4-Coumarate:CoA ligase from cell suspension cultures of Petroselinum hortense Hoffm. Partial purification, substrate specificity, and further properties.
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Cations, Divalent↗
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BRCA1-A and LIG4 complexes mediate ecDNA biogenesis and cancer drug resistance.
Extrachromosomal circular DNA (ecDNA) is frequently generated within the nucleus, contributing to genome dynamics and heterogeneity, thereby promoting cancer cell evolution and adaptation. However, the mechanisms underlying ecDNA biogenesis remain poorly understood. Here, using genome-wide CRISPR screening in human cells, we identified the BRCA1-A and the LIG4 complexes as key drivers of ecDNA production. Following DNA segmentation, the upstream BRCA1-A complex protects DNA ends from excessive resection, promoting end-joining for circularization. Conversely, the MRN complex, which mediates end resection and thus antagonizes the BRCA1-A complex, suppresses ecDNA formation. Downstream, LIG4 conservatively mediates ecDNA production by joining the free ends of the DNA fragments. Furthermore, ecDNA from patient tumors harbors junction sites with a LIG4 signature. Notably, disruption of either LIG4 or the BRCA1-A complex in cancer cells impairs ecDNA-mediated adaptation, hindering the development of resistance to both chemotherapy and targeted therapies. Together, our study reveals the roles of the LIG4 and BRCA1-A complexes in ecDNA biogenesis, and uncovers therapeutic targets to block ecDNA-mediated adaptation for cancer treatment.
Humans↗
Pan-genome characterization of the maize 4CL gene family and its dynamic responses to abiotic stress.
1.Pan-genome analysis across 26 maize inbred lines identified 13 Zm4CL genes (nine core and four near-core) classified into three evolutionary clades.2.Structural variations (SVs) are significantly associated with the expression and altered conserved protein domains of key Zm4CL genes.3.Zm4CL genes exhibit distinct tissue-specific expression patterns and dynamic enzymatic and transcriptional responses to stresses, particularly cold and drought.4-Coumarate:CoA ligase (4CL) is a key enzyme in the phenylpropanoid pathway and plays important roles in plant growth, development, and responses to environmental stresses. However, a comprehensive pan-genome analysis of the 4CL gene family in maize is still lacking. In this study, 13 Zm4CL genes were identified from a maize pan-genome comprising 26 diverse inbred lines, including nine core genes and four near-core genes. Phylogenetic analysis classified these genes into three evolutionary clades, while Ka/Ks analysis indicated that most members have been maintained under purifying selection, although several genes exhibited greater evolutionary divergence and relatively relaxed evolutionary constraints. Structural variation (SV) analysis revealed significant associations between SVs and the expression of Zm4CL2 and Zm4CL3, while sequence comparisons suggested that SVs were also associated with alterations in conserved protein domains in some genotypes. Transcriptome analyses revealed distinct tissue-specific expression patterns and diverse transcriptional responses to abiotic and biotic stresses. Enzyme activity assays showed that cold stress significantly increased 4CL activity at 12 h, whereas heat, salt, and alkali stresses caused an initial decrease followed by recovery, while drought had no significant effect. Time-course RT-qPCR further validated dynamic expression changes of representative Zm4CL genes under cold and drought stresses. Overall, this study provides a comprehensive pan-genome framework for understanding the evolutionary conservation, regulatory diversification, and stress-responsive characteristics of the maize Zm4CL gene family, providing valuable resources for future functional studies and the genetic improvement of stress tolerance in maize.
Zea mays↗
Potential inhibitors of collagen biosynthesis, 4,4-Difluoro-L-proline and 4,4-dimethyl-DL-proline and their activation by prolyl-tRNA ligase.
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Amino Acyl-tRNA Synthetases↗
RNF4 and USP7 cooperate in ubiquitin-regulated steps of DNA replication.
DNA replication requires precise regulation achieved through post-translational modifications, including ubiquitination and SUMOylation. These modifications are linked by the SUMO-targeted E3 ubiquitin ligases (STUbLs). Ring finger protein 4 (RNF4), one of only two mammalian STUbLs, participates in double-strand break repair and resolving DNA-protein cross-links. However, its role in DNA replication has been poorly understood. Using CRISPR/Cas9 genetic screens, we discovered an unexpected dependency of RNF4 mutants on ubiquitin specific peptidase 7 (USP7) for survival in TP53-null retinal pigment epithelial cells. TP53-/-/RNF4-/-/USP7-/- triple knockout (TKO) cells displayed defects in DNA replication that cause genomic instability. These defects were exacerbated by the proteasome inhibitor bortezomib, which limited the nuclear ubiquitin pool. A shortage of free ubiquitin suppressed the ataxia telangiectasia and Rad3-related (ATR)-mediated checkpoint response, leading to increased cell death. In conclusion, RNF4 and USP7 work cooperatively to sustain a functional level of nuclear ubiquitin to maintain the integrity of the genome.
Animals↗
The occurrence of enzymes involved in phenylpropanoid metabolism in the tapetum fraction of anthers.
The contents of another loculus were separated in a pollen and tapetum fraction. The following enzymes involved in phenylpropanoid metabolism were present in the tapetum fraction: shikimate dehydrogenase; phenylalanine ammonialyase; cinnamic acid 4-hydroxylase; SAM (S-adenosylmethionine): caffeate 3-O-methyltransferase; hydroxycinnamate: CoA ligase; "flavanone synthase"; chalcone-flavanone isomerase; SAM: 3',4'-dihydroxyflavonoid 3'-O-methyltransferase; O-glucosyltransferase. It is postulated, that these enzymes derived from the tapetum catalyze the different steps of phenylpropanoid metabolism at or in cavities of the exine after their transfer into the loculus.
Acyltransferases↗
Evidence for a single species of DNA ligase localized in nuclei of rat liver.
DNA ligase activity was distributed mainly in the extract of nuclei rapidly isolated from rat liver and about 20% of the nuclear activity in the cytoplasmic fraction. The DNA ligases of nuclear extract and cytoplasmic fraction showed similar gel-filtration patterns and the same sedimentation coefficient of 5.5 S. The appearance of 4-S enzyme from nuclei or nuclear extract was partly inhibited by phenylmethylsulfonyl fluoride. These results suggest that the major activity of DNA ligase in rat liver is due to a single species of the enzyme localized in nuclei and that 4-S nuclear DNA ligase reported previously is an artifact arising from 5.5-S enzyme during the isolation procedure.
Animals↗
Metabolic changes associated with the occurrence of fatty liver and kidney syndrome in chicks.
1. The changes in a number of metabolic measurements brought about by low-biotin diets associated with high and low incidences of fatty liver and kidney syndrome (FLKS) were studied in healthy 4-week-old broiler chicks. 2. Liver pyruvate carboxylase (pyruvate: CO2 ligase (ADP); EC 6.4.1.1) activity was low in birds fed on a diet causing a high incidence FLKS but the addition of fat or protein to this diet, to decrease the incidence of FLKS, increased enzyme activity. 3. Liver weights, blood lactate concentrations, plasma lactate dehydrogenase (L-lactate: NAD oxidoreductase; EC 1.1.1.27) activitvities and values for C16:1 : C18:0 fatty acid in liver, adipose tissue and plasma triglyceride were highest in birds fed on the high-FLKS diet and all measurements were negatively correlated with pyruvate carboxylase activity. 4. Birds with high plasma lactate dehydrogenase activity or triglyceride C16:1 : C18:0 values were the most likely to develop FLKS when fasted. 5. There was no evidence that increased liver weight was associated with increase activities of certain other liver enzymes. 6. It is concluded that FLKS occurs in birds with little or no hepatic gluconeogenic capacity via pyruvate carboxylase as a result of a dietary insufficiency of biotin but that the initiation of the syndrome in probably associated with the inhibition of other pathways of gluconeogenesis.
Animals↗
Synaptic and non-synaptic mitochondria from rat brain: isolation and characterization.
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4-Aminobutyrate Transaminase↗
Total synthesis of the structural gene for the precursor of a tyrosine suppressor transfer RNA from Escherichia coli. 12. Synthesis of a DNA duplex corresponding to a sequence of 23 nucleotide units adjoining the C-C-A end.
In continuing the work on the total synthesis of the gene for an Escherichia coli tyrosine suppressor tRNA (accompanying papers) and as a part of a study of the mechanism of transcription of this gene, a 23-nucleotide unit-long DNA corresponding to the previously determined (Loewen, P., Sekiya, T., and Khorana, H. G. (1974) J. Biol. Chem. 249, 217) sequence has been synthesized. The synthesis was carried out by dividing the total duplex into the following five deoxyribooligonucleotide segments, all of which were chemically synthesized: (a) the undecanucleotide, d(A-G-T-G-A-T-G-G-T-G-G); (b)the undecanucleotide, d(T-C-A-C-T-T-T-C-A-A-A); (c) the undecanucleotide, d(G-G-A-C-T-T-T-T-G-A-A); (d) the dodecanucleotide, d(A-G-T-C-C-C-T-G-A-A-C-T); and (e) the heptanucleotide, d(A-G-T-T-C-A-G). All the five synthetic oligonucleotides were characterized by chromatographic and radioactive fingerprinting methods after labeling the 5'-ends with a 32P-phosphate group. Synthesis of the double-stranded DNA duplex was completed by joining 5'-phosphorylated segments 1, 3, and 4 in the presence of segments 2 and 5 using T4-polynucleotide ligase. The DNA duplex was characterized.
Base Sequence↗
Enzymes of agmatine degradation and the control of their synthesis in Klebsiella aerogenes.
The degradation of agmatine to succinate by Klebsiella aerogenes occurs in five steps. The enzyme catalyzing the first step, agmatinase, is induced by agmatine. The enzymes catalyzing the second and third steps, putrescine aminotransferase and 4-aminobutyraldehyde dehydrogenase, are induced by putrescine and also by their product, 4-aminobutyrate. The enzymes catalyzing the fourth and fifth steps, 4-aminobutyrate aminotransferase and succinate semialdehyde dehydrogenase, are induced by 4-aminobutyrate. This compound also serves as gratuitous inducer of the catabolic acetylornithine aminotransferase. The formation of the enzymes responsible for agmatine degradation is regulated not only by induction, but also by catabolite repression and activation by glutamine synthetase.
4-Aminobutyrate Transaminase↗
Pleiotropic effects of a DNA adenine methylation mutation (dam-3) in Escherichia coli K12.
The dam-3 mutation results in a five-fold reduction in the number of 6-methyl-adenine (6-meA) residues in the DNA of E. coli K12 or phage lambda. The DNA of phage fd appears to be devoid of 6-meA when propagated on dam-3 bacteria. The phenotypic differences between dam-3 and dam+ bacteria include: (i) increased free phage in lysogenic dam-3 cultures, (2) increased sensitivity to methyl methanesulfonate (MMS), (3) inviability of dam-3 lex-I strains, (4) lower molecular weight of DNA in dam-3 bacteria in the absence of DNA ligase and (5) increased rate of DNA degradation in dam-3 recA strains.
Adenine↗
Fatty acid transport by the lipophilic bacterium Nocardia asteroides.
Hexadecanoate was translocated in Nocardia asteroides by a constitutive transport system(s), which transported short, medium, and long-chain fatty acids. Inhibition of hexadenocanoate transport by homologues suggested that at least two systems are present: one specific for short-chain fatty acids and the other specific for medium- and long-chain fatty acids. Saturation kinetics typical of a carrier-mediated transport system (Kt = 870 muM)were observed, and concentration of fatty acids against a gradient was achieved. Inhibitor studies indicated that free sulfhydryl groups, a functional respiratory chain, and energy are required for translocation. Efflux of [14C]hexadecanoate in the presence of excess unlabeled hexadecanoate or 2,4-dinitrophenol and the cytoplasmic localization of acyl-coenzyme A synthetase (acid:coenzyme A ligase [adenosine monophosphate]; EC 6.2.1.3) (Calmes and Deal, 1973) are consistent with the hypothesis that fatty acids are transported and released intracellularly as free fatty acids.
Adsorption↗
Polymyxin synthetase:L-2,4-diaminobutyrate activating enzyme,.
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Amino Acids, Diamino↗
Mapping and cloning of Eco RI-fragments of bacteriophage T5+ DNA.
The Eco RI-fragments of bacteriophage T5 DNA were mapped using a technique which involves primarily length measurements of molecules observed in the electron microscope. Since Eco RI cleavage generates termini with 4-nucleotide long cohesive ends, fragments of complete and partial Eco RI digests were covalently circularized with DNA ligase at dilute DNA concentrations before measuring relative to internal length standards. This established the order of the internal Eco RI fragments. The two external Eco RI fragments, which had only one Eco RI terminus, were positioned relative to the internal fragments by identifying the location of some of the naturally-occurring nicks in partially denatured linear Eco RI fragments. An attempt was made to clone each of the internal Eco RI-fragments of T5 DNA via transformation into E. coli after ligation in vitro with the plasmid pMB 9. Only one fragment could be cloned and this fragment did not specify any new polypeptides in minicells of either the E. coli EK1 host, X1411, or the EK 2 host, X1776.
Chromosome Mapping↗
The effects of spermine and Mg2+ on the catalytic mechanism of isoleucine: tRNA ligase.
Isoleucyl-tRNA formation catalysed by isoleucine: tRNA ligase is stimulated by both Mg2+ and spermine in the pH-range 7.0 to 8.0 at 310 K. At low [Mg2+] the acceleration caused by both cations together exceeds the sum of their individual effects. 2. The spermine-stimulated reaction has a steeper temperature-dependence than reaction in the presence of Mg2+. Two phases in the kinetics of isoleucyl-tRNA formation are detected in the presence of Mg2+ plus or minus spermine, but only a single step is observed in the presence of spermine alone. Thus the rate-limiting steps under normal assay conditions are different for the two cations. 3. Enzyme-bound isoleucyl-AMP can be formed in the absence of Mg-2+ and plus or minus spermine. 4. It is concluded that there is no evidence for cation-dependent differences in the reaction mechanism of isoleucine: tRNA ligase, though there are certainly differences in the relative rates of some of the individual steps.
Enzyme Activation↗
Cell surface lipids and adhesion. II. The turnover of lipid components of the plasmalemma in relation to cell adhesion.
The preceding paper showed that those conditions that ought to stimulate reacylation of lysolipids in cells can increase cell adhesions. Similarly we found that conditions that would be expected to lead to the accumulation of lysolipids in the cell surface diminish cell adhesion. This paper reports on the answers to the following questions. (1) Is reacylation of lysolipids in the cells stimulated by an external supply of CoA, ATP and a fatty acid? (2) Does this reacylation lead to the incorporation of exogenous fatty acid in the plasmlemma? (3) What range of fatty acids can be incorporated into the plasmalemma and into what compounds? (4) Does the plasmalemma contain the enzyme systems to effect this turnover, namely phospholipase A2, a CoA-ligase and an appropriate acyl transferase(s)? (5) Do lysolipids accumulate in the plasmalemma under conditions which diminish cell adhesion? We find that saturated fatty acids in the range C14--C18, and some unsaturated fatty acids are incorporated into the plasmalemmae of these neural retina cells. About 20% of the plasmlemma content of fatty acids can be turned over in 30'. Incorporation is mainly into phosphatidyl choline, serine and ethanolamine in both R1 and R2 positions. The plasmalemmae contain the enzymes to effect the turnover. Isolated plasmalemmae are active in this turnover. Incubation of the plasmalemmae with phospholipase A2 leads to an accumulation of lysolipids. Very low levels of phospholipase stimulate turnover, possibly endogenous phospholipase activity is the rate-limiting step in the system. These findings are discussed in relation to the possible mechanisms by which lipids might affect adhesion.
Acyltransferases↗
Growth inhibition of Escherichia coli W by D-norvalyl-D-alanine: an analogue of D-alanine in position 4 of the peptide subunit of peptidoglycan.
Position 4 analogues of d-alanine in the peptide subunit of uridine 5'-diphosphate-N-acetylmuramyl-Ala(1)-dGlu(2)- Lys(3)-dAla(4)-dAla(5) have a significant inhibitory effect on penicillin-sensitive peptidoglycan synthesis in Gaffkya homari (C. V. Carpenter, S. Goyer, and F. C. Neuhaus, 1976). The specificity profile of this in vitro system has been used as a basis for designing analogues with potential antibacterial activity. To circumvent the specificity determinants exerted by d-alanine:d-alanine ligase (adenosine 5'-diphosphate), attention was directed to dd-dipeptides of the type d-alanyl-analogue-d-alanine as a method for incorporating analogues into position 4 of the peptide subunit in vivo. Of the three dipeptides, dAbu-dAla, dNva-dAla, and dVal-dAla, only dNva-dAla (5 x 10(-4) M) inhibited the growth of Escherichia coli W in the presence of 5 x 10(-6) M d-cycloserine. This concentration of d-cycloserine did not inhibit growth, but it potentiated the bactericidal activity of the dipeptide. The lack of antibacterial activity observed with dAbu-dAla and dVal-dAla was correlated with the poor ability of these dipeptides to be taken up via the dipeptide transport system of this organism. Prevention of lysis induced by dNva-dAla plus d-cycloserine by certain dipeptides and not by others supported this correlation. It is proposed that the d-norvalyl residue of the dipeptide is incorporated in vivo into position 4 of the peptide subunit of peptidoglycan, and that this subunit is not an effective substrate in the reaction(s) catalyzed by the penicillin-susceptible enzyme(s) of cross-linked peptidoglycan synthesis.
Alanine↗