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Methods in Molecular Biology (Clifton,... 2022With improvements in biophysical approaches, there is growing interest in characterizing large, flexible multi-protein complexes. The use of recombinant baculoviruses to...
With improvements in biophysical approaches, there is growing interest in characterizing large, flexible multi-protein complexes. The use of recombinant baculoviruses to express heterologous genes in cultured insect cells has advantages for the expression of human protein complexes because of the ease of co-expressing multiple proteins in insect cells and the presence of a conserved post-translational machinery that introduces many of the same modifications found in human cells. Here we describe the preparation of recombinant baculoviruses expressing DNA ligase IIIα, XRCC1, and TDP1, their subsequent co-expression in cultured insect cells, the purification of complexes containing DNA ligase IIIα from insect cell lysates, and their characterization by multi-angle light scattering linked to size exclusion chromatography and negative stain electron microscopy.
Topics: Animals; DNA Ligase ATP; DNA Ligases; DNA-Binding Proteins; Humans; Insecta; Poly-ADP-Ribose Binding Proteins; X-ray Repair Cross Complementing Protein 1; Xenopus Proteins
PubMed: 35290642
DOI: 10.1007/978-1-0716-2063-2_15 -
Biology Direct Dec 2009Eukaryotic Nucleo-Cytoplasmic Large DNA Viruses (NCLDV) encode most if not all of the enzymes involved in their DNA replication. It has been inferred that genes for...
BACKGROUND
Eukaryotic Nucleo-Cytoplasmic Large DNA Viruses (NCLDV) encode most if not all of the enzymes involved in their DNA replication. It has been inferred that genes for these enzymes were already present in the last common ancestor of the NCLDV. However, the details of the evolution of these genes that bear on the complexity of the putative ancestral NCLDV and on the evolutionary relationships between viruses and their hosts are not well understood.
RESULTS
Phylogenetic analysis of the ATP-dependent and NAD-dependent DNA ligases encoded by the NCLDV reveals an unexpectedly complex evolutionary history. The NAD-dependent ligases are encoded only by a minority of NCLDV (including mimiviruses, some iridoviruses and entomopoxviruses) but phylogenetic analysis clearly indicated that all viral NAD-dependent ligases are monophyletic. Combined with the topology of the NCLDV tree derived by consensus of trees for universally conserved genes suggests that this enzyme was represented in the ancestral NCLDV. Phylogenetic analysis of ATP-dependent ligases that are encoded by chordopoxviruses, most of the phycodnaviruses and Marseillevirus failed to demonstrate monophyly and instead revealed an unexpectedly complex evolutionary trajectory. The ligases of the majority of phycodnaviruses and Marseillevirus seem to have evolved from bacteriophage or bacterial homologs; the ligase of one phycodnavirus, Emiliana huxlei virus, belongs to the eukaryotic DNA ligase I branch; and ligases of chordopoxviruses unequivocally cluster with eukaryotic DNA ligase III.
CONCLUSIONS
Examination of phyletic patterns and phylogenetic analysis of DNA ligases of the NCLDV suggest that the common ancestor of the extant NCLDV encoded an NAD-dependent ligase that most likely was acquired from a bacteriophage at the early stages of evolution of eukaryotes. By contrast, ATP-dependent ligases from different prokaryotic and eukaryotic sources displaced the ancestral NAD-dependent ligase at different stages of subsequent evolution. These findings emphasize complex routes of viral evolution that become apparent through detailed phylogenomic analysis but not necessarily in reconstructions based on phyletic patterns of genes.
REVIEWERS
This article was reviewed by: Patrick Forterre, George V. Shpakovski, and Igor B. Zhulin.
Topics: Asfarviridae; Biological Evolution; Cell Nucleus; Cytoplasm; DNA Ligase ATP; DNA Ligases; DNA Viruses; Eukaryota; Genome, Viral; Iridoviridae; Phycodnaviridae; Phylogeny; Poxviridae
PubMed: 20021668
DOI: 10.1186/1745-6150-4-51 -
Nucleic Acids Research 2006Allosteric nucleic acid ligases have been used previously to transform analyte-binding into the formation of oligonucleotide templates that can be amplified and...
Allosteric nucleic acid ligases have been used previously to transform analyte-binding into the formation of oligonucleotide templates that can be amplified and detected. We have engineered binary deoxyribozyme ligases whose two components are brought together by bridging oligonucleotide effectors. The engineered ligases can 'read' one sequence and then 'write' (by ligation) a separate, distinct sequence, which can in turn be uniquely amplified. The binary deoxyribozymes show great specificity, can discriminate against a small number of mutations in the effector, and can read and recode DNA information with high fidelity even in the presence of excess obscuring genomic DNA. In addition, the binary deoxyribozymes can read non-natural nucleotides and write natural sequence information. The binary deoxyribozyme ligases could potentially be used in a variety of applications, including the detection of single nucleotide polymorphisms in genomic DNA or the identification of short nucleic acids such as microRNAs.
Topics: Allosteric Regulation; Base Pairing; Base Sequence; Catalysis; Catalytic Domain; DNA Ligases; DNA, Catalytic; Enzyme Activation; Nucleic Acid Conformation; Oligonucleotides; Polymerase Chain Reaction; Protein Engineering; Substrate Specificity
PubMed: 16648360
DOI: 10.1093/nar/gkl176 -
DNA Repair May 2014Non-homologous end joining (NHEJ) repairs DNA double-strand breaks generated by DNA damage and also those occurring in V(D)J recombination in immunoglobulin and T cell...
Non-homologous end joining (NHEJ) repairs DNA double-strand breaks generated by DNA damage and also those occurring in V(D)J recombination in immunoglobulin and T cell receptor production in the immune system. In NHEJ DNA-PKcs assembles with Ku heterodimer on the DNA ends at double-strand breaks, in order to bring the broken ends together and to assemble other proteins, including DNA ligase IV (LigIV), required for DNA repair. Here we focus on structural aspects of the interactions of LigIV with XRCC4, XLF, Artemis and DNA involved in the bridging and end-joining steps of NHEJ. We begin with a discussion of the role of XLF, which interacts with Ku and forms a hetero-filament with XRCC4; this likely forms a scaffold bridging the DNA ends. We then review the well-defined interaction of XRCC4 with LigIV, and discuss the possibility of this complex interrupting the filament formation, so positioning the ligase at the correct positions close to the broken ends. We also describe the interactions of LigIV with Artemis, the nuclease that prepares the ends for ligation and also interacts with DNA-PK. Lastly we review the likely affects of Mendelian mutations on these multiprotein assemblies and their impacts on the form of inherited disease.
Topics: Binding Sites; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA Ligases; DNA Repair Enzymes; DNA-Activated Protein Kinase; DNA-Binding Proteins; Endonucleases; Humans; Models, Molecular; Multiprotein Complexes; Nuclear Proteins; Protein Binding
PubMed: 24636752
DOI: 10.1016/j.dnarep.2014.02.010 -
The Journal of Biological Chemistry Jan 2008Double-strand breaks are common in all living cells, and there are two major pathways for their repair. In eukaryotes, homologous recombination is restricted to late S... (Review)
Review
Double-strand breaks are common in all living cells, and there are two major pathways for their repair. In eukaryotes, homologous recombination is restricted to late S or G(2), whereas nonhomologous DNA end joining (NHEJ) can occur throughout the cell cycle and is the major pathway for the repair of double-strand breaks in multicellular eukaryotes. NHEJ is distinctive for the flexibility of the nuclease, polymerase, and ligase activities that are used. This flexibility permits NHEJ to function on the wide range of possible substrate configurations that can arise when double-strand breaks occur, particularly at sites of oxidative damage or ionizing radiation. NHEJ does not return the local DNA to its original sequence, thus accounting for the wide range of end results. Part of this heterogeneity arises from the diversity of the DNA ends, but much of it arises from the many alternative ways in which the nuclease, polymerases, and ligase can act during NHEJ. Physiologic double-strand break processes make use of the imprecision of NHEJ in generating antigen receptor diversity. Pathologically, the imprecision of NHEJ contributes to genome mutations that arise over time.
Topics: DNA Breaks, Double-Stranded; DNA Helicases; DNA Ligases; DNA Repair; DNA Repair Enzymes; DNA-Directed DNA Polymerase; Humans; Models, Biological
PubMed: 17999957
DOI: 10.1074/jbc.R700039200 -
Cellular and Molecular Life Sciences :... Mar 2009DNA double-strand breaks (DSBs) arise in cells from endogenous and exogenous attacks on the DNA backbone, but also as a direct consequence of replication failures.... (Review)
Review
DNA double-strand breaks (DSBs) arise in cells from endogenous and exogenous attacks on the DNA backbone, but also as a direct consequence of replication failures. Proper repair of all these DSBs is essential for genome stability. Repair of broken chromosomes is a challenge for dividing cells that need to distribute equal genetic information to daughter cells. Consequently, eukaryotic organisms have evolved multi-potent and efficient mechanisms to repair DSBs that are primarily divided into two types of pathways: nonhomologous end joining (NHEJ) and homologous recombination (HR). Here we briefly describe how eukaryotic cells sense DSBs and trigger cell cycle arrest to allow repair, and we review the mechanisms of both NHEJ and HR pathways and the choice between them. (Part of a Multi-author Review).
Topics: Cell Cycle; DNA Breaks, Double-Stranded; DNA Damage; DNA Ligases; DNA Repair; DNA Replication; Genomic Instability; Recombination, Genetic
PubMed: 19153654
DOI: 10.1007/s00018-009-8740-3 -
Genomics Nov 2023Ligase IV is a key enzyme involved during DNA double-strand breaks (DSBs) repair through nonhomologous end joining (NHEJ). However, in contrast to Ligase IV deficient...
Ligase IV is a key enzyme involved during DNA double-strand breaks (DSBs) repair through nonhomologous end joining (NHEJ). However, in contrast to Ligase IV deficient mouse cells, which are embryonic lethal, Ligase IV deficient human cells, including pre-B cells, are viable. Using CRISPR-Cas9 mediated genome editing, we have generated six different LIG4 mutants in cervical cancer and normal kidney epithelial cell lines. While the LIG4 mutant cells showed a significant reduction in NHEJ, joining mediated through microhomology-mediated end joining (MMEJ) and homologous recombination (HR) were significantly high. The reduced NHEJ joining activity was restored by adding purified Ligase IV/XRCC4. Accumulation of DSBs and reduced cell viability were observed in LIG4 mutant cells. LIG4 mutant cells exhibited enhanced sensitivity towards DSB-inducing agents such as ionizing radiation (IR) and etoposide. More importantly, the LIG4 mutant of cervical cancer cells showed increased sensitivity towards FDA approved drugs such as Carboplatin, Cisplatin, Paclitaxel, Doxorubicin, and Bleomycin used for cervical cancer treatment. These drugs, in combination with IR showed enhanced cancer cell death in the background of LIG4 gene mutation. Thus, our study reveals that mutation in LIG4 results in compromised NHEJ, leading to sensitization of cervical cancer cells towards currently used cancer therapeutics.
Topics: Animals; Female; Humans; Mice; DNA Damage; DNA End-Joining Repair; DNA Ligase ATP; DNA Ligases; DNA Repair; Ligases; Uterine Cervical Neoplasms
PubMed: 37871849
DOI: 10.1016/j.ygeno.2023.110731 -
Molecular Microbiology Feb 2006DNA ligases join the ends of DNA molecules during replication, repair and recombination. ATP-dependent ligases are found predominantly in the eukarya and archaea whereas...
DNA ligases join the ends of DNA molecules during replication, repair and recombination. ATP-dependent ligases are found predominantly in the eukarya and archaea whereas NAD+-dependent DNA ligases are found only in the eubacteria and in entomopoxviruses. Using the genetically tractable halophile Haloferax volcanii as a model system, we describe the first genetic analysis of archaeal DNA ligase function. We show that the Hfx. volcanii ATP-dependent DNA ligase family member, LigA, is non-essential for cell viability, raising the question of how DNA strands are joined in its absence. We show that Hfx. volcanii also encodes an NAD+-dependent DNA ligase family member, LigN, the first such enzyme to be identified in the archaea, and present phylogenetic analysis indicating that the gene encoding this protein has been acquired by lateral gene transfer (LGT) from eubacteria. As with LigA, we show that LigN is also non-essential for cell viability. Simultaneous inactivation of both proteins is lethal, however, indicating that they now share an essential function. Thus the LigN protein acquired by LGT appears to have been co-opted as a back-up for LigA function, perhaps to provide additional ligase activity under conditions of high genotoxic stress.
Topics: Archaeal Proteins; DNA Damage; DNA Ligase ATP; DNA Ligases; Evolution, Molecular; Gene Transfer, Horizontal; Haloferax volcanii; Phylogeny
PubMed: 16420348
DOI: 10.1111/j.1365-2958.2005.04975.x -
Cell Cycle (Georgetown, Tex.) Sep 2010Okazaki fragment processing is an integral part of DNA replication. For a long time, we assumed that the maturation of these small RNA-primed DNA fragments did not...
Okazaki fragment processing is an integral part of DNA replication. For a long time, we assumed that the maturation of these small RNA-primed DNA fragments did not necessarily have to occur during S phase, but could be postponed to late in S phase after the bulk of DNA synthesis had been completed. This view was primarily based on the arrest phenotype of temperature-sensitive DNA ligase I mutants in yeast, which accumulated with an almost fully duplicated set of chromosomes. However, many temperature-sensitive alleles can be leaky and the re-evaluation of DNA ligase I-deficient cells has offered new and unexpected insights into how cells keep track of lagging strand synthesis. It turns out that if Okazaki fragment joining goes awry, cells have their own alarm system in the form of ubiquitin that is conjugated to the replication clamp PCNA. Although this modification results in mono- and poly-ubiquitination of PCNA, it is genetically distinct from the known post-replicative repair mark at lysine 164. In this Extra View, we discuss the possibility that eukaryotic cells utilize different enzymatic pathways and ubiquitin attachment sites on PCNA to alert the replication machinery to the accumulation of single-stranded gaps or nicks behind the fork.
Topics: DNA; DNA Damage; DNA Ligase ATP; DNA Ligases; DNA Repair; DNA Replication; Proliferating Cell Nuclear Antigen; S Phase; SUMO-1 Protein; Ubiquitination
PubMed: 20930510
DOI: 10.4161/cc.9.18.13121 -
Nature Communications Feb 2024Nonhomologous end joining (NHEJ), the primary pathway of vertebrate DNA double-strand-break (DSB) repair, directly re-ligates broken DNA ends. Damaged DSB ends that...
Nonhomologous end joining (NHEJ), the primary pathway of vertebrate DNA double-strand-break (DSB) repair, directly re-ligates broken DNA ends. Damaged DSB ends that cannot be immediately re-ligated are modified by NHEJ processing enzymes, including error-prone polymerases and nucleases, to enable ligation. However, DSB ends that are initially compatible for re-ligation are typically joined without end processing. As both ligation and end processing occur in the short-range (SR) synaptic complex that closely aligns DNA ends, it remains unclear how ligation of compatible ends is prioritized over end processing. In this study, we identify structural interactions of the NHEJ-specific DNA Ligase IV (Lig4) within the SR complex that prioritize ligation and promote NHEJ fidelity. Mutational analysis demonstrates that Lig4 must bind DNA ends to form the SR complex. Furthermore, single-molecule experiments show that a single Lig4 binds both DNA ends at the instant of SR synapsis. Thus, Lig4 is poised to ligate compatible ends upon initial formation of the SR complex before error-prone processing. Our results provide a molecular basis for the fidelity of NHEJ.
Topics: DNA Ligase ATP; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA Repair; DNA Ligases; DNA
PubMed: 38341432
DOI: 10.1038/s41467-024-45553-z