periodicals, Inc.
Topics: Animals; Cell Cycle; Cell Line, Tumor; Cell Proliferation; DNA Ligase ATP; DNA Replication; Drug Design; Enzyme Inhibitors; HEK293 Cells; Humans; Male; Molecular Docking Simulation; Rats; Rats, Wistar; Small Molecule Libraries
PubMed: 27312791
DOI: 10.1002/mc.22516 -
Cold Spring Harbor Protocols Mar 2017This protocol describes an automated procedure for constructing a nonindexed Illumina DNA library and relies on the use of a CyBi-SELMA automated pipetting machine, the...
This protocol describes an automated procedure for constructing a nonindexed Illumina DNA library and relies on the use of a CyBi-SELMA automated pipetting machine, the Covaris E210 shearing instrument, and the epMotion 5075. With this method, genomic DNA fragments are produced by sonication, using high-frequency acoustic energy to shear DNA. Here, double-stranded DNA is fragmented when exposed to the energy of adaptive focused acoustic shearing (AFA). The resulting DNA fragments are ligated to adaptors, amplified by polymerase chain reaction (PCR), and subjected to size selection using magnetic beads. The product is suitable for use as template in whole-genome sequencing.
Topics: Automation, Laboratory; DNA; DNA Ligases; Gene Library; Genomics; Molecular Biology; Polymerase Chain Reaction; Sequence Analysis, DNA; Sonication
PubMed: 27803279
DOI: 10.1101/pdb.prot094623