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1.
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.  相似文献   

2.
We have previously identified a DNA ligase (LigTk) from a hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1. The enzyme is the only characterized ATP-dependent DNA ligase from a hyperthermophile, and allows the analysis of enzymatic DNA ligation reactions at temperatures above the melting point of the substrates. Here we have focused on the interactions of LigTk with various DNA substrates, and its specificities toward metal cations. LigTk could utilize Mg2+, Mn2+, Sr2+ and Ca2+ as a metal cation, but not Co2+, Zn2+, Ni2+, or Cu2+. The enzyme displayed typical Michaelis-Menten steady-state kinetics with an apparent Km of 1.4 microm for nicked DNA. The kcat value of the enzyme was 0.11*s-1. Using various 3' hydroxyl group donors (L-DNA) and 5' phosphate group donors (R-DNA), we could detect ligation products as short as 16 nucleotides, the products of 7 + 9 nucleotide or 8 + 8 nucleotide combinations at 40 degrees C. An elevation in temperature led to a decrease in reaction efficiency when short oligonucleotides were used, suggesting that the formation of a nicked, double-stranded DNA substrate preceded enzyme-substrate recognition. LigTk was not inhibited by the addition of excess duplex DNA, implying that the enzyme did not bind strongly to the double-stranded ligation product after nick-sealing. In terms of reaction fidelity, LigTk was found to ligate various substrates with mismatched base-pairing at the 5' end of the nick, but did not show activity towards the 3' mismatched substrates. LigTk could not seal substrates with a 1-nucleotide or 2-nucleotide gap. Small amounts of ligation products were detected with DNA substrates containing a single nucleotide insertion, relatively more with the 5' insertions. The results revealed the importance of proper base-pairing at the 3' hydroxyl side of the nick for the ligation reaction by LigTk.  相似文献   

3.
Duplex strand joining reactions catalyzed by vaccinia virus DNA polymerase   总被引:1,自引:0,他引:1  
Vaccinia virus DNA polymerase catalyzes duplex-by-duplex DNA joining reactions in vitro and many features of these recombination reactions are reprised in vivo. This can explain the intimate linkage between virus replication and genetic recombination. However, it is unclear why these apparently ordinary polymerases exhibit this unusual catalytic capacity. In this study, we have used different substrates to perform a detailed investigation of the mechanism of duplex-by-duplex recombination catalyzed by vaccinia DNA polymerase. When homologous, blunt-ended linear duplex substrates are incubated with vaccinia polymerase, in the presence of Mg2+ and dNTPs, the appearance of joint molecules is preceded by the exposure of complementary single-stranded sequences by the proofreading exonuclease. These intermediates anneal to form a population of joint molecules containing hybrid regions flanked by nicks, 1–5 nt gaps, and/or short overhangs. The products are relatively resistant to exonuclease (and polymerase) activity and thus accumulate in joining reactions. Surface plasmon resonance (SPR) measurements showed the enzyme has a relative binding affinity favoring blunt-ended duplexes over molecules bearing 3′-recessed gaps. Recombinant duplexes are the least favored ligands. These data suggest that a particular combination of otherwise ordinary enzymatic and DNA-binding properties, enable poxvirus DNA polymerases to promote duplex joining reactions.  相似文献   

4.
Chlorella virus DNA ligase (ChVLig) has pluripotent biological activity and an intrinsic nick-sensing function. ChVLig consists of three structural modules that envelop nicked DNA as a C-shaped protein clamp: a nucleotidyltransferase (NTase) domain and an OB domain (these two are common to all DNA ligases) as well as a distinctive β-hairpin latch module. The NTase domain, which performs the chemical steps of ligation, binds the major groove flanking the nick and the minor groove on the 3'-OH side of the nick. Here we performed a structure-guided mutational analysis of the NTase domain, surveying the effects of 35 mutations in 19 residues on ChVLig activity in vivo and in vitro, including biochemical tests of the composite nick sealing reaction and of the three component steps of the ligation pathway (ligase adenylylation, DNA adenylylation, and phosphodiester synthesis). The results highlight (i) key contacts by Thr-84 and Lys-173 to the template DNA strand phosphates at the outer margins of the DNA ligase footprint; (ii) essential contacts of Ser-41, Arg-42, Met-83, and Phe-75 with the 3'-OH strand at the nick; (iii) Arg-176 phosphate contacts at the nick and with ATP during ligase adenylylation; (iv) the role of Phe-44 in forming the protein clamp around the nicked DNA substrate; and (v) the importance of adenine-binding residue Phe-98 in all three steps of ligation. Kinetic analysis of single-turnover nick sealing by ChVLig-AMP underscored the importance of Phe-75-mediated distortion of the nick 3'-OH nucleoside in the catalysis of DNA 5'-adenylylation (step 2) and phosphodiester synthesis (step 3). Induced fit of the nicked DNA into a distorted conformation when bound within the ligase clamp may account for the nick-sensing capacity of ChVLig.  相似文献   

5.
A novel DNA joining activity catalyzed by T4 DNA ligase.   总被引:1,自引:1,他引:1       下载免费PDF全文
The use of T4 and E. coli DNA ligases in genetic engineering technology is usually associated with nick-closing activity in double stranded DNA or ligation of 'sticky-ends' to produce recombinant DNA molecules. We describe in this communication the ability of T4 DNA ligase to catalyze intramolecular loop formation between annealed oligodeoxyribonucleotides wherein Watson-Crick base pairing is absent on one side of the ligation site. Enzyme concentration, loop size, substrate specificity, and base composition were explored in an effort to maximize yield. Amounts of T4 DNA ligase in large molar excess to DNA template and ligated product are necessary to achieve high yields.  相似文献   

6.
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.  相似文献   

7.
Repair of DNA double strand breaks by non-homologous end joining   总被引:25,自引:0,他引:25  
Lees-Miller SP  Meek K 《Biochimie》2003,85(11):1161-1173
DNA double strand breaks (DSB) are the most serious form of DNA damage. If not repaired they can lead to cell death. If misrepaired DSBs contribute to chromosomal aberrations and genomic instability. Non-homologous end joining (NHEJ) is one of two major pathways for the repair of DSBs in human cells. Proteins known to be required for NHEJ include the DNA-dependent protein kinase (DNA-PK), XRCC4, DNA ligase IV, and Artemis. This review discusses how these and other accessory proteins may function in the repair of DSBs produced by ionizing radiation (IR) and by V(D)J recombination.  相似文献   

8.
We have tested the ability of T4 DNA ligase to rejoin radiation-induced DNA strand breaks in living hamster cells (CHO-K1, EM9, xrs-5). T4 DNA ligase was introduced into cells by electroporation prior to x-irradiation. Single- and double-strand breaks were measured by the alkaline comet assay technique, and double-strand breaks (DSBs) were evaluated by the pulsed-field gel electrophoresis method. In the comet assay, the three cell lines showed reduced tail moments following pretreatment with T4 DNA ligase, both directly after irradiation and after repair incubation for 4 h. Similarly, the results obtained from pulsed-field gel electrophoresis showed reduced DSB frequencies after pretreatment with T4 DNA ligase. We conclude that exogeneous T4 ligase contributes to rejoining of radiation-induced strand breaks.  相似文献   

9.
10.
Previous studies (1–5) have shown that several nucleotidyl transferases are metalloenzymes and in a few cases (1–3) the metal has been identified as zinc. In all instances these enzymes are specifically inhibited by incubation with the chelating agent 1,10-phenanthroline but are not affected by the structurally similar 1,7-phenanthroline which does not chelate metals. We report here that tRNA nucleotidyl transferase from E. coli is inhibited by 1,10-phenanthroline and that only the initial rate of incorporation of AMP is affected; CMP incorporation is not specifically inhibited by this chelator. This finding is in conflict with a previous study (5) in which it was claimed that tRNA nucleotidyl transferase from Rous sarcoma virus and from yeast was unaffected by 1,10-phenanthroline and suggests that the E. coli tRNA nucleotidyl transferase is a metalloenzyme.  相似文献   

11.
12.
DNA ligase IV is an essential protein that functions in DNA non-homologous end-joining, the major mechanism that rejoins DNA double-strand breaks in mammalian cells. LIG4 syndrome represents a human disorder caused by mutations in DNA ligase IV that lead to impaired but not ablated activity. Thus far, five conserved motifs in DNA ligases have been identified. We previously reported G469E as a mutational change in a LIG4 syndrome patient. G469 does not lie in any of the previously reported motifs. A sequence comparison between DNA ligases led us to identify residues 468-476 of DNA ligase IV as a further conserved motif, designated motif Va, present in eukaryotic DNA ligases. We carried out mutational analysis of residues within motif Va examining the impact on adenylation, double-stranded ligation, and DNA binding. We interpret our results using the DNA ligase I:DNA crystal structure. Substitution of the glycine at position 468 with an alanine or glutamic acid severely compromises protein activity and stability. Substitution of G469 with an alanine or glutamic acid is better tolerated but still impacts upon activity and protein stability. These finding suggest that G468 and G469 are important for protein stability and provide insight into the hypomorphic nature of the G469E mutation identified in a LIG4 syndrome patient. In contrast, residues 470, 473 and 476 within motif Va can be changed to alanine residues without any impact on DNA binding or adenylation activity. Importantly, however, such mutational changes do impact upon double-stranded ligation activity. Considered in light of the DNA ligase I:DNA crystal structure, our findings suggest that residues 470-476 function as part of a molecular pincer that maintains the DNA in a conformation that is required for ligation.  相似文献   

13.
Mammalian DNA ligases are composed of a conserved catalytic domain flanked by unrelated sequences. At the C-terminal end of the catalytic domain, there is a 16-amino acid sequence, known as the conserved peptide, whose role in the ligation reaction is unknown. Here we show that conserved positively charged residues at the C-terminal end of this motif are required for enzyme-AMP formation. These residues probably interact with the triphosphate tail of ATP, positioning it for nucleophilic attack by the active site lysine. Amino acid residues within the sequence RFPR, which is invariant in the conserved peptide of mammalian DNA ligases, play critical roles in the subsequent nucleotidyl transfer reaction that produces the DNA-adenylate intermediate. DNA binding by the N-terminal zinc finger of DNA ligase III, which is homologous with the two zinc fingers of poly(ADP-ribose) polymerase, is not required for DNA ligase activity in vitro or in vivo. However, this zinc finger enables DNA ligase III to interact with and ligate nicked DNA at physiological salt concentrations. We suggest that in vivo the DNA ligase III zinc finger may displace poly(ADP-ribose) polymerase from DNA strand breaks, allowing repair to occur.  相似文献   

14.
mRNA capping entails GMP transfer from GTP to a 5' diphosphate RNA end to form the structure G(5')ppp(5')N. A similar reaction involving AMP transfer to the 5' monophosphate end of DNA or RNA occurs during strand joining by polynucleotide ligases. In both cases, nucleotidyl transfer occurs through a covalent lysyl-NMP intermediate. Sequence conservation among capping enzymes and ATP-dependent ligases in the vicinity of the active site lysine (KxDG) and at five other co-linear motifs suggests a common structural basis for covalent catalysis. Mutational studies support this view. We propose that the cellular and DNA virus capping enzymes and ATP-dependent ligases constitute a protein superfamily evolved from a common ancestral enzyme. Within this superfamily, the cellular capping enzymes display more extensive similarity to the ligases than they do to the poxvirus capping enzymes. Recent studies suggest that eukaryotic RNA viruses have evolved alternative pathways of cap metabolism catalysed by structurally unrelated enzymes that nonetheless employ a phosphoramidate intermediate. Comparative analysis of these enzymes, particularly at the structural level, should illuminate the shared reaction mechanism while clarifying the basis for nucleotide specificity and end recognition. The capping enzymes merit close attention as potential targets for antiviral therapy.  相似文献   

15.
We have characterized an enzymatic activity from human cell nuclei which is capable of catalyzing strand exchange between homologous DNA sequences. The strand exchange activity was Mg2+ dependent and required ATP hydrolysis. In addition, it was capable of promoting reannealing of homologous DNA sequences and could form nucleoprotein networks in a fashion reminiscent of purified bacterial RecA protein. Using an in vitro recombination assay, we also showed that the strand exchange activity was biologically important. The factor(s) responsible for the activity has been partially purified.  相似文献   

16.
DNA polymerase mu (pol mu), which is related to terminal deoxynucleotidyl transferase and DNA polymerase beta, is thought to be involved in non-homologous end joining and V(D)J recombination. Pol mu is induced by ionizing radiation and exhibits low fidelity. Analysis of translesion replication by purified human pol mu revealed that it bypasses a synthetic abasic site with high efficiency, using primarily a misalignment mechanism. It can also replicate across two tandem abasic sites, using the same mechanism. Pol mu extends primers whose 3'-terminal nucleotides are located opposite the abasic site. Most remarkably, this extension occurs via a mode of nucleotidyl transferase activity, which does not depend on the sequence of the template. This is not due to simple terminal nucleotidyl transferase activity, because pol mu is unable to add dNTPs to an oligo(dT)29 primer or to a blunt end duplex oligonucleotide under standard conditions. Thus, pol mu is a dual mode DNA-synthesizing enzyme, which can act as either a classical DNA polymerase or as a non-canonical, template-dependent, but sequence-independent nucleotidyl transferase. To our knowledge, this is the first report on a DNA-synthesizing enzyme with such properties. These activities may be required for its function in non-homologous end joining in the processing of DNA ends prior to ligation.  相似文献   

17.
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.  相似文献   

18.
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.  相似文献   

19.
In the current model of DNA SSBR, PARP1 is regarded as the sensor of single-strand breaks (SSBs). However, biochemical studies have implicated LIG3 as another possible SSB sensor. Using a laser micro-irradiation protocol that predominantly generates SSBs, we were able to demonstrate that PARP1 is dispensable for the accumulation of different single-strand break repair (SSBR) proteins at sites of DNA damage in live cells. Furthermore, we show in live cells for the first time that LIG3 plays a role in mediating the accumulation of the SSBR proteins XRCC1 and PNKP at sites of DNA damage. Importantly, the accumulation of LIG3 at sites of DNA damage did not require the BRCT domain-mediated interaction with XRCC1. We were able to show that the N-terminal ZnF domain of LIG3 plays a key role in the enzyme''s SSB sensing function. Finally, we provide cellular evidence that LIG3 and not PARP1 acts as the sensor for DNA damage caused by the topoisomerase I inhibitor, irinotecan. Our results support the existence of a second damage-sensing mechanism in SSBR involving the detection of nicks in the genome by LIG3.  相似文献   

20.
Branched nucleic acid strands exist as intermediates in certain biological reactions, and bifurcating DNA also presents interesting opportunities in biotechnological applications. We describe here how T4 DNA ligase can be used for efficient construction of DNA molecules having one 5′ end but two distinct 3′ ends that extend from the 2′ and 3′ carbons, respectively, of an internal nucleotide. The nature of the reaction products is investigated, and optimal reaction conditions are reported for the construction of branched oligonucleotides. We discuss the utility of these branched DNA nanostructures for gene detection.  相似文献   

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