Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:6.5.1.2 (DNA ligase)
 2,749 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The RAD6 gene from the yeast Saccharomyces cerevisiae encodes a ubiquitin carrier protein (E2) required for a variety of cellular processes including DNA repair, induced mutagenesis, and sporulation. Here we identify an E2 from a higher plant, wheat, that is similar to RAD6 with respect to both structure and in vitro substrate specificity. The protein was purified from wheat germ by a combination of ubiquitin covalent affinity chromatography and anion-exchange HPLC and has an apparent molecular mass of 23 kDa [referred to as E2(23 kDa)]. E2(23 kDa) was capable of binding ubiquitin by means of a thiol ester linkage in an ATP-dependent and ubiquitin-activating enzyme-dependent reaction. In the presence of a variety of target proteins, E2(23 kDa), like the RAD6 gene product, formed covalent ubiquitin-protein conjugates in vitro only with histones in a ubiquitin protein ligase-independent reaction. E2(23 kDa) recognized both core and linker histones with an apparent order of preference of H2A greater than or equal to H1 greater than H2B greater than H3 greater than H4. This E2 protein was approximately 17-fold more effective at conjugating ubiquitin to histones than three other purified wheat germ E2 proteins tested. Mouse anti-E2(23 kDa) antibodies were used to isolate E2(23 kDa) DNA sequences from a wheat cDNA expression library. Antibody-positive clones were confirmed by amino acid identity of the sequence deduced from the cDNA to the peptide sequence of an E2(23 kDa) tryptic fragment. Protein expressed in Escherichia coli by the E2(23 kDa) cDNA was capable of both thiol ester adduct formation and conjugation of ubiquitin to histones. Analysis of the E2(23 kDa) cDNA shows that it encodes a protein with considerable amino acid sequence similarity to the yeast RAD6 gene product. Similarities exist at the amino terminus, the region surrounding the putative ubiquitin binding site, and at the carboxyl terminus, which is unusually acidic. Based on both the structural and enzymatic similarities to the RAD6 gene product, E2(23 kDa) may represent the first DNA repair enzyme identified in higher plants.
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PMID:A ubiquitin carrier protein from wheat germ is structurally and functionally similar to the yeast DNA repair enzyme encoded by RAD6. 255 33

Numerous environmental carcinogens involve radical formation interacting with DNA to produce 2-deoxyribonolactone (dL), a major type of oxidized abasic site, implicated in DNA strand breaks, mutagenesis, and formation of covalent DNA-protein cross-links (DPC). Studies showed major dL-specific DPC occurred due to reactions with DNA polymerase beta (Polbeta) dependent on native conformation, while other DPC formed involved nonenzymatic reactions of DNA binding proteins with dL lesions. Polbeta appeared to play a major role in alleviating the cytotoxic effects of neocarzinostatin, which was used as a dL-producing agent. When a duplex DNA containing a dL at a site-specific position was incubated with purified histones, DPC were formed between dL and each histone protein, including H1, H2A, H2B, H3, and H4. Comparative kinetic analysis of DPC formation with histones and Polbeta revealed two distinct mechanisms of dL-mediated DPC formation. The rate of DPC formation with Polbeta was approximately two orders of magnitude higher than that with various histone proteins. These results indicate that catalytic activity of Polbeta mediates rapid DPC formation between dL and this DNA repair enzyme, whereas nonenzymatic reactions of dL with histones form DPC more slowly. The abundance of histones and their constant interaction with DNA may nevertheless yield significant levels of DPC with dL, as biomarkers of dL-induced cytotoxicity. Overall, data suggest that occurrence of dL-mediated DPC with histones may contribute to the genotoxic effects of dL in DNA.
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PMID:Biochemical evaluation of genotoxic biomarkers for 2-deoxyribonolactone-mediated cross-link formation with histones. 2007 2

Advances in high-throughput characterization of protein networks in vivo have resulted in large databases of unexplored protein interactions that occur during normal cell function. Their further characterization requires quantitative experimental strategies that are easy to implement in laboratories without specialized equipment. We have overcome many of the previous limitations to thermodynamic quantification of protein interactions, by developing a series of in-solution fluorescence-based strategies. These methods have high sensitivity, a broad dynamic range, and can be performed in a high-throughput manner. In three case studies we demonstrate how fluorescence (de)quenching and fluorescence resonance energy transfer can be used to quantitatively probe various high-affinity protein-DNA and protein-protein interactions. We applied these methods to describe the preference of linker histone H1 for nucleosomes over DNA, the ionic dependence of the DNA repair enzyme PARP1 in DNA binding, and the interaction between the histone chaperone Nap1 and the histone H2A-H2B heterodimer.
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PMID:Fluorescence strategies for high-throughput quantification of protein interactions. 2212 Dec 11