Structure-function analysis of the OB and latch domains of chlorella virus DNA ligase

Chlorella virus DNA ligase (ChVLig) is a minimized eukaryal ATP-dependent DNA sealing enzyme with an intrinsic nick-sensing function. ChVLig consists of three structural domains, nucleotidyltransferase (NTase), OB-fold, and latch, that envelop the nicked DNA as a C-shaped protein clamp. The OB domain engages the DNA minor groove on the face of the duplex behind the nick, and it makes contacts to amino acids in the NTase domain surrounding the ligase active site. The latch module occupies the DNA major groove flanking the nick. Residues at the tip of the latch contact the NTase domain to close the ligase clamp. Here we performed a structure-guided mutational analysis of the OB and latch domains. Alanine scanning defined seven individual amino acids as essential in vivo (Lys-274, Arg-285, Phe-286, and Val-288 in the OB domain; Asn-214, Phe-215, and Tyr-217 in the latch), after which structure-activity relations were clarified by conservative substitutions. Biochemical tests of the composite nick sealing reaction and of each of the three chemical steps of the ligation pathway highlighted the importance of Arg-285 and Phe-286 in the catalysis of the DNA adenylylation and phosphodiester synthesis reactions. Phe-286 interacts with the nick 5'-phosphate nucleotide and the 3'-OH base pair and distorts the DNA helical conformation at the nick. Arg-285 is a key component of the OB-NTase interface, where it forms a salt bridge to the essential Asp-29 side chain, which is imputed to coordinate divalent metal catalysts during the nick sealing steps.     

Kinetic Analysis of DNA Strand Joining by Chlorella Virus DNA Ligase and the Role of Nucleotidyltransferase Motif VI in Ligase Adenylylation

Chlorella virus DNA ligase (ChVLig) is an instructive model for mechanistic studies of the ATP-dependent DNA ligase family. ChVLig seals 3'-OH and 5'-PO(4) termini via three chemical steps: 1) ligase attacks the ATP α phosphorus to release PP(i) and form a covalent ligase-adenylate intermediate; 2) AMP is transferred to the nick 5'-phosphate to form DNA-adenylate; 3) the 3'-OH of the nick attacks DNA-adenylate to join the polynucleotides and release AMP. Each chemical step requires Mg(2+). Kinetic analysis of nick sealing by ChVLig-AMP revealed that the rate constant for phosphodiester synthesis (k(step3) = 25 s(-1)) exceeds that for DNA adenylylation (k(step2) = 2.4 s(-1)) and that Mg(2+) binds with similar affinity during step 2 (K(d) = 0.77 mm) and step 3 (K(d) = 0.87 mm). The rates of DNA adenylylation and phosphodiester synthesis respond differently to pH, such that step 3 becomes rate-limiting at pH ≤ 6.5. The pH profiles suggest involvement of one and two protonation-sensitive functional groups in catalysis of steps 2 and 3, respectively. We suggest that the 5'-phosphate of the nick is the relevant protonation-sensitive moiety and that a dianionic 5'-phosphate is necessary for productive step 2 catalysis. Motif VI, located at the C terminus of the OB-fold domain of ChVLig, is a conserved feature of ATP-dependent DNA ligases and GTP-dependent mRNA capping enzymes. Presteady state and burst kinetic analysis of the effects of deletion and missense mutations highlight the catalytic contributions of ChVLig motif VI, especially the Asp-297 carboxylate, exclusively during the ligase adenylylation step.     

How an RNA ligase discriminates RNA versus DNA damage

T4 RNA ligase 2 (Rnl2) exemplifies a family of RNA-joining enzymes that includes protozoan RNA-editing ligases. Rnl2 efficiently seals 3'-OH/5'-PO4 RNA nicks in either a duplex RNA or an RNA:DNA hybrid but cannot seal DNA nicks. RNA specificity arises from a requirement for at least two ribonucleotides immediately flanking the 3'-OH of the nick; the rest of the nicked duplex can be replaced by DNA. The terminal 2'-OH at the nick is important for the attack of the 3'-OH on the 5'-adenylated strand to form a phosphodiester, but dispensable for nick recognition and adenylylation of the 5'-PO4 strand. The penultimate 2'-OH is important for nick recognition. Stable binding of Rnl2 at a nick depends on contacts to both the N-terminal adenylyltransferase domain and its signature C-terminal domain. Nick sensing also requires adenylylation of Rnl2. These results provide insights to the evolution of nucleic acid repair systems.     

Structural basis for nick recognition by a minimal pluripotent DNA ligase

Chlorella virus DNA ligase, the smallest eukaryotic ligase known, has pluripotent biological activity and an intrinsic nick-sensing function, despite having none of the accessory domains found in cellular ligases. A 2.3-A crystal structure of the Chlorella virus ligase-AMP intermediate bound to duplex DNA containing a 3'-OH-5'-PO4 nick reveals a new mode of DNA envelopment, in which a short surface loop emanating from the OB domain forms a beta-hairpin 'latch' that inserts into the DNA major groove flanking the nick. A network of interactions with the 3'-OH and 5'-PO4 termini in the active site illuminates the DNA adenylylation mechanism and the crucial roles of AMP in nick sensing and catalysis. Addition of a divalent cation triggered nick sealing in crystallo, establishing that the nick complex is a bona fide intermediate in the DNA repair pathway.     

Deinococcus radiodurans RNA ligase exemplifies a novel ligase clade with a distinctive N-terminal module that is important for 5'-PO4 nick sealing and ligase adenylylation but dispensable for phosphodiester formation at an adenylylated nick

Deinococcus radiodurans RNA ligase (DraRnl) is a template-directed ligase that seals nicked duplexes in which the 3′-OH strand is RNA. DraRnl is a 342 amino acid polypeptide composed of a C-terminal adenylyltransferase domain fused to a distinctive 126 amino acid N-terminal module (a putative OB-fold). An alanine scan of the C domain identified 9 amino acids essential for nick ligation, which are located within nucleotidyltransferase motifs I, Ia, III, IIIa, IV and V. Seven mutants were dysfunctional by virtue of defects in ligase adenylylation: T163A, H167A, G168A, K186A, E230A, F281A and E305A. Four of these were also defective in phosphodiester formation at a preadenylylated nick: G168A, E230A, F281A and E305A. Two nick sealing-defective mutants were active in ligase adenylylation and sealing a preadenylylated nick, thereby implicating Ser185 and Lys326 in transfer of AMP from the enzyme to the nick 5′-PO₄. Whereas deletion of the N-terminal domain suppressed overall nick ligation and ligase adenylylation, it did not compromise sealing at a preadenylylated nick. Mutational analysis of 15 residues of the N domain identified Lys26, Gln31 and Arg79 as key constituents. Structure–activity relationships at the essential residues were determined via conservative substitutions. We propose that DraRnl typifies a new clade of polynucleotide ligases. DraRnl homologs are detected in several eukaryal proteomes.     

Sequence-specific 1HN, 13C, and 15N backbone resonance assignments of the 34 kDa Paramecium bursaria Chlorella virus 1 (PBCV1) DNA ligase

Chlorella virus DNA ligase (ChVLig) is a minimal (298-amino acid) pluripotent ATP-dependent ligase composed of three structural modules—a nucleotidyltransferase domain, an OB domain, and a β-hairpin latch—that forms a circumferential clamp around nicked DNA. ChVLig provides an instructive model to understand the chemical and conformational steps of nick repair. Here we report the assignment of backbone ¹³C, ¹⁵N, ¹H^N resonances of this 34.2 kDa protein, the first for a DNA ligase in full-length form.     

Mutational analysis of Chlorella virus DNA ligase: catalytic roles of domain I and motif VI.

A conserved catalytic core of the ATP-dependent DNA ligases is composed of an N-terminal domain (domain 1, containing nucleotidyl transferase motifs I, III, IIIa and IV) and a C-terminal domain (domain 2, containing motif VI) with an intervening cleft. Motif V links the two structural domains. Deletion analysis of the 298 amino acid Chlorella virus DNA ligase indicates that motif VI plays a critical role in the reaction of ligase with ATP to form ligase-adenylate, but is dispensable for the two subsequent steps in the ligation pathway; DNA-adenylate formation and strand closure. We find that formation of a phosphodiester at a pre-adenylated nick is subject to a rate limiting step that does not apply during the sealing of nicked DNA by ligase-adenylate. This step, presumably conformational, is accelerated or circumvented by deleting five amino acids of motif VI. The motif I lysine nucleophile (Lys27) is not required for strand closure by wild-type ligase, but this residue enhances the closure rate by a factor of 16 when motif VI is truncated. We find that a more extensively truncated ligase consisting of only N-terminal domain 1 and motif V is inert in ligase--adenylate formation, but competent to catalyze strand closure at a pre-adenylated nick. These results suggest that different enzymic catalysts facilitate the three steps of the DNA ligase reaction.     

NAD+-dependent DNA ligase encoded by a eukaryotic virus.

We report the production, purification, and characterization of an NAD(+)-dependent DNA ligase encoded by the Amsacta moorei entomopoxvirus (AmEPV), the first example of an NAD(+) ligase from a source other than eubacteria. AmEPV ligase lacks the zinc-binding tetracysteine domain and the BRCT domain that are present in all eubacterial NAD(+) ligases. Nonetheless, the monomeric 532-amino acid AmEPV ligase catalyzed strand joining on a singly nicked DNA in the presence of a divalent cation and NAD(+). Neither ATP, dATP, nor any other nucleoside triphosphate could substitute for NAD(+). Structure probing by limited proteolysis showed that AmEPV ligase is punctuated by a surface-accessible loop between the nucleotidyltransferase domain, which is common to all ligases, and the N-terminal domain Ia, which is unique to the NAD(+) ligases. Deletion of domain Ia of AmEPV ligase abolished the sealing of 3'-OH/5'-PO(4) nicks and the reaction with NAD(+) to form ligase-adenylate, but had no effect on phosphodiester formation at a pre-adenylated nick. Alanine substitutions at residues within domain Ia either reduced (Tyr(39), Tyr(40), Asp(48), and Asp(52)) or abolished (Tyr(51)) sealing of a 5'-PO(4) nick and adenylyl transfer from NAD(+) without affecting ligation of DNA-adenylate. We conclude that: (i) NAD(+)-dependent ligases exist in the eukaryotic domain of the phylogenetic tree; and (ii) ligase structural domain Ia is a determinant of cofactor specificity and is likely to interact directly with the nicotinamide mononucleotide moiety of NAD(+).     

Dual mechanisms whereby a broken RNA end assists the catalysis of its repair by T4 RNA ligase 2

T4 RNA ligase 2 (Rnl2) efficiently seals 3'-OH/5'-PO4 RNA nicks via three nucleotidyl transfer steps. Here we show that the terminal 3'-OH at the nick accelerates the second step of the ligase pathway (adenylylation of the 5'-PO4 strand) by a factor of 1000, even though the 3'-OH is not chemically transformed during the reaction. Also, the terminal 2'-OH at the nick accelerates the third step (attack of the 3'-OH on the 5'-adenylated strand to form a phosphodiester) by a factor of 25-35, even though the 2'-OH is not chemically reactive. His-37 of Rnl2 is uniquely required for step 3, providing a approximately 10(2) rate acceleration. Biochemical epistasis experiments show that His-37 and the RNA 2'-OH act independently. We conclude that the broken RNA end promotes catalysis of its own repair by Rnl2 via two mechanisms, one of which (enhancement of step 3 by the 2'-OH) is specific to RNA ligation. Substrate-assisted catalysis provides a potential biochemical checkpoint during nucleic acid repair.     

Kinetic mechanism of human DNA ligase I reveals magnesium-dependent changes in the rate-limiting step that compromise ligation efficiency

DNA ligase I (LIG1) catalyzes the ligation of single-strand breaks to complete DNA replication and repair. The energy of ATP is used to form a new phosphodiester bond in DNA via a reaction mechanism that involves three distinct chemical steps: enzyme adenylylation, adenylyl transfer to DNA, and nick sealing. We used steady state and pre-steady state kinetics to characterize the minimal mechanism for DNA ligation catalyzed by human LIG1. The ATP dependence of the reaction indicates that LIG1 requires multiple Mg(2+) ions for catalysis and that an essential Mg(2+) ion binds more tightly to ATP than to the enzyme. Further dissection of the magnesium ion dependence of individual reaction steps revealed that the affinity for Mg(2+) changes along the reaction coordinate. At saturating concentrations of ATP and Mg(2+) ions, the three chemical steps occur at similar rates, and the efficiency of ligation is high. However, under conditions of limiting Mg(2+), the nick-sealing step becomes rate-limiting, and the adenylylated DNA intermediate is prematurely released into solution. Subsequent adenylylation of enzyme prevents rebinding to the adenylylated DNA intermediate comprising an Achilles' heel of LIG1. These ligase-generated 5'-adenylylated nicks constitute persistent breaks that are a threat to genomic stability if they are not repaired. The kinetic and thermodynamic framework that we have determined for LIG1 provides a starting point for understanding the mechanism and specificity of mammalian DNA ligases.     

Footprinting of Chlorella virus DNA ligase bound at a nick in duplex DNA.

The 298-amino acid ATP-dependent DNA ligase of Chlorella virus PBCV-1 is the smallest eukaryotic DNA ligase known. The enzyme has intrinsic specificity for binding to nicked duplex DNA. To delineate the ligase-DNA interface, we have footprinted the enzyme binding site on DNA and the DNA binding site on ligase. The size of the exonuclease III footprint of ligase bound a single nick in duplex DNA is 19-21 nucleotides. The footprint is asymmetric, extending 8-9 nucleotides on the 3'-OH side of the nick and 11-12 nucleotides on the 5'-phosphate side. The 5'-phosphate moiety is essential for the binding of Chlorella virus ligase to nicked DNA. Here we show that the 3'-OH moiety is not required for nick recognition. The Chlorella virus ligase binds to a nicked ligand containing 2',3'-dideoxy and 5'-phosphate termini, but cannot catalyze adenylation of the 5'-end. Hence, the 3'-OH is important for step 2 chemistry even though it is not itself chemically transformed during DNA-adenylate formation. A 2'-OH cannot substitute for the essential 3'-OH in adenylation at a nick or even in strand closure at a preadenylated nick. The protein side of the ligase-DNA interface was probed by limited proteolysis of ligase with trypsin and chymotrypsin in the presence and absence of nicked DNA. Protease accessible sites are clustered within a short segment from amino acids 210-225 located distal to conserved motif V. The ligase is protected from proteolysis by nicked DNA. Protease cleavage of the native enzyme prior to DNA addition results in loss of DNA binding. These results suggest a bipartite domain structure in which the interdomain segment either comprises part of the DNA binding site or undergoes a conformational change upon DNA binding. The domain structure of Chlorella virus ligase inferred from the solution experiments is consistent with the structure of T7 DNA ligase determined by x-ray crystallography.     

Chlorella virus DNA ligase: nick recognition and mutational analysis.

Chlorella virus PBCV-1 DNA ligase seals nicked DNA substrates consisting of a 5'-phosphate-terminated strand and a 3'-hydroxyl-terminated strand annealed to a bridging DNA template strand. The enzyme discriminates at the DNA binding step between substrates containing a 5'-phosphate versus a 5'-hydroxyl at the nick. Mutational analysis of the active site motif KxDGxR (residues 27-32) illuminates essential roles for the conserved Lys, Asp and Arg moieties at different steps of the ligase reaction. Mutant K27A is unable to form the covalent ligase-(Lys-straightepsilonN-P)-adenylate intermediate and hence cannot activate a nicked DNA substrate via formation of the DNA-adenylate intermediate. Nonetheless, K27A catalyzes phosphodiester bond formation at a pre-adenylated nick. This shows that the active site lysine is not required for the strand closure reaction. K27A binds to nicked DNA-adenylate, but not to a standard DNA nick. This suggests that occupancy of the AMP binding pocket of DNA ligase is important for nick recognition. Mutant D29A is active in enzyme-adenylate formation and binds readily to nicked DNA, but is inert in DNA-adenylate formation. R32A is unable to catalyze any of the three reactions of the ligation pathway and does not bind to nicked DNA.     

Mutational analysis of bacteriophage T4 RNA ligase 1. Different functional groups are required for the nucleotidyl transfer and phosphodiester bond formation steps of the ligation reaction

T4 RNA ligase 1 (Rnl1) exemplifies an ATP-dependent RNA ligase family that includes fungal tRNA ligase (Trl1) and a putative baculovirus RNA ligase. Rnl1 acts via a covalent enzyme-AMP intermediate generated by attack of Lys-99 N zeta on the alpha phosphorus of ATP. Mutation of Lys-99 abolishes ligase activity. Here we tested the effects of alanine mutations at 19 conserved positions in Rnl1 and thereby identified 9 new residues essential for ligase activity: Arg-54, Lys-75, Phe-77, Gly-102, Lys-119, Glu-227, Gly-228, Lys-240, and Lys-242. Seven of the essential residues are located within counterparts of conserved nucleotidyltransferase motifs I (99KEDG102), Ia (118SK119), IV (227EGYVA231), and V (238HFKIK242) that comprise the active sites of DNA ligases, RNA capping enzymes, and T4 RNA ligase 2. Three other essential residues, Arg-54, Lys-75 and Phe-77, are located upstream of the AMP attachment site within a conserved domain unique to the Rnl1-like ligase family. We infer a shared evolutionary history and active site architecture in Rnl1 (a tRNA repair enzyme) and Trl1 (a tRNA splicing enzyme). We determined structure-activity relationships via conservative substitutions and examined mutational effects on the isolated steps of Rnl1 adenylylation (step 1) and phosphodiester bond formation (step 3). Lys-75, Lys-240, and Lys-242 were found to be essential for step 1 and overall ligation of 5'-phosphorylated RNA but not for phosphodiester bond formation. These results suggest that the composition of the Rnl1 active site is different during steps 1 and 3. Mutations at Arg-54 and Lys-119 abolished the overall RNA ligation reaction without affecting steps 1 and 3. Arg-54 and Lys-119 are thereby implicated as specific catalysts of the RNA adenylation reaction (step 2) of the ligation pathway.     

Nick sensing by vaccinia virus DNA ligase requires a 5' phosphate at the nick and occupancy of the adenylate binding site on the enzyme.

Vaccinia virus DNA ligase has an intrinsic nick-sensing function. The enzyme discriminates at the substrate binding step between a DNA containing a 5' phosphate and a DNA containing a 5' hydroxyl at the nick. Further insights into nick recognition and catalysis emerge from studies of the active-site mutant K231A, which is unable to form the covalent ligase-adenylate intermediate and hence cannot activate a nicked DNA substrate via formation of the DNA-adenylate intermediate. Nonetheless, K231A does catalyze phosphodiester bond formation at a preadenylated nick. Hence, the active-site lysine of DNA ligase is not required for the strand closure step of the ligation reaction. The K231A mutant binds tightly to nicked DNA-adenylate but has low affinity for a standard DNA nick. The wild-type vaccinia virus ligase, which is predominantly ligase-adenylate, binds tightly to a DNA nick. This result suggests that occupancy of the AMP binding pocket of DNA ligase is essential for stable binding to DNA. Sequestration of an extrahelical nucleotide by DNA-bound ligase is reminiscent of the base-flipping mechanism of target-site recognition and catalysis used by other DNA modification and repair enzymes.     

Structure-guided mutational analysis of the OB, HhH, and BRCT domains of Escherichia coli DNA ligase

NAD(+)-dependent DNA ligases (LigAs) are ubiquitous in bacteria and essential for growth. LigA enzymes have a modular structure in which a central catalytic core composed of nucleotidyltransferase and oligonucleotide-binding (OB) domains is linked via a tetracysteine zinc finger to distal helix-hairpin-helix (HhH) and BRCT (BRCA1-like C-terminal) domains. The OB and HhH domains contribute prominently to the protein clamp formed by LigA around nicked duplex DNA. Here we conducted a structure-function analysis of the OB and HhH domains of Escherichia coli LigA by alanine scanning and conservative substitutions, entailing 43 mutations at 22 amino acids. We thereby identified essential functional groups in the OB domain that engage the DNA phosphodiester backbone flanking the nick (Arg(333)); penetrate the minor grove and distort the nick (Val(383) and Ile(384)); or stabilize the OB fold (Arg(379)). The essential constituents of the HhH domain include: four glycines (Gly(455), Gly(489), Gly(521), Gly(553)), which bind the phosphate backbone across the minor groove at the outer margins of the LigA-DNA interface; Arg(487), which penetrates the minor groove at the outer margin on the 3 (R)-OH side of the nick; and Arg(446), which promotes protein clamp formation via contacts to the nucleotidyltransferase domain. We find that the BRCT domain is required in its entirety for effective nick sealing and AMP-dependent supercoil relaxation.     

Specificity and fidelity of strand joining by Chlorella virus DNA ligase.

Chlorella virus PBCV-1 DNA ligase seals nicked duplex DNA substrates consisting of a 5'-phosphate-terminated strand and a 3'-hydroxyl-terminated strand annealed to a bridging template strand, but cannot ligate a nicked duplex composed of two DNAs annealed on an RNA template. Whereas PBCV-1 ligase efficiently joins a 3'-OH RNA to a 5'-phosphate DNA, it is unable to join a 3'-OH DNA to a 5'-phosphate RNA. The ligase discriminates at the substrate binding step between nicked duplexes containing 5'-phosphate DNA versus 5'-phosphate RNA strands. PBCV-1 ligase readily seals a nicked duplex DNA containing a single ribonucleotide substitution at the reactive 5'-phosphate end. These results suggest a requirement for a B-form helical conformation of the polynucleotide on the 5'-phosphate side of the nick. Single base mismatches at the nick exert disparate effects on DNA ligation efficiency. PBCV-1 ligase tolerates mismatches involving the 5'-phosphate nucleotide, with the exception of 5'-A:G and 5'-G:A mispairs, which reduce ligase activity by two orders of magnitude. Inhibitory configurations at the 3'-OH nucleotide include 3'-G:A, 3'-G:T, 3'-T:T, 3'-A:G, 3'-G:G, 3'-A:C and 3'-C:C. Our findings indicate that Chlorella virus DNA ligase has the potential to affect genome integrity by embedding ribonucleotides in viral DNA and by sealing nicked molecules with mispaired ends, thereby generating missense mutations.     

Kinetic characterization of single strand break ligation in duplex DNA by T4 DNA ligase

T4 DNA ligase catalyzes phosphodiester bond formation between juxtaposed 5'-phosphate and 3'-hydroxyl termini in duplex DNA in three steps: 1) enzyme-adenylylate formation by reaction with ATP; 2) adenylyl transfer to a 5'-phosphorylated polynucleotide to generate adenylylated DNA; and 3) phosphodiester bond formation with release of AMP. This investigation used synthetic, nicked DNA substrates possessing either a 5'-phosphate or a 5'-adenylyl phosphate. Steady state experiments with a nicked substrate containing juxtaposed dC and 5'-phosphorylated dT deoxynucleotides (substrate 1) yielded kcat and kcat/Km values of 0.4±0.1 s(-1) and 150±50 μm(-1) s(-1), respectively. Under identical reaction conditions, turnover of an adenylylated version of this substrate (substrate 1A) yielded kcat and kcat/Km values of 0.64±0.08 s(-1) and 240±40 μm(-1) s(-1). Single turnover experiments utilizing substrate 1 gave fits for the forward rates of Step 2 (k2) and Step 3 (k3) of 5.3 and 38 s(-1), respectively, with the slowest step ～10-fold faster than the rate of turnover seen under steady state conditions. Single turnover experiments with substrate 1A produced a Step 3 forward rate constant of 4.3 s(-1), also faster than the turnover rate of 1A. Enzyme self-adenylylation was confirmed to also occur on a fast time scale (～6 s(-1)), indicating that the rate-limiting step for T4 DNA ligase nick sealing is not a chemical step but rather is most likely product release. Pre-steady state reactions displayed a clear burst phase, consistent with this conclusion.     

RNA 3'-phosphate cyclase (RtcA) catalyzes ligase-like adenylylation of DNA and RNA 5'-monophosphate ends

RNA 3'-phosphate cyclase (Rtc) enzymes are a widely distributed family that catalyze the synthesis of RNA 2',3'-cyclic phosphate ends via an ATP-dependent pathway comprising three nucleotidyl transfer steps: reaction of Rtc with ATP to form a covalent Rtc-(histidinyl-N)-AMP intermediate and release PP(i); transfer of AMP from Rtc to an RNA 3'-phosphate to form an RNA(3')pp(5')A intermediate; and attack by the terminal nucleoside O2' on the 3'-phosphate to form an RNA 2',3'-cyclic phosphate product and release AMP. The chemical transformations of the cyclase pathway resemble those of RNA and DNA ligases, with the key distinction being that ligases covalently adenylylate 5'-phosphate ends en route to phosphodiester synthesis. Here we show that the catalytic repertoire of RNA cyclase overlaps that of ligases. We report that Escherichia coli RtcA catalyzes adenylylation of 5'-phosphate ends of DNA or RNA strands to form AppDNA and AppRNA products. The polynucleotide 5' modification reaction requires the His(309) nucleophile, signifying that it proceeds through a covalent RtcA-AMP intermediate. We established this point directly by demonstrating transfer of [(32)P]AMP from RtcA to a pDNA strand. RtcA readily adenylylated the 5'-phosphate at a 5'-PO(4)/3'-OH nick in duplex DNA but was unable to covert the nicked DNA-adenylate to a sealed phosphodiester. Our findings raise the prospect that cyclization of RNA 3'-ends might not be the only biochemical pathway in which Rtc enzymes participate; we discuss scenarios in which the 5'-adenylyltransferase of RtcA might play a role.     

Conserved residues in domain Ia are required for the reaction of Escherichia coli DNA ligase with NAD+.

NAD(+)-dependent DNA ligases are present in all bacteria and are essential for growth. Their unique substrate specificity compared with ATP-dependent human DNA ligases recommends the NAD(+) ligases as targets for the development of new broad-spectrum antibiotics. A plausible strategy for drug discovery is to identify the structural components of bacterial DNA ligase that interact with NAD(+) and then to isolate small molecules that recognize these components and thereby block the binding of NAD(+) to the ligase. The limitation to this strategy is that the structural determinants of NAD(+) specificity are not known. Here we show that reactivity of Escherichia coli DNA ligase (LigA) with NAD(+) requires N-terminal domain Ia, which is unique to, and conserved among, NAD(+) ligases but absent from ATP-dependent ligases. Deletion of domain Ia abolished the sealing of 3'-OH/5'-PO(4) nicks and the reaction with NAD(+) to form ligase-adenylate but had no effect on phosphodiester formation at a preadenylated nick. Alanine substitutions at conserved residues within domain Ia either reduced (His-23, Tyr-35) or abolished (Tyr-22, Asp-32, Asp-36) sealing of a 5'-PO(4) nick and adenylyl transfer from NAD(+) without affecting ligation of pre-formed DNA-adenylate. We suggest that these five side chains comprise a binding site for the nicotinamide mononucleotide moiety of NAD(+). Structure-activity relationships were clarified by conservative substitutions.     

Nick recognition by DNA ligases

Phage T7 DNA ligase seals nicked DNA substrates and is a representative member of the ATP-dependent class of DNA ligases. Although the catalytic mechanism of DNA ligases has been delineated, little is known about the nature of nick recognition by these enzymes. Here, we show that T7 ligase discriminates, at the nick-binding step, between nicks containing either a 5'-phosphate or a 5'-OH. T7 ligase binds preferentially to phosphorylated nicks and catalyses the sealing reaction. We also show using DNA footprinting studies, that T7 ligase binds asymmetrically to nicks as a monomer, with the protein interface covering between 12 and 14 bp of DNA. Based on molecular modelling studies we propose a structural model of the ligase-DNA complex consistent with these and other data. Using photo-crosslinking and site-directed mutagenesis we have identified two residues, K238 and K240, critical for the transadenylation and nick-sealing reactions. Sequence conservation and structural analysis supports the premise that these two lysine residues are critical for both nucleotide binding and DNA nick recognition. The implications of these results on the ligation mechanism are discussed.