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Stewart Shuman - One of the best experts on this subject based on the ideXlab platform.
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structures of atp bound dna ligase d in a closed domain conformation reveal a network of amino acid and metal contacts to the atp phosphates
Journal of Biological Chemistry, 2019Co-Authors: Mihaelacarmen Unciuleac, Yehuda Goldgur, Stewart ShumanAbstract:DNA Ligases are the sine qua non of genome integrity and essential for DNA replication and repair in all organisms. DNA Ligases join 3'-OH and 5'-PO4 ends via a series of three nucleotidyl transfer steps. In step 1, ligase reacts with ATP or NAD+ to form a covalent ligase-(lysyl-Nζ)-AMP intermediate and release pyrophosphate (PPi) or nicotinamide mononucleotide. In step 2, AMP is transferred from ligase-adenylate to the 5'-PO4 DNA end to form a DNA-adenylate intermediate (AppDNA). In step 3, ligase catalyzes attack by a DNA 3'-OH on the DNA-adenylate to seal the two ends via a phosphodiester bond and release AMP. Eukaryal, archaeal, and many bacterial and viral DNA Ligases are ATP-dependent. The catalytic core of ATP-dependent DNA Ligases consists of an N-terminal nucleotidyltransferase domain fused to a C-terminal OB domain. Here we report crystal structures at 1.4-1.8 A resolution of Mycobacterium tuberculosis LigD, an ATP-dependent DNA ligase dedicated to nonhomologous end joining, in complexes with ATP that highlight large movements of the OB domain (∼50 A), from a closed conformation in the ATP complex to an open conformation in the covalent ligase-AMP intermediate. The LigD·ATP structures revealed a network of amino acid contacts to the ATP phosphates that stabilize the transition state and orient the PPi leaving group. A complex with ATP and magnesium suggested a two-metal mechanism of lysine adenylylation driven by a catalytic Mg2+ that engages the ATP α phosphate and a second metal that bridges the ATP β and γ phosphates.
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crucial role for dna ligase iii in mitochondria but not in xrcc1 dependent repair
Nature, 2011Co-Authors: Stewart Shuman, Deniz Simsek, Amy M Furda, Jerome Artus, Erika Brunet, Annakaterina Hadjantonakis, Bennett Van HoutenAbstract:Mammalian cells contain three different DNA ligase enzymes, each with different properties but all involved in DNA replication and repair. Ligase III (Lig3) is known to form a complex with the nuclear DNA repair protein Xrcc1, and Lig3 null animals cannot be made. This raises the question of whether this nuclear role in base-excision repair (BER) is the critical function of Lig3 that maintains viability. Two groups reporting in this issue of Nature investigate different aspects of Lig3 function in vivo, both concluding that the catalytic activity of Lig3 is critical for mitochondrial DNA maintenance and viability, but unexpectedly, is dispensable for Xrcc1-mediated nuclear BER. These findings suggest that Lig3 mutations might cause some of the human syndromes associated with defects in the replication and/or repair of mitochondrial DNA. Eukaryotic cells have several DNA Ligases. DNA ligase III (Lig3) forms a complex with Xrcc1 that can function in nuclear repair. But, Lig3 null animals cannot be made; is this nuclear role in base excision repair its critical function? This is one of two papers showing that the role of Lig3 in the nucleus is non-essential. Rather, the catalytic activity of Lig3, but not Xrcc1, is essential for the maintenance of mitochondria. Mammalian cells have three ATP-dependent DNA Ligases, which are required for DNA replication and repair1. Homologues of ligase I (Lig1) and ligase IV (Lig4) are ubiquitous in Eukarya, whereas ligase III (Lig3), which has nuclear and mitochondrial forms, appears to be restricted to vertebrates. Lig3 is implicated in various DNA repair pathways with its partner protein Xrcc1 (ref. 1). Deletion of Lig3 results in early embryonic lethality in mice, as well as apparent cellular lethality2, which has precluded definitive characterization of Lig3 function. Here we used pre-emptive complementation to determine the viability requirement for Lig3 in mammalian cells and its requirement in DNA repair. Various forms of Lig3 were introduced stably into mouse embryonic stem (mES) cells containing a conditional allele of Lig3 that could be deleted with Cre recombinase. With this approach, we find that the mitochondrial, but not nuclear, Lig3 is required for cellular viability. Although the catalytic function of Lig3 is required, the zinc finger (ZnF) and BRCA1 carboxy (C)-terminal-related (BRCT) domains of Lig3 are not. Remarkably, the viability requirement for Lig3 can be circumvented by targeting Lig1 to the mitochondria or expressing Chlorella virus DNA ligase, the minimal eukaryal nick-sealing enzyme3, or Escherichia coli LigA, an NAD+-dependent ligase1. Lig3-null cells are not sensitive to several DNA-damaging agents that sensitize Xrcc1-deficient cells4,5,6. Our results establish a role for Lig3 in mitochondria, but distinguish it from its interacting protein Xrcc1.
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DNA Ligases: Progress and Prospects
Journal of Biological Chemistry, 2009Co-Authors: Stewart ShumanAbstract:DNA Ligases seal 5′-PO4 and 3′-OH polynucleotide ends via three nucleotidyl transfer steps involving ligase-adenylate and DNA-adenylate intermediates. DNA Ligases are essential guardians of genomic integrity, and ligase dysfunction underlies human genetic disease syndromes. Crystal structures of DNA Ligases bound to nucleotide and nucleic acid substrates have illuminated how ligase reaction chemistry is catalyzed, how Ligases recognize damaged DNA ends, and how protein domain movements and active-site remodeling are used to choreograph the end-joining pathway. Although a shared feature of DNA Ligases is their envelopment of the nicked duplex as a C-shaped protein clamp, they accomplish this feat by using remarkably different accessory structural modules and domain topologies. As structural, biochemical, and phylogenetic insights coalesce, we can expect advances on several fronts, including (i) pharmacological targeting of Ligases for antibacterial and anticancer therapies and (ii) the discovery and design of new strand-sealing enzymes with unique substrate specificities.
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Structure and Mechanism of RNA Ligase
Structure, 2004Co-Authors: Li Kai Wang, Christopher D. Lima, Stewart ShumanAbstract:T4 RNA ligase 2 (Rnl2) exemplifies an RNA ligase family that includes the RNA editing Ligases (RELs) of Trypanosoma and Leishmania . The Rnl2/REL enzymes are defined by essential signature residues and a unique C-terminal domain, which we show is essential for sealing of 3′-OH and 5′-PO 4 RNA ends by Rnl2, but not for ligase adenylation or phosphodiester bond formation at a preadenylated AppRNA end. The N-terminal segment Rnl2(1-249) of the 334 aa Rnl2 protein comprises an autonomous adenylyltransferase/AppRNA ligase domain. We report the 1.9 A crystal structure of the ligase domain with AMP bound at the active site, which reveals a shared fold, catalytic mechanism, and evolutionary history for RNA Ligases, DNA Ligases, and mRNA capping enzymes.
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Structure-function analysis of T4 RNA ligase 2.
Journal of Biological Chemistry, 2003Co-Authors: Shenmin Yin, Stewart ShumanAbstract:Abstract Bacteriophage T4 RNA ligase 2 (Rnl2) exemplifies a polynucleotide ligase family that includes the trypanosome RNA-editing Ligases and putative RNA Ligases encoded by eukaryotic viruses and archaea. Here we analyzed 12 individual amino acids of Rnl2 that were identified by alanine scanning as essential for strand joining. We determined structure-activity relationships via conservative substitutions and examined mutational effects on the isolated steps of ligase adenylylation and phosphodiester bond formation. The essential residues of Rnl2 are located within conserved motifs that define a superfamily of nucleotidyl transferases that act via enzyme-(lysyl-N)-NMP intermediates. Our mutagenesis results underscore a shared active site architecture in Rnl2-like Ligases, DNA Ligases, and mRNA capping enzymes. They also highlight two essential signature residues, Glu34 and Asn40, that flank the active site lysine nucleophile (Lys35) and are unique to the Rnl2-like ligase family.
Alan E. Tomkinson - One of the best experts on this subject based on the ideXlab platform.
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Altered DNA ligase activity in human disease.
Mutagenesis, 2019Co-Authors: Alan E. Tomkinson, Tasmin Naila, Seema Khattri BhandariAbstract:The joining of interruptions in the phosphodiester backbone of DNA is critical to maintain genome stability. These breaks, which are generated as part of normal DNA transactions, such as DNA replication, V(D)J recombination and meiotic recombination as well as directly by DNA damage or due to DNA damage removal, are ultimately sealed by one of three human DNA Ligases. DNA Ligases I, III and IV each function in the nucleus whereas DNA ligase III is the sole enzyme in mitochondria. While the identification of specific protein partners and the phenotypes caused either by genetic or chemical inactivation have provided insights into the cellular functions of the DNA Ligases and evidence for significant functional overlap in nuclear DNA replication and repair, different results have been obtained with mouse and human cells, indicating species-specific differences in the relative contributions of the DNA Ligases. Inherited mutations in the human LIG1 and LIG4 genes that result in the generation of polypeptides with partial activity have been identified as the causative factors in rare DNA ligase deficiency syndromes that share a common clinical symptom, immunodeficiency. In the case of DNA ligase IV, the immunodeficiency is due to a defect in V(D)J recombination whereas the cause of the immunodeficiency due to DNA ligase I deficiency is not known. Overexpression of each of the DNA Ligases has been observed in cancers. For DNA ligase I, this reflects increased proliferation. Elevated levels of DNA ligase III indicate an increased dependence on an alternative non-homologous end-joining pathway for the repair of DNA double-strand breaks whereas elevated level of DNA ligase IV confer radioresistance due to increased repair of DNA double-strand breaks by the major non-homologous end-joining pathway. Efforts to determine the potential of DNA ligase inhibitors as cancer therapeutics are on-going in preclinical cancer models.
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scr7 is neither a selective nor a potent inhibitor of human dna ligase iv
DNA Repair, 2016Co-Authors: George E Greco, Yoshihiro Matsumoto, Rhys Brooks, Zhengfei Lu, Michael R Lieber, Alan E. TomkinsonAbstract:DNA Ligases are attractive therapeutics because of their involvement in completing the repair of almost all types of DNA damage. A series of DNA ligase inhibitors with differing selectivity for the three human DNA Ligases were identified using a structure-based approach with one of these inhibitors being used to inhibit abnormal DNA ligase IIIα-dependent repair of DNA double-strand breaks (DSB)s in breast cancer, neuroblastoma and leukemia cell lines. Raghavan and colleagues reported the characterization of a derivative of one of the previously identified DNA ligase inhibitors, which they called SCR7 (designated SCR7-R in our experiments using SCR7). SCR7 appeared to show increased selectivity for DNA ligase IV, inhibit the repair of DSBs by the DNA ligase IV-dependent non-homologous end-joining (NHEJ) pathway, reduce tumor growth, and increase the efficacy of DSB-inducing therapeutic modalities in mouse xenografts. In attempting to synthesize SCR7, we encountered problems with the synthesis procedures and discovered discrepancies in its reported structure. We determined the structure of a sample of SCR7 and a related compound, SCR7-G, that is the major product generated by the published synthesis procedure for SCR7. We also found that SCR7-G has the same structure as the compound (SCR7-X) available from a commercial vendor (XcessBio). The various SCR7 preparations had similar activity in DNA ligation assay assays, exhibiting greater activity against DNA Ligases I and III than DNA ligase IV. Furthermore, SCR7-R failed to inhibit DNA ligase IV-dependent V(D)J recombination in a cell-based assay. Based on our results, we conclude that SCR7 and the SCR7 derivatives are neither selective nor potent inhibitors of DNA ligase IV.
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Eukaryotic DNA Ligases: structural and functional insights.
Annual Review of Biochemistry, 2008Co-Authors: Tom Ellenberger, Alan E. TomkinsonAbstract:DNA Ligases are required for DNA replication, repair, and recombination. In eukaryotes, there are three families of ATP-dependent DNA Ligases. Members of the DNA ligase I and IV families are found in all eukaryotes, whereas DNA ligase III family members are restricted to vertebrates. These enzymes share a common catalytic region comprising a DNA-binding domain, a nucleotidyltransferase (NTase) domain, and an oligonucleotide/oligosaccharide binding (OB)-fold domain. The catalytic region encircles nicked DNA with each of the domains contacting the DNA duplex. The unique segments adjacent to the catalytic region of eukaryotic DNA Ligases are involved in specific protein-protein interactions with a growing number of DNA replication and repair proteins. These interactions determine the specific cellular functions of the DNA ligase isozymes. In mammals, defects in DNA ligation have been linked with an increased incidence of cancer and neurodegeneration.
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rational design of human dna ligase inhibitors that target cellular dna replication and repair
Cancer Research, 2008Co-Authors: Xi Chen, Shijun Zhong, Barbara Dziegielewska, Tom Ellenberger, Gerald M Wilson, Alexander D Mackerell, Alan E. TomkinsonAbstract:Based on the crystal structure of human DNA ligase I complexed with nicked DNA, computer-aided drug design was used to identify compounds in a database of 1.5 million commercially available low molecular weight chemicals that were predicted to bind to a DNA-binding pocket within the DNA-binding domain of DNA ligase I, thereby inhibiting DNA joining. Ten of 192 candidates specifically inhibited purified human DNA ligase I. Notably, a subset of these compounds was also active against the other human DNA Ligases. Three compounds that differed in their specificity for the three human DNA Ligases were analyzed further. L82 inhibited DNA ligase I, L67 inhibited DNA Ligases I and III, and L189 inhibited DNA Ligases I, III, and IV in DNA joining assays with purified proteins and in cell extract assays of DNA replication, base excision repair, and nonhomologous end-joining. L67 and L189 are simple competitive inhibitors with respect to nicked DNA, whereas L82 is an uncompetitive inhibitor that stabilized complex formation between DNA ligase I and nicked DNA. In cell culture assays, L82 was cytostatic whereas L67 and L189 were cytotoxic. Concordant with their ability to inhibit DNA repair in vitro, subtoxic concentrations of L67 and L189 significantly increased the cytotoxicity of DNA-damaging agents. Interestingly, the ligase inhibitors specifically sensitized cancer cells to DNA damage. Thus, these novel human DNA ligase inhibitors will not only provide insights into the cellular function of these enzymes but also serve as lead compounds for the development of anticancer agents. [Cancer Res 2008;68(9):3169–77]
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human dna ligase i completely encircles and partially unwinds nicked dna
Nature, 2004Co-Authors: John M Pascal, Alan E. Tomkinson, Patrick J Obrien, Tom EllenbergerAbstract:The end-joining reaction catalysed by DNA Ligases is required by all organisms and serves as the ultimate step of DNA replication, repair and recombination processes. One of three well characterized mammalian DNA Ligases, DNA ligase I, joins Okazaki fragments during DNA replication. Here we report the crystal structure of human DNA ligase I (residues 233 to 919) in complex with a nicked, 5' adenylated DNA intermediate. The structure shows that the enzyme redirects the path of the double helix to expose the nick termini for the strand-joining reaction. It also reveals a unique feature of mammalian Ligases: a DNA-binding domain that allows ligase I to encircle its DNA substrate, stabilizes the DNA in a distorted structure, and positions the catalytic core on the nick. Similarities in the toroidal shape and dimensions of DNA ligase I and the proliferating cell nuclear antigen sliding clamp are suggestive of an extensive protein-protein interface that may coordinate the joining of Okazaki fragments.
Judit Ovadi - One of the best experts on this subject based on the ideXlab platform.
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halotag targeted sirtuin rearranging ligand sirreal for the development of proteolysis targeting chimeras protacs against the lysine deacetylase sirtuin 2 sirt2
ChemBioChem, 2020Co-Authors: Matthias Schiedel, Nathalie Wossner, Attila Lehotzky, Sandor Szunyogh, Soren Hammelmann, Dina Robaa, Wolfgang Sippl, Judit Oláh, Oliver Einsle, Judit OvadiAbstract:We have discovered the sirtuin rearranging ligands (SirReals) as a novel class of highly potent and selective inhibitors of the NAD+-dependent lysine deacetylase sirtuin 2 (Sirt2). In previous studies, conjugation of a SirReal with a ligand for the E3 ubiquitin ligase cereblon to form a so-called proteolysis targeting chimera (PROTAC), enabled small molecule-induced degradation of Sirt2. Here, we report the structure-based development of a chloroalkylated SirReal that induces the degradation of Sirt2 mediated by Halo-tagged E3 ubiquitin Ligases. Using this orthogonal approach for Sirt2 degradation, we show that also other E3 Ligases than cereblon, such as the E3 ubiquitin ligase parkin, can be harnessed for small molecule-induced Sirt2 degradation, thereby emphasizing the great potential of parkin to be utilized as an E3 ligase for new PROTACs approaches. Thus, our study provides new insights into targeted protein degradation in general and Sirt2 degradation in particular.
Matthias Schiedel - One of the best experts on this subject based on the ideXlab platform.
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halotag targeted sirtuin rearranging ligand sirreal for the development of proteolysis targeting chimeras protacs against the lysine deacetylase sirtuin 2 sirt2
ChemBioChem, 2020Co-Authors: Matthias Schiedel, Nathalie Wossner, Attila Lehotzky, Sandor Szunyogh, Soren Hammelmann, Dina Robaa, Wolfgang Sippl, Judit Oláh, Oliver Einsle, Judit OvadiAbstract:We have discovered the sirtuin rearranging ligands (SirReals) as a novel class of highly potent and selective inhibitors of the NAD+-dependent lysine deacetylase sirtuin 2 (Sirt2). In previous studies, conjugation of a SirReal with a ligand for the E3 ubiquitin ligase cereblon to form a so-called proteolysis targeting chimera (PROTAC), enabled small molecule-induced degradation of Sirt2. Here, we report the structure-based development of a chloroalkylated SirReal that induces the degradation of Sirt2 mediated by Halo-tagged E3 ubiquitin Ligases. Using this orthogonal approach for Sirt2 degradation, we show that also other E3 Ligases than cereblon, such as the E3 ubiquitin ligase parkin, can be harnessed for small molecule-induced Sirt2 degradation, thereby emphasizing the great potential of parkin to be utilized as an E3 ligase for new PROTACs approaches. Thus, our study provides new insights into targeted protein degradation in general and Sirt2 degradation in particular.
Tomas Lindahl - One of the best experts on this subject based on the ideXlab platform.
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dna ligase iv from hela cell nuclei
Journal of Biological Chemistry, 1996Co-Authors: Peter Robins, Tomas LindahlAbstract:Abstract A human cDNA encoding a previously unrecognized DNA ligase IV has been identified (Wei, Y.-F., Robins, P., Carter, K., Caldecott, K., Pappin, D. J. C., Yu, G.-L., Wang, R.-P., Shell, B. K., Nash, R. A., Schar, P., Barnes, D. E., Haseltine, W. A., and Lindahl, T. (1995) Mol. Cell. Biol. 15, 3206-3216). Antibodies have been raised against predicted peptide sequences of DNA ligase IV and used to identify the enzyme during purification from HeLa cell nuclei. The 96-kDa DNA ligase IV and the 103-kDa DNA ligase III co-migrate during SDS-polyacrylamide gel electrophoresis and have similar column fractionation properties, which complicates the distinction between the two enzymes, but they have been separated by Mono S liquid chromatography. During initial size fractionation by gel chromatography in 1 M NaCl, DNA ligase IV elutes in the same position as the DNA ligase III-XRCC1 protein complex, indicating that DNA ligase IV is also bound to another protein or occurs as a dimer. DNA ligase IV has been purified free from other DNA Ligases, and its enzymatic properties have been examined. The purified protein effectively joins single-strand breaks in a double-stranded polydeoxynucleotide in an ATP-dependent reaction. The substrate specificity of DNA ligase IV differs from those of the other two cloned human DNA Ligases, I and III, with regard to their ability to join the hybrid substrates oligo(dT)·poly(rA) and oligo(rA)·poly(dT). DNA ligase IV occurs in part as an enzyme-adenylate complex in HeLa cell nuclear extracts.
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19 DNA Ligases
Cold Spring Harbor Monograph Archive, 1996Co-Authors: R A Nash, Tomas LindahlAbstract: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). The first step in the reaction is the formation of a covalent enzyme/adenylate intermediate. DNA Ligases from eukaryotes, archaea, and viruses employ ATP as cofactor, whereas eubacterial DNA Ligases use NAD to generate the adenylyl group. The ATP is cleaved to AMP and pyrophosphate with the adenylyl residue linked by a phosphoramidate bond to the ɛ-amino group of a specific lysine residue at the active site of the protein. The reaction is readily reversed in vitro by addition of pyrophosphate. Since DNA Ligases contain an unusually reactive lysine residue in their active site, a Schiff base can be formed with pyridoxal phosphate. In consequence, the activities of DNA Ligases, e.g., mammalian DNA Ligases I and II, are inhibited in vitro by pyridoxal phosphate. ATP-dependent DNA Ligases can employ certain cofactor analogs such as dATP, but the anomalous enzyme/nucleotide complexes formed by some Ligases appear to function poorly in subsequent steps of the DNA-joining reaction. The activated AMP residue of the DNA ligase/adenylate intermediate is transferred to the 5′-phosphate terminus of a single-strand break in double-stranded DNA to generate a covalent DNA-AMP complex with a 5′ –5′ phosphoanhydride bond. In the final step of DNA ligation, unadenylylated DNA ligase is required for the generation of a phosphodiester bond and catalyzes displacement of the AMP residue through...
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Mammalian DNA Ligases.
Annual Review of Biochemistry, 1992Co-Authors: Tomas Lindahl, Deborah E. BarnesAbstract:DNA LIGASE I .... ... ....... ...... . . . . . .. . . . .. ........ . 255 Structure. . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 255 Gene Structure and Chromosome Mapping . . . . ......... . . . . . .. . . . . . . . . . . . . 258 Catalytic Properties 259 DNA Ligase I is a Phosphoprotein 259 Subcellular Localization and Functional Properties 261 Activators and Inhibitors 262 DNA LIGASE II . . . . . . . .. . . . . . . ..... . . . . . . . . . . ... . . . . . . . . . .... . . . . . . . . ...... . . . . . . . . . . ..... 263 DNA LIGASE III. ... ...... . . . . . ......... . . . . . ...... . . . . . . . . . . .. ...... . . . . ......... . . . . . . .. . 265 DNA Ligases IN OTHER EUKARYOTIC SySTEMS... . . . . . .. . . .. . . . . . . . ....... . . . . . . . . . 267 Schizosaccharomyces pombe and Saccharomyces cerevisiae DNA Ligases . . . . ....... 268 Drosophila melanogaster DNA Ligases. ...... . . . . . . . . . . . . . ........ .. . . . . . .... . . 269 Xenopus laevis DNA Ligase 271 Vaccinia Virus DNA Ligase 272 HUMAN CELL LINES DEFICIEN T IN DN A LIGATION . .. .. . . . . . . . . ..... . . . .. . . . ..... . . . 273 The DNA Ligase I-Defective Cell Line 46BR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 274 Bloom's Syndrome ..... 276 Acute Lymphoblastic Leukemia 277 Concluding Statement 277
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Three distinct DNA Ligases in mammalian cells.
Journal of Biological Chemistry, 1991Co-Authors: A E Tomkinson, Edward Roberts, N F Totty, Graham Daly, Tomas LindahlAbstract:Abstract The major DNA ligase of proliferating mammalian cells, DNA ligase I, catalyzes the joining of single strand breaks in double stranded DNA and is active on a synthetic substrate of oligo(dT) hybridized to poly(dA). DNA ligase I does not catalyze the joining of an oligo(dT).poly(rA) substrate. Two additional DNA Ligases, II and III, which can act on the latter substrate have been purified from calf thymus. DNA ligase II, which has been described previously, is a 72-kDa protein. DNA ligase III migrates as a 100-kDa protein in denaturing gel electrophoresis. Structural, immunochemical, and catalytic studies on the three DNA ligase activities strongly indicate that they are the products of three different genes.
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Location of the active site for enzyme-adenylate formation in DNA Ligases.
Proceedings of the National Academy of Sciences of the United States of America, 1991Co-Authors: A E Tomkinson, N F Totty, M Ginsburg, Tomas LindahlAbstract:Abstract The enzyme-AMP reaction intermediate of the 102-kDa bovine DNA ligase I was digested with trypsin, and the adenylylated peptide was isolated by chromatography under conditions that maintain the acid-labile phosphoramidate bond. Microsequencing of the peptide showed that it contains an internal trypsin-resistant lysine residue, as expected for the site of adenylylation. Inhibition of DNA ligase I activity by pyridoxal 5'-phosphate also indicated the presence of a reactive lysine residue in the catalytic domain of the enzyme. Comparison of the known primary structures of several other DNA Ligases with the adenylylated region of mammalian DNA ligase I allows their active sites to be tentatively assigned by sequence homology. The ATP-dependent DNA Ligases of mammalian cells, fission yeast, budding yeast, vaccinia virus, and bacteriophages T3, T4, and T7 contain the active site motif Lys-Tyr/Ala-Asp-Gly-(Xaa)-Arg, with the reactive lysine residue flanked by hydrophobic amino acids. The distance between the postulated adenylylation site and the carboxyl terminus of the polypeptide is very similar in these ATP-dependent DNA Ligases, whereas the size of the amino-terminal region is highly variable.