2a). Mutagen. The source data underlying all main text Figs. Unauthorized use of these marks is strictly prohibited. DNA ligase: structure, mechanism, and function - PubMed Our results support a model whereby LIG1 fidelity is governed by a high-fidelity (HiFi) interface between LIG1, Mg2+, and the DNA substrate that tunes the enzyme to release pro-mutagenic DNA nicks. Williams, J. S., Lujan, S. A. DNA polymerase synthesizes a copy of the lagging strand from 3'-to-5' direction with help from two enzymes: DNA primase and DNA ligase. 6d and 6E7,8,9. Using a gel-based ligation assay, the concentration of active enzyme was determined and the extent of adenylylation of the purified enzyme was comparable to that of the LIG1WT (Supplementary Fig. Structure 21, 672679 (2013). We thank Dr. Tom Kunkel and Dr. Lars Pedersen for comments on the manuscript. Structural analysis of bacteriophage T4 DNA replication: a review in Moreover, the toxicity of anti-cancer MutT homolog 1 (MTH1) inhibitors that increase concentrations of oxidatively damaged nucleotide pools is hypothesized to act in part by promoting DNA repair polymerase incorporation of oxidized nucleotides and subsequent ligation by DNA ligases21. 8) but retains the ability to bind adenylylated ligation intermediates24. 7, e2341 (2017). The 28-mer-C:G oligo was formed by annealing oligos 7, 8, and 9 (Supplementary Table1). DNA unwinds at the origin of replication. In control reactions with the 28-mer-C:G substrate, both LIG1WT and LIG1EE/AA remained highly committed to ligating the substrate (Fig. Cell 154, 157168 (2013). Wallace, B. D. & Williams, R. S. Ribonucleotide triggered DNA damage and RNA-DNA damage responses. DNA replication involves key enzymes like topoisomerase, helicase, DNA primase, DNA polymerase, and DNA ligase. Visual Connection Figure 9.10 A replication fork is formed by the opening of the origin of replication, and helicase separates the DNA strands. P01 CA092584/CA/NCI NIH HHS/United States, R01 GM057479/GM/NIGMS NIH HHS/United States, NCI CPTC Antibody Characterization Program. 2a). b Domain structure of human LIG1. DNA Repair 35, 116125 (2015). High-fidelity DNA ligation enforces accurate Okazaki fragment - Nature Mutant 232 LIG1 vectors were constructed using site-directed mutagenesis with synthetic primers and confirmed by sequencing of the coding region. A similar trend was observed for the 8oxoG:A substrate, with APTX effectively suppressing the reaction catalyzed by LIG1WT, whereas not affecting the reaction catalyzed by the LIG1E346A/E592A mutant (Fig. 4e and Supplementary Fig. It has a very important role in DNA Replication in E.Coli. The N-terminal domain (gray) contains a nuclear localization signal and a PIP box motif that mediates interactions with PCNA. Crystallization and cryoprotection were performed as follows: (1) LIG1WT 3ddC 200mM Mg2+ complex: nicked 18mer was assembled by annealing oligos 1, 2, and 3 (Supplementary Table1), and the cryoprotectant was supplemented with 200mM MgCl2. 29, 189193 (2001). Two-tiered enforcement of high-fidelity DNA ligation. It unzips the double-stranded DNA. Google Scholar. 2007;35(5):1624-37. doi: 10.1093/nar/gkm006. Nature 431, 217221 (2004). 4b and Supplementary Fig. For both the Mg2+ and ATP dependences, 500nM of the 28-mer substrate was used along with either 200M ATP or 20mM MgCl2, respectively. The fraction of observed abortive ligation events was determined using Eq. 7C, D). Cherry, A. L. et al. 5B). 13, 10811093 (2004). Nucleic Acids Res. Interaction between PCNA and DNA ligase I is critical for joining of Okazaki fragments and long-patch base-excision repair. sharing sensitive information, make sure youre on a federal LIG1E346A/E592A structures in complex with damaged 8oxoG:A DNA. 3e, f). Drive syringes were loaded with the standard reaction buffer and reactions were quenched using a solution of 1N H2SO4 and 0.25% SDS. Ligase | DNA replication, Enzymatic activity, Protein synthesis Tumbale, P. et al. (5) LIG1E346A/E592A 3OH EDTA complex: nicked 18mer from annealing oligos 1, 3, and 4 (Supplementary Table1), and the cryoprotectant was supplemented with 2mM EDTA. The location of four coordinated Mg2+ ions (Sites 14, Fig. Mol. They catalyze the joining of two molecules, deriving the needed energy from the cleavage of an . DNA ligase I is required for fetal liver erythropoiesis but is not essential for mammalian cell viability. Adams, P. D. et al. This interaction, which is regulated by phosphorylation of the non-catalytic N-terminus of DNA ligase I, also appears to be critical for DNA replication. WT and H260N human APTX catalytic domain (cat, residues 165342) were expressed from pET15b as N-terminal 6 His-tagged proteins in E.coli BL21(DE3) codon-plus cells (Novagen). -Helicase begins to unwind the DNA at the ORIGIN OF REPLICATION (a specific DNA nucleotide sequence) It's common to only show one strands sequence of bases, since the other can be . Every cell completes the entire process . DNA binding induces a large, Figure 17.3. The similar size and shape of the rings formed by the PCNA trimer and the DNA ligase I catalytic region when it engages a DNA nick suggest that these proteins interact to form a double-ring structure during the joining of Okazaki fragments. CAS X-ray diffraction data was collected on Beamline 22-ID of the Advanced Photon Source at a wavelength of 1.000. X-ray diffraction data were processed and scaled using the HKL2000 suite36. 2 and Supplementary Table2). ISSN 2041-1723 (online). However, the 5-AMP moiety must be removed to restore a 5-phosphate for ligation after 3-proofreading and gap filling. By performing rapid-quenching experiments over a range of Mg2+ concentrations with the 28-mer-C:G substrate, we obtained the maximal rates of both adenylyl transfer (ktransfer) and nick sealing (kseal). This intrinsic fidelity hardwires LIG1 to prevent deleterious abortive and mutagenic ligations. For the DNA substrate dependences at saturating levels of MgCl2 and ATP, reactions contained 20mM MgCl2 and 200M ATP. Given that Site 4 ligands are not conserved, we focused our attention on the functional role of Site 1. Bio Chapter 22 DNA Flashcards | Quizlet 2.1 ). However, appropriate molecular models that define the basis of DNA ligase fidelity have been lacking. Rates were obtained from fits to the single-turnover data using a two-step irreversible published BerkeleyMadonna model30. Extensive efforts have established how DNA polymerases discriminate against the incorporation of incorrect nucleotides to varying degrees on the basis of base pairing (correct versus incorrect) and/or sugar identity (deoxyribonucleotides versus ribonucleotides). 1998 Feb;407(1):1-9. doi: 10.1016/s0921-8777(97)00050-5. DNA ligases in the repair and replication of DNA Authors D J Timson 1 , M R Singleton , D B Wigley Affiliation 1 Sir William Dunn School of Pathology, The University of Oxford, South Parks Road, OX1 3RE, Oxford, UK. Topoisomerase unwinds the DNA, helicase separates the strands, and primase adds RNA primers. We find that this site is dispensable for catalysis of step 2 and step 3, but it is critical for discrimination against incorrect base pairs at the 3-end of the nick. and transmitted securely. We next characterized the single-turnover ligation kinetics of the LIG1E346A/E592A mutant using a rapid chemical quench approach30. Bethesda, MD 20894, Web Policies Removal of MgHiFi causes rearrangements in proteinDNA van der Waals contacts proximal to the mutation, creating a cavity that allows for alternative conformations of the DNA backbone and dynamic binding of the phosphodiester backbone between the -1 and -2 nucleotides (Supplementary Fig. The element that transformed the bacteria in Griffith's . Purified DNA ligases have proved to be useful reagents in the construction in vitro of . DNA ligases in the repair and replication of DNA - PubMed & Lindahl, T. Delayed DNA joining at 3 mismatches by human DNA ligases. In the absence of DNA, these domains adopt an extended structure but transition into a compact ring structure when they engage a DNA nick, with each of the domains contacting the DNA. d Active site architecture of the 8oxoG:A complex. (6) LIG1E346A/E592A 38oxoG EDTA complex: nicked 18mer was assembled by annealing oligos 3, 5, and 6 (Supplementary Table1), and the cryoprotectant was supplemented with 2mM EDTA. was supported in part by a training fellowship from the Cellular Biotechnology Training Program at the University of Michigan (T32GM130763). Intriguingly, the delay in nick sealing is prolonged in LIG1WT-catalyzed reactions compared with those containing LIG1E346A/E592A (Fig. Time courses for reactions with the C:G and 8oxoG:A substrates are shown in Supplementary Fig. Although this hypothesis is yet to be tested, the physiological roles of APTX are considered to be complex as it also interacts with several proteins related to the DNA damage response, including XRCC1 and XRCC435. DNA ligases IIIa (922 amino acids) and IIIb (862 amino acids) are the products of a single gene; they differ in amino acid sequence only at their carboxyl termini as . AboutTranscript. DNA ligases are essential guardians of genomic integrity, and ligase dysfunction underlies human genetic disease syndromes. To obtain b APTX increases fidelity of LIG1 by capturing and hydrolyzing the AMPDNA intermediate, preventing accumulation of abortive, and mutagenic ligation products. Ligase - an overview | ScienceDirect Topics Nature Communications (Nat Commun) In this work, we establish a foundation for understanding high-fidelity DNA ligation by human LIG1. doi: 10.1016/s0960-9822(01)00500-0. 32, 59285934 (2004). e Cartoon display of LIG1 comparisons with LIG3 and LIG4. The 5' and 3' ends of DNA (pronounced five prime and three prime) are the two ends of single strand of DNA. Genet 13, 489491 (1996). Our study reveals that LIG1 discriminates against damaged 3 termini at both steps 2 (adenylyl transfer) and 3 (nick sealing) of the ligase reaction. J. Clin. The positions of the nuclear localization signals (NLS, blue) of DNA ligase I and DNA ligase III, and the mitochondrial leader sequence (MLS, cyan) of DNA ligase III are shown. DNA Ligase - Synopsis Bookshelf Subsequent purification was achieved by size exclusion chromatography (Superdex 75, GE healthcare in 50mM Tris, pH 7.5, 500mM NaCl, 5% glycerol, 0.1% -mercaptoethanol) and cation exchange chromatography on a 5mL Hi-Trap SP HP (GE Healthcare). Steady-state reactions contained 0 or 10nM APTX, 1nM LIG1WT, or LIG1E346A/E592A, and 500nM nicked DNA substrate. In the context of 3-blocked ends, LIG1 has been shown to release the AMPDNA species in a process referred to as abortive ligation23,24,25,26. In addition, DNA ligase III is essential for DNA replication in the absence of the replicative DNA ligase, DNA ligase I. DNA ligase III is a component of an alternative non-homologous end joining (NHEJ) pathway for DNA double-strand break (DSB) repair that is more active when the major DNA ligase IV-dependent pathway is defective. For replicative polymerases, the overall fidelity is dictated by intrinsic selectivity for the correct nucleotides, exonucleolytic proofreading, and post-replicative repair pathways such as mismatch DNA repair and ribonucleotide excision repair1,2. Unless noted otherwise, reactions were quenched in the standard loading buffer (50mM EDTA/90% formamide/0.01% xylene cyanol/0.01% bromophenol blue). Anurag M, Jaehnig EJ, Krug K, Lei JT, Bergstrom EJ, Kim BJ, Vashist TD, Huynh AMT, Dou Y, Gou X, Huang C, Shi Z, Wen B, Korchina V, Gibbs RA, Muzny DM, Doddapaneni H, Dobrolecki LE, Rodriguez H, Robles AI, Hiltke T, Lewis MT, Nangia JR, Nemati Shafaee M, Li S, Hagemann IS, Hoog J, Lim B, Osborne CK, Mani DR, Gillette MA, Zhang B, Echeverria GV, Miles G, Rimawi MF, Carr SA, Ademuyiwa FO, Satpathy S, Ellis MJ. (4) LIG1WT 3OH EDTA complex: nicked 18mer was assembled by annealing oligos 1, 3, and 4 (Supplementary Table1), and the cryoprotectant was supplemented with 2mM EDTA. 2023 Feb 24;18(2):e0282236. PMID: 10946235 DOI: 10.1016/s0921-8777 (00)00033-1 Abstract DNA ligases are critical enzymes of DNA metabolism. Consistent with previous reports, we find LIG1WT is greatly compromised in its ability to complete ligation of the 8oxoG:C substrate, instead aborting ligation and leading to an accumulation of the AMPDNA intermediate (Fig. Symp. Parsons, J. L., Dianova, I. I. 5a). This cavity is reduced relative to that which is present in LIG1E346A/E592A (Fig. The maintenance of genomic integrity requires high fidelity in all DNA transactions, including DNA replication, recombination, and repair. In molecular biology, DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule. The non-catalytic N-terminal region of eukaryotic DNA ligase I is responsible for the specific participation of these enzymes in DNA replication. The column was washed with buffer 1, followed by buffer 2 (buffer 1 with 50mM imidazole). DNA Ligase. Nature Communications DNA ligases are Mg++-dependent enzymes that catalyze the formation of phosphodiester bonds at single-strand breaks in double-stranded DNA (for review and main references up to 1992, see Engler and Richardson 1982; Lindahl and Barnes 1992). c The fraction of abortive ligation events determined from steady-state ligation reactions containing 1 or 10nM LIG1, 2M DNA substrate, 1mM ATP and 2mM MgCl2 (1mM Mg2+free). Congruent with this decrease in abortive ligation, the LIG1E346A/E592A mutation substantially increases the catalytic efficiency for ligation of both 8oxoG-containing substrates (Fig. By submitting a comment you agree to abide by our Terms and Community Guidelines. DNA replication: Mechanisms and therapeutic interventions for diseases. doi: 10.1002/mco2.210. Summary of two-tiered fidelity of DNA ligation by LIG1and APTX. Both residues are stringently conserved in LIG1 homologs, including budding yeast Cdc9p and fission yeast cdc17 (Fig. 2020 Dec 4;432(24):166698. doi: 10.1016/j.jmb.2020.10.032. X-ray crystal structures of the three human DNA ligases (LIG1, LIG3, and LIG4) in complex with DNA have revealed a conserved ligase three-domain core architecture that encircles the DNA nick6,7,8,9, induces partial unwinding and alignment of the 3- and 5-DNA ends, and distorts the 3-OH strand into a C3-endo sugar conformation to adopt an A-form likegeometry7,8,9. W-31109-Eng-38. It folds the DNA into a coiled structure. Although LIG1WT can catalyze ligation of the 8oxoG:A substrate, it does so slowly, with a significant (~70%) accumulation of the adenylylated DNA intermediate. Reactions contained 5nM of 232 LIG1WT or LIG1E346A/E592A and 500nM DNA substrate with a C:G, 8oxoG:C or 8oxoG:A pair at the 1 nt position. 23, 34523461 (2004). Peer reviewer reports are available. It breaks the hydrogen bonds that hold the two strands of the DNA together. There, short pieces of DNA (called Okazaki fragments) are made by DNA polymerase with the help of a short RNA primer and then joined together by another enzyme called DNA ligase. Publishers note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Disabling the high-fidelity mechanism via removal of the MgHiFi site significantly reduces the ability of the enzyme to discriminate against improper nicks, nonspecifically increases ligation efficiency on both undamaged and damaged nicks, and permits the enzyme to evade APTX surveillance (Fig. All data are the meanS.D. It matches new bases to the old strand by complementary base pairing. Despite inclusion of EDTA, we found that the 5-termini are fully adenylylated in the crystals and poised for step 3 (nick sealing; Fig. Environ. 5). 4f and Supplementary Fig. DNA ligase I, the replicative DNA ligase - PubMed Cells were lysed by three rounds of passaging through an EmulsiFlex-C3 (Avestin) with 20kpsi pressure. DNA ligase - Wikipedia In this structure, the R592 substitution occupies the approximate position of E592 plus the HiFi magnesium ligand, and forms a salt bridge to the phosphodiester backbone in place of the metal-DNA contact. ; writingreviewing and editing, R.S.W., P.J.O., T.J.J., P.T., A.R., M.J.S. 4c). Human DNA ligases in replication and repair - PubMed PubMedGoogle Scholar. To define the molecular basis of LIG1 substrate recognition and fidelity, we sought proteinDNA crystallization conditions suitable for high-resolution structural determination. Flipping of 8oxoG into the major groove coincides with three key structural alterations (Figures5BD): disruption of the local hydration structure, concerted rearrangements of active site residues N590 and R589, and movement of the pyrophosphate towards R589. The N2 and N3 atoms of the 8oxoG make additional contacts with the phosphate backbone. Partial dissociation of LIG1 gives the opportunity for APTX to capture the intermediate. FOIA We speculate that discontinuous 3-strand binding contributes to the relaxed fidelity for 3 mismatches observed for human LIG3 compared to LIG117, and the mutagenic DNA repair reactions performed during DSB repair by LIG410,11. Right: stick model superposition. As ablation of the MgHiFi site had significant impacts on substrate interactions, we examined the structural consequences of this mutation (Supplementary Fig. In the first of three chemical steps, an active site lysine (K568 in LIG1) is adenylylated by ATP, releasing pyrophosphate. 4). Step 1: Replication Fork Formation Before DNA can be replicated, the double stranded molecule must be "unzipped" into two single strands. PMID: 22918593 PMCID: PMC3881551 All structures were solved by molecular replacement using PDB entry1X9N as a search model with PHASER37. 18, 11891195 (2011). To protect the integrity of the genome, DNA ligase 1 (LIG1) discriminates against DNA junctions harboring mutagenic 3-DNA mismatches or oxidative DNA damage, but how such high-fidelity ligation is enforced is unknown. These data demonstrate that LIG1E346A/E592A, which disrupts the MgHiFi site, has increased commitment to nick-sealing of an 8oxoG:C substrate relative to LIGWT. Cell Biol. DNA ligases seal 5-PO4 and 3-OH polynucleotide ends via three nucleotidyl transfer steps involving ligase-adenylate and DNA-adenylate intermediates. Mechanism of APTX nicked DNA sensing and pleiotropic inactivation in neurodegenerative disease. 44, 22982309 (2016). What Is DNA Ligase? Definition & Role - Excedr 4b and Supplementary Fig. ; investigation P.T., M.J.S., T.J.J., A.R.R,; writingoriginal draft, R.S.W., P.J.O., T.J.J., P.T. EMBO J. These data reveal that LIG1WT discriminates strongly against 8oxoG:C at both the adenylyl transfer and nick-sealing steps (Fig. The first step in the reaction is the formation of a covalent enzyme/adenylate intermediate. d The high-fidelity metal site of LIG1 is scaffolded by glutamate side chains from the AdD (E592, cyan) and the DBD (E346, blue). c Comparison of WT and mutant LIG1 3-strand binding. Both steady-state and single-turnover reactions for measuring the effect of APTX on ligation were carried out under physiological ATP and Mg2+ concentrations. It is able to join the two DNA fragments as a result of the formation of a phosphodiester bond between them with the help of a molecule of energy. 2022 Oct 19;13:1033338. doi: 10.3389/fimmu.2022.1033338. We report a series of high-resolution structures of wild-type and mutant LIG1 bound to either correctly base-paired or damaged DNA. LIG1E346A/E592A exhibited a two-to- threefold reduction in kcat and approximately twofold reduction in the KM value for ATP relative to WT LIG1 (Supplementary Fig. 1). Pre-steady-state reactions containing 800nM LIG1, 80nM 8oxoG:C, 1mM Mg2+free, and 1mM ATP were performed in the absence d or presence e of APTXH260N. Mechanistic comparison of high-fidelity and error-prone DNA polymerases and ligases involved in DNA repair. It is known that DNA ligases, the key enzymes responsible for completing replication and repair pathways, also contribute to overall fidelity3,4,5. Patrick J OBrien or R. Scott Williams. An In-depth Look at the 7 Major Steps of DNA Replication The position of the active site lysine residue within motif I that forms the covalent bond with AMP is indicated for each DNA ligase. Vijayakumar S, Chapados BR, Schmidt KH, Kolodner RD, Tainer JA, Tomkinson AE. In a second tier of protection, LIG1 activity is surveilled by Aprataxin (APTX), which suppresses mutagenic and abortive ligation at sites of oxidative DNA damage. Webster, A. D., Barnes, D. E., Arlett, C. F., Lehmann, A. R. & Lindahl, T. Growth retardation and immunodeficiency in a patient with mutations in the DNA ligase I gene. b X-ray structure of LIG1E592R (purple) overlaid upon LIGWT (grey). McCoy, A. J. et al. DNA helicase DNA ligase DNA polymerase all of these. Nature 443, 713716 (2006). HHS Vulnerability Disclosure, Help Structure of an aprataxin-DNA complex with insights into AOA1 neurodegenerative disease. Molecular mechanism of DNA replication - Khan Academy If adenylylation of an improper nick occurs, then the MgHiFi site architecture leads to enhanced partitioning away from erroneous ligation and towards abortive ligation. DNA ligases catalyze the joining of DNA strands to complete DNA replication, recombination and repair transactions. DNA Ligases: Progress and Prospects* - Journal of Biological Chemistry Previous studies with an NAD-dependent enzyme Escherichia coli DNA ligase (LigA) provided evidence for discrimination in both steps22. DNA Ligase is widely utilized in laboratories for carrying out recombinant DNA experiments. Cotner-Gohara, E. et al. a Stereo view of final model-phased 2Fo-Fc electron density contoured at 2.0 and displayed for the 8oxoG:A nucleotide pair bound in the E346A/E592A double mutant structure shows the pairing is in a Hoogsteen configuration (A anti:8oxoG syn). This gives an opportunity for other forms of DNA repair, such as exonucleolytic proofreading. Cite this article. Epub 2020 Nov 4. Dynamic DNA-bound PCNA complexes co-ordinate Okazaki fragment synthesis, processing and ligation. 5C, D). DNA replication. To protect the integrity of the genome, DNA ligase 1 (LIG1 . Biol. 37, e98875 (2018). The 232 LIG1 constructs were transformed and expressed in E. coli BL21(DE3) Rosetta 2 cells. The fidelity of the ligation step determines how ends are resolved during nonhomologous end joining. See this image and copyright information in PMC. Reynolds, J. J. et al. Unexpectedly, the kinetics of adenylyl transfer and nick sealing for ligation of the 8oxoG substrates are not significantly altered for LIG1E346A/E592A (Fig. 2, in which V is the initial rate for formation of intermediate or product. Conlin, M. P. et al. Hsu, G. W., Ober, M., Carell, T. & Beese, L. S. Error-prone replication of oxidatively damaged DNA by a high-fidelity DNA polymerase. of 3 replicates. Microbiol. ; supervision R.S.W., P.J.O. Figure 17.1. The DNA ligase is functional in joining the Okazaki fragments that take shape on the lagging strand while the DNA replicates. Together with K746, these lysines are suitably positioned to stabilize the AMP leaving group during nick sealing. Matsumoto Y, Brooks RC, Sverzhinsky A, Pascal JM, Tomkinson AE. This process gives us two identical sets of genes, which will then be passed on to two daughter cells. Abortive ligation on damaged nicks could provide additional time for other DNA repair pathways to operate. Following purification, 232 LIG1WT and LIG1E346A/E592A proteins were dialyzed into storage buffer (25mM Tris-Cl, pH 7.5, 150mM NaCl, 1mM DTT, and 0.1mM EDTA and stored at 80oC). For reasons further elaborated below we refer to this cation as the high-fidelity Mg2+ (MgHiFi). The octahedral coordination of the partially hydrated MgHiFi involves interactions with the phosphodiester backbone between the 3 and 4 nucleotides (relative to the 3-OH of the nick) as well as two glutamate residues from the protein, Glu 346 in the DBD and Glu 592 in the AdD (Fig. APTX suppresses LIG1-catalyzed ligation of 8oxoG-containing DNA. official website and that any information you provide is encrypted A reporting summary for this article is available as a Supplementary Information file. The DNA-binding domain (DBD, blue), Adenylation domain (AdD, green), and OB-fold domain (OBD, yellow) comprise the catalytic core, mapping to residues 262909. 56, 121 (2015). Amino acids, glutamine 566 and arginine 771, are replaced with lysine and tryptophan, respectively in the polypeptides encoded by the mutant. DNA ligase is an enzyme that joins DNA strands through the formation of phosphodiester bonds in a process called DNA ligation.
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