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PubMed2005

Using molecular beacon to monitor activity of E. coli DNA ligase.

NAD(+)-dependent DNA ligase has been widely used in gene diagnostics for disease-associated mutation detection and has proved to be necessary for screening bactericidal drugs targeted to DNA ligases. However, further research has been restricted since conventional ligase assay technology is limited to gel electrophoresis, which is discontinuous, time-consuming and laborious. An innovative approach is developed for monitoring the activity of E. coli DNA ligase catalyzing nucleic acid ligation in the report. This approach utilizes a molecular beacon hybridized with two single-stranded DNA (ssDNA) segments to be ligated to form a hybrid with a nick, and could therefore be recognized by the enzyme. Ligation of the two ssDNA segments would cause conformation changes of the molecular beacon, leading to significant fluorescence enhancement. Compared to gel electrophoresis, this approach can provide real time information about ligase, is more time efficient, and is easier to use. The effect of quinacrine, a drug for malaria, on the activity of the ligase is detected, thereby certifying the capability of the method for developing novel antibacterial drugs targeted at NAD(+)-dependent ligase. The fidelity of strand joining by the ligase is examined based on this approach. The effects of external factors on activity of the ligase are analyzed, and then an assay of E. coli DNA ligase is performed with a broad linear range of 4.0 x 10(-4) Weiss Unit mL(-1) to 0.4 Weiss Unit mL(-1) and the detection limit of 4.0 x 10(-4) Weiss Unit mL(-1).

DNA Ligase ATP↗
PubMed2000

Arabidopsis DNA ligase IV is induced by gamma-irradiation and interacts with an Arabidopsis homologue of the double strand break repair protein XRCC4.

Rejoining of single- and double-strand breaks (DSBs) introduced in DNA during replication, recombination, and DNA damage is catalysed by DNA ligase enzymes. Eukaryotes possess multiple DNA ligase enzymes, each having distinct roles in cellular metabolism. Double-strand breaks in DNA, which can occur spontaneously in the cell or be induced experimentally by gamma-irradiation, represent one of the most serious threats to genomic integrity. Non-homologous end joining (NHEJ) rather than homologous recombination is the major pathway for repair of DSBs in organisms with complex genomes, including humans and plants. DNA ligase IV in Saccharomyces cerevisiae and humans catalyses the final step in the NHEJ pathway of DSB repair. In this study we identify an Arabidopsis thaliana homologue (AtLIG4) of human and S. cerevisiae DNA ligase IV which is shown to encode an ATP-dependent DNA ligase with a theoretical molecular mass of 138 kDa and 48% similarity in amino-acid sequence to the human DNA ligase IV. Yeast two-hybrid analysis demonstrated a strong interaction between A. thaliana DNA ligase IV and the A. thaliana homologue of the human DNA ligase IV-binding protein XRCC4. This interaction is shown to be mediated via the tandem BRCA C-terminal domains of A. thaliana DNA ligase IV protein. Expression of AtLIG4 is induced by gamma-irradiation but not by UVB irradiation, consistent with an in vivo role for the A. thaliana DNA ligase IV in DSB repair.

Amino Acid Sequence↗
PubMed1997

An interaction between DNA ligase I and proliferating cell nuclear antigen: implications for Okazaki fragment synthesis and joining.

Although three human genes encoding DNA ligases have been isolated, the molecular mechanisms by which these gene products specifically participate in different DNA transactions are not well understood. In this study, fractionation of a HeLa nuclear extract by DNA ligase I affinity chromatography resulted in the specific retention of a replication protein, proliferating cell nuclear antigen (PCNA), by the affinity resin. Subsequent experiments demonstrated that DNA ligase I and PCNA interact directly via the amino-terminal 118 aa of DNA ligase I, the same region of DNA ligase I that is required for localization of this enzyme at replication foci during S phase. PCNA, which forms a sliding clamp around duplex DNA, interacts with DNA pol delta and enables this enzyme to synthesize DNA processively. An interaction between DNA ligase I and PCNA that is topologically linked to DNA was detected. However, DNA ligase I inhibited PCNA-dependent DNA synthesis by DNA pol delta. These observations suggest that a ternary complex of DNA ligase I, PCNA and DNA pol delta does not form on a gapped DNA template. Consistent with this idea, the cell cycle inhibitor p21, which also interacts with PCNA and inhibits processive DNA synthesis by DNA pol delta, disrupts the DNA ligase I-PCNA complex. Thus, we propose that after Okazaki fragment DNA synthesis is completed by a PCNA-DNA pol delta complex, DNA pol delta is released, allowing DNA ligase I to bind to PCNA at the nick between adjacent Okazaki fragments and catalyze phosphodiester bond formation.

Chromatography, Affinity↗
PubMed2003

Specific and potent inhibition of NAD+-dependent DNA ligase by pyridochromanones.

Pyridochromanones were identified by high throughput screening as potent inhibitors of NAD+-dependent DNA ligase from Escherichia coli. Further characterization revealed that eubacterial DNA ligases from Gram-negative and Gram-positive sources were inhibited at nanomolar concentrations. In contrast, purified human DNA ligase I was not affected (IC50 > 75 microm), demonstrating remarkable specificity for the prokaryotic target. The binding mode is competitive with the eubacteria-specific cofactor NAD+, and no intercalation into DNA was detected. Accordingly, the compounds were bactericidal for the prominent human pathogen Staphylococcus aureus in the low microg/ml range, whereas eukaryotic cells were not affected up to 60 microg/ml. The hypothesis that inhibition of DNA ligase is the antibacterial principle was proven in studies with a temperature-sensitive ligase-deficient E. coli strain. This mutant was highly susceptible for pyridochromanones at elevated temperatures but was rescued by heterologous expression of human DNA ligase I. A physiological consequence of ligase inhibition in bacteria was massive DNA degradation, as visualized by fluorescence microscopy of labeled DNA. In summary, the pyridochromanones demonstrate that diverse eubacterial DNA ligases can be addressed by a single inhibitor without affecting eukaryotic ligases or other DNA-binding enzymes, which proves the value of DNA ligase as a novel target in antibacterial therapy.

Adenosine Monophosphate↗
PubMed1985

DNA ligase activities during hepatocarcinogenesis induced by N-2-acetylaminofluorene.

A progressive accumulation of DNA breaks has been reported to occur in nuclear DNA obtained from putative premalignant hepatic lesions induced by carcinogens. To determine if this alteration resulted from a defect in the level of, or functional activity of DNA ligases, we compared these enzymes in normal rat liver, 24-h regenerating liver, and hepatic nodules at intervals after cessation of N-2-acetylaminofluorene (AAF) treatment. Nuclear extracts of hepatocytes were separated into soluble and chromatin fractions, and multiple forms of DNA ligase activity were obtained by AcA34 gel filtration chromatography. In activities of the two largest species, DNA ligase Ia (480 kd) and DNA ligase Ib (240 kd), were present exclusively in soluble, nuclear fractions and were increased 4-fold and 2-fold, respectively, in 24-h regenerating livers. In AAF-induced nodules, these species were increased 3-fold and 1.5-fold, respectively, above those of normal rat liver, somewhat higher than predicted from the rate of cell division. In all of the test tissues, these ligase species demonstrated identical sensitivity to inhibition with 0.1 M NaCl or heating at 50 degrees C. DNA ligase II (80 kd) was found in both soluble nuclear fractions and chromatin at approximately identical levels in all tissues tested. Ligase II from all tissues also demonstrated identical responses to salt and heat. These data support the concept that DNA ligases Ia and Ib are related to DNA replication and suggest that ligase II may be a repair enzyme. The failure to detect significant alterations from expected values in the hepatic nodules and the lack of alteration in sensitivity to salt and heat indicate that the accumulation of DNA damage (presumably breaks) previously observed in carcinogen-induced altered hepatocytes is not due to an alteration in the level or the biochemical properties of DNA ligase.

2-Acetylaminofluorene↗
PubMed1997

Two distinct DNA ligase activities in mitotic extracts of the yeast Saccharomyces cerevisiae.

Four biochemically distinct DNA ligases have been identified in mammalian cells. One of these enzymes, DNA ligase I, is functionally homologous to the DNA ligase encoded by the Saccharomyces cerevisiae CDC9 gene. Cdc9 DNA ligase has been assumed to be the only species of DNA ligase in this organism. In the present study we have identified a second DNA ligase activity in mitotic extracts of S. cerevisiae with chromatographic properties different from Cdc9 DNA ligase, which is the major DNA joining activity. This minor DNA joining activity, which contributes 5-10% of the total cellular DNA joining activity, forms a 90 kDa enzyme-adenylate intermediate which, unlike the Cdc9 enzyme-adenylate intermediate, reacts with an oligo (pdT)/poly (rA) substrate. The levels of the minor DNA joining activity are not altered by mutation or by overexpression of the CDC9 gene. Furthermore, the 90 kDa polypeptide is not recognized by a Cdc9 antiserum. Since this minor species does not appear to be a modified form of Cdc9 DNA ligase, it has been designated as S. cerevisiae DNA ligase II. Based on the similarities in polynucleotide substrate specificity, this enzyme may be the functional homolog of mammalian DNA ligase III or IV.

Animals↗
PubMed1991

Two types of DNA ligase I activity in lymphoblastoid cells from patients with Bloom's syndrome.

DNA ligases I and II were separated by hydroxylapatite (HA) column chromatography in cell-free extracts of lymphoblastoid cell lines (LCLs) derived from two unrelated patients with Bloom's syndrome (BS) and two healthy individuals. The specific activity of ligase I from the crude extract was consistently lower in GM3403, a BS LCL from an Ashkenazi Jewish patient, than in normal control LCLs. By contrast, the level of ligase I activity in BSL-2KA, another BS LCL derived from a Japanese patient, was equivalent to those in normal LCLs, although GM3403 and BSL-2KA shared the feature of exceedingly high frequency of spontaneous sister-chromatid exchange. The levels of total ligase activity in crude extracts without the separation into the two forms, however, were approximately two-fold higher for the two BS LCLs than for the normal LCLs. Partial purification by chromatography on a DEAE-cellulose 23 column and a phosphocellulose column did not affect the superiority of the two BS LCLs over the normal LCLs in the specific activity of the total ligases. Nonetheless, subsequent application to an HA column again resulted in much less elevation of the specific activity of ligase I for GM3403 than for BSL-2KA and control LCLs. The levels of ligase II activity, accounting for 4-13% of total ligase activity, were similar among the LCLs examined. Irrespective of the extent of purification, essentially no difference in the heat lability of DNA ligase I was detected among the four LCLs. These findings suggest that there may exist among BS LCLs at least two types of subtle abnormality of DNA ligase I itself and/or a putative substance modulating the enzyme function.

Bloom Syndrome↗
PubMed1988

A possible effect of heme on the fate of DNA ligase activity extracted from differentiating mouse erythroleukemia cells.

When mouse erythroleukemia (MEL) cells were induced to differentiate by growth in the presence of dimethyl sulfoxide, hexamethylene bisacetamide (HMBA), or hemin, the apparent activity of DNA ligase extractable from inducer-treated cells decreased 70 to 80% when compared to untreated cells. Earlier work had indicated that these changes did not occur in a differentiation-resistant MEL cell variant and suggested that the decrease in the level of DNA ligase activity might be related to the differentiation process. Since the MEL cells accumulate high levels of both hemoglobin-bound and non-hemoglobin-bound heme, the effect of both hemoglobin and hemin on DNA ligase activity of MEL cell extracts was tested. When cell-free extracts containing DNA ligase activity were preincubated with hemin at concentrations up to 150 microM, an 80% or greater inhibition of the DNA ligase activity resulted. The ATP-dependent DNA ligase from bacteriophage T4 was also inhibited by hemin, but the NAD-dependent DNA ligase from Escherichia coli was not sensitive to this treatment. Preincubation of these same extracts with hemoglobin at levels comparable to those present in differentiating cells did not result in inhibition of any of the ATP-dependent DNA ligases tested. Culturing the cells with dimethyl sulfoxide in the presence of imidazole resulted in a marked decrease in globin chain accumulation but did not reverse the dimethyl sulfoxide-related decrease in DNA ligase activity. These data suggest the possibility that heme or its metabolites, but not globin or hemoglobin, could serve to modify the process of DNA replication and/or repair in differentiating MEL cells via inhibition of DNA ligase activity. These data are consistent with the findings of Lo et al. (S.C. Lo, R. Aft, and G.C. Mueller, Cancer Res., 41: 864-870, 1981) which correlated the onset of differentiation-related terminal cell division in MEL cells with the levels of nonhemoglobin heme present in these cells.

Acetamides↗
PubMed1994

Characterization of the Shope fibroma virus DNA ligase gene.

The Shope fibroma virus (SFV) DNA ligase gene has been cloned and sequenced, and the biochemical requirements of the gene product have been determined in vitro. The SFV ligase gene maps to the BamHI L1/L2 boundary and spans 1.7 kb. The gene is predicted to encode a 559-amino-acid protein of M(r) = 63,139 which shares 45% amino acid identity with Orthopoxvirus ligases. The C-terminal two-thirds of the protein appears to encode the catalytic domain and shares distant homology with many ligases. The N-terminal homology is shared between only Orthopoxviruses and Leporipoxviruses and suggests that DNA ligases may be composite structures consisting of two independently evolved protein domains. Although the the gene encodes features characteristic of both early and late poxviral genes, Northern analysis showed that SFV ligase is expressed as a late gene product. In order to prove the identity of the protein it was expressed as a glutathione S-transferase fusion in Escherichia coli, affinity purified, and shown to be a Mg2+.ATP-dependent ligase in vitro. The recombinant protein can also form a covalent ligase.AMP complex characteristic of ATP-dependent DNA ligases. The SFV ligase gene can be disrupted and is thus not essential for viral growth in culture. This was shown by recombining a PCR product, encoding a P7.5 promoter and E. coli guanine phosphoribosyltransferase gene (gpt) into the open reading frame, and selecting for gpt+ viruses. This work provides insights into the evolution of Orthopoxviruses and Leporipoxviruses and strains suitable for a detailed analysis of the role DNA ligases play in poxviral recombination.

Amino Acid Sequence↗
PubMed2001

Completion of base excision repair by mammalian DNA ligases.

Three mammalian genes encoding DNA ligases--LIG1, LIG3, and LIG4--have been identified. Genetic, biochemical, and cell biology studies indicate that the products of each of these genes play a unique role in mammalian DNA metabolism. Interestingly, cell lines deficient in either DNA ligase I (46BR.1G1) or DNA ligase III (EM9) are sensitive to simple alkylating agents. One interpretation of these observations is that DNA ligases I and III participate in functionally distinct base excision repair (BER) subpathways. In support of this idea, extracts from both DNA ligase-deficient cell lines are defective in catalyzing BER in vitro and both DNA ligases interact with other BER proteins. DNA ligase I interacts directly with proliferating cell nuclear antigen (PCNA) and DNA polymerase beta (Pol beta), linking this enzyme with both short-patch and long-patch BER. In somatic cells, DNA ligase III alpha forms a stable complex with the DNA repair protein Xrcc1. Although Xrcc1 has no catalytic activity, it also interacts with Pol beta and poly(ADP-ribose) polymerase (PARP), linking DNA ligase III alpha with BER and single-strand break repair, respectively. Biochemical studies suggest that the majority of short-patch base excision repair events are completed by the DNA ligase III alpha/Xrcc1 complex. Although there is compelling evidence for the participation of PARP in the repair of DNA single-strand breaks, the role of PARP in BER has not been established.

Animals↗
PubMed1998

DNA ligase gene disruptions can depress viral growth and replication in poxvirus-infected cells.

Poxvirus-encoded DNA ligases are assumed to play a role in viral DNA replication; however mutational inactivation of vaccinia ligase has not been reported to affect viral growth rates in culture. This communication re-examines this surprising aspect of poxviral biology using both Shope fibroma virus (SFV) and vaccinia virus. SFV and vaccinia ligase deficiencies create essentially identical phenotypes. In particular, ligase-deficient SFV strains are mildly UV sensitive and etoposide resistant, phenotypes previously shown to characterize ligase-deficient vaccinia strains. Moreover, we find that ligase mutations can inhibit the growth of both SFV and vaccinia virus in vitro. The poor growth observed in the absence of a viral ligase is correlated with a two- to tenfold reduction in viral and extragenomic DNA synthesis. This phenotype is host dependent. No differences in viral growth or DNA yield were seen when vaccinia strains were cultured on rabbit (SIRC) cells, but ligase deficiencies reduced growth and DNA yields when vaccinia was plated on BSC-40 cells or SFV on SIRC cells. Despite these replicative defects, mutational inactivation of SFV ligase produced no detectable increase in the number of viral DNA breaks and had no effect on virus-catalyzed extragenomic DNA recombination or UV repair. We conclude that poxviral ligases do play a role in viral DNA replication, but the replicative defect is obscured in some cell lines.

Animals↗
PubMed2006

Amplification and disruption of the phenylacetyl-CoA ligase gene of Penicillium chrysogenum encoding an aryl-capping enzyme that supplies phenylacetic acid to the isopenicillin N-acyltransferase.

A gene, phl, encoding a phenylacetyl-CoA ligase was cloned from a phage library of Penicillium chrysogenum AS-P-78. The presence of five introns in the phl gene was confirmed by reverse transcriptase-PCR. The phl gene encoded an aryl-CoA ligase closely related to Arabidopsis thaliana 4-coumaroyl-CoA ligase. The Phl protein contained most of the amino acids defining the aryl-CoA (4-coumaroyl-CoA) ligase substrate-specificity code and differed from acetyl-CoA ligase and other acyl-CoA ligases. The phl gene was not linked to the penicillin gene cluster. Amplification of phl in an autonomous replicating plasmid led to an 8-fold increase in phenylacetyl-CoA ligase activity and a 35% increase in penicillin production. Transformants containing the amplified phl gene were resistant to high concentrations of phenylacetic acid (more than 2.5 g/l). Disruption of the phl gene resulted in a 40% decrease in penicillin production and a similar reduction of phenylacetyl-CoA ligase activity. The disrupted mutants were highly susceptible to phenylacetic acid. Complementation of the disrupted mutants with the phl gene restored normal levels of penicillin production and resistance to phenylacetic acid. The phenylacetyl-CoA ligase encoded by the phl gene is therefore involved in penicillin production, although a second aryl-CoA ligase appears to contribute partially to phenylacetic acid activation. The Phl protein lacks a peptide-carrier-protein domain and behaves as an aryl-capping enzyme that activates phenylacetic acid and transfers it to the isopenicillin N acyltransferase. The Phl protein contains the peroxisome-targeting sequence that is also present in the isopenicillin N acyltransferase. The peroxisomal co-localization of these two proteins indicates that the last two enzymes of the penicillin pathway form a peroxisomal functional complex.

Acyltransferases↗
PubMed2004

Characterization of bacteriophage T3 DNA ligase.

DNA ligases of bacteriophage T4 and T7 have been widely used in molecular biology for decades, but little is known about bacteriophage T3 DNA ligase. Here is the first report on the cloning, expression and biochemical characterization of bacteriophage T3 DNA ligase. The polyhistidine-tagged recombinant T3 DNA ligase was shown to be an ATP-dependent enzyme. The enzymatic activity was not affected by high concentration of monovalent cations up to 1 M, whereas 2 mM ATP could inhibit its activity by 50%. Under optimal conditions (pH 8.0, 0.5 mM ATP, 5 mM DTT, 1 mM Mg(2+) and 300 mM Na(+)), 1 fmol of T3 DNA ligase could achieve 90% ligation of 450 fmol of cohesive dsDNA fragments in 30 min. T3 DNA ligase was shown to be over 5-fold more efficient than T4 DNA ligase for ligation of cohesive DNA fragments, but less active for blunt-ended DNA fragments. Phylogenetic analysis showed that T3 DNA ligase is more closely related to T7 DNA ligase than to T4 DNA ligase.

Amino Acid Sequence↗
PubMed1999

Biochemical properties of a high fidelity DNA ligase from Thermus species AK16D.

NAD+-dependent DNA ligases from thermophilic bacteria Thermus species are highly homologous with amino acid sequence identities ranging from 85 to 98%. Thermus species AK16D ligase, the most divergent of the seven Thermus isolates collected worldwide, was cloned, expressed in Escherichia coli and purified to homogeneity. This Thermus ligase is similar to Thermus thermophilus HB8 ligase with respect to pH, salt, NAD+, divalent cation profiles and steady-state kinetics.However, the former is more discriminative toward T/G mismatches at the 3'-side of the ligation junction, as judged by the ratios of initial ligation rates of matched and mismatched substrates. The two wild-type Thermus ligases and a Tth ligase mutant (K294R) demonstrate 1-2 orders of magnitude higher fidelity than viral T4 DNA ligase. Both Thermus ligases are active with either the metal cofactor Mg2+, Mn2+or Ca2+but not with Co2+, Ni2+, Cu2+or Zn2+. While the nick closure step with Ca2+becomes rate-limiting which results in the accumulation of DNA-adenylate intermediate, Ni2+only supports intermediate formation to a limited extent. Both Thermus ligases exhibit enhanced mismatch ligation when Mn2+is substituted for Mg2+, but the Tsp. AK16D ligase remains more specific toward perfectly matched substrate.

Amino Acid Sequence↗
PubMed1975

Mammalian DNA ligases. Serological evidence for two separate enzymes.

Mammalian cells contain two DNA ligase activities with different chromatographic properties, referred to as DNA ligase I and II. The major ligase activity present in calf thymus cell extracts, DNA ligase I, has been purified 1000-fold. After repeated injections of this enzyme with complete Freund's adjuvant into a rabbit, antibodies were induced that inhibit DNA ligase I from calf, human, mouse, and rabbit tissues. This antiserum did not affect DNA ligase II from the same sources to a detectable extent, even at a concentration 10-fold higher than that required for 98% inhibition of DNA ligase I. These data strongly indicate that the two mammalian DNA ligase activities are due to two separate enzymes, and not to two forms of the same enzyme. Both enzymes are present in the nuclear fraction, but are also found in the cytoplasmic fraction. Rapidly dividing cells (mouse ascites tumor cells and calf thymus) contain higher amounts of DNA ligase I than other cells (calf liver and spleen, human placenta, and rabbit spleen), while no such correlation was observed for DNA ligase II.

Animals↗
PubMed1995

Phytanic acid oxidation: topographical localization of phytanoyl-CoA ligase and transport of phytanic acid into human peroxisomes.

To understand the possible role of phytanoyl-CoA ligase, present in the membrane, in the oxidation of phytanic acid in the matrix of peroxisomes (Pahan, K. and I. Singh. 1993. FEBS Lett. 333: 154-158) we examined the transport of phytanic acid/phytanoyl-CoA into peroxisomes and the topology of the active site of phytanoyl-CoA ligase in the peroxisomal membrane. The increase in lignoceroyl-CoA ligase as compared to no change in the activities of palmitoyl-CoA and phytanoyl-CoA ligases when peroxisomes were disrupted with detergent or sonication and inhibition of the activities of both palmitoyl-CoA and phytanoyl-CoA ligase by impermeable inhibitor of acyl-CoA ligases (mercury-dextran) and trypsin treatment in the intact peroxisomes. On the other hand, the lignoceroyl-CoA ligase activity was inhibited by mercury-dextran and trypsin only in the disrupted peroxisomes. Taken together, these studies support the conclusion that the enzymatic site of phytanoyl-CoA ligase is on the cytoplasmic surface of peroxisomal membrane. This implies that phytanoyl-CoA is synthesized on the cytoplasmic surface of peroxisomal membrane and is translocated through the membrane for its alpha-oxidation to pristanic acid in the matrix of peroxisomes. To delineate the transport for phytanic acid through the peroxisomal membrane, we examined cofactors and energy requirements for its transport into peroxisomes. The similar rates of transport of phytanoyl-CoA and phytanic acid under conditions favorable for fatty acid activation (presence of ATP, CoASH, and MgCl2) and the lack of transport of phytanic acid when ATP and/or CoASH were removed or replaced with their inactive analogues (ATP and/or CoASH) from assay medium clearly demonstrates that the transport of phytanic acid requires prior synthesis of phytanoyl-CoA by phytanoyl-CoA ligase. The prerequisite activation of phytanic acid to phytanoyl-CoA for its alpha-oxidation only in intact peroxisomes, and oxidation of free phytanic acid in digitonin-permealized peroxisomes or isolated matrix, suggests that phytanoyl-CoA ligase (in peroxisomal membrane) regulates the oxidation of phytanic acid in peroxisomes by providing phytanoyl-CoA for its transport into peroxisomes.

Binding Sites↗
PubMedDate not supplied

[Use of Cibacron blue F 3GA-CL-sepharose 6B for the purification of T4 DNA- and RNA-ligases].

The sorption capacity of the dye cibacron blue F3GA, immobilized on CL-Sepharose 6B and other support matrices, in respect to DNA- and RNA-ligases T4 was being studies. Cibacron blue F3GA immobilized on CL-Sepharose 6B binds a three-fold amount of DNA-ligase in comparison to RNA-ligase. The enzyme chromatography on cibbacron blue F3GA-CL-Sepharose 6B revealed a stronger linkage between DNA-ligase T4 and the sorbent than between RNA-ligase T4 and the sorbent. Elution was performed with potassium chloride. DNA-ligase T4 was eluted with 0.25-0.5 M KCl and RNA-ligase T4 with 0.08-0.18 M KCl. Since deoxyexonuclease contaminants possess stronger bonds with the sorbent than ligases, elution of deoxyexonucleases occurs at higher concentrations of KCl. Chromatography of enzymes on cibacron blue F3GA-CL-Sepharose 6B allows one to obtain DNA- and RNA-ligases essentially free of DNase and RNase contaminants.

Absorption↗
PubMed1994

Altered DNA ligase III activity in the CHO EM9 mutant.

Delayed joining of DNA strand breaks and a high spontaneous level of sister-chromatid exchanges (SCEs) are characteristics of the mutant cell strain EM9 of Chinese hamster ovary (CHO) cells. The introduction of the human gene XRCC1 into EM9 cells reverts the phenotypic properties of EM9 to those of the wild type. We have investigated both DNA ligase activities and a protein which stimulates DNA ligase activity in mutant EM9 cells, XRCC1-transfectant H9T3-7-1 cells and wild-type AA8 cells. Our results, which demonstrate both a decreased DNA ligase activity in EM9 cells using poly(rA).oligo(dT) as substrate and a decreased ability of DNA ligase III to form a covalent DNA ligase III-adenylate intermediate with AMP, clearly indicate an altered DNA ligase III activity in the mutant. Furthermore, the AMP-binding capacity of DNA ligase III and its enzymatic activity with the synthetic polymer were restored after transfection of EM9 with the human XRCC1 gene. Immunoblotting data suggest that the XRCC1 gene does not code for DNA ligase III. In conclusion, the data indicate that the EM9 cell strain has an altered DNA ligase III activity that can be restored by the XRCC1 gene product.

Adenosine Monophosphate↗