Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.30.2 (endonuclease)
 18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Rad25 protein in yeast is a DNA helicase and a subunit of the general transcription factor TFIIH. While in vitro studies have led to the hypothesis that TFIIH helicase activity plays a role in promoter melting, in vivo tests are lacking. Using potassium permanganate, which preferentially modifies single-stranded DNA, we show that a temperature-sensitive rad25(ts) mutant severely reduces the normally extensive promoter melting observed in vivo on the highly expressed genes TDH2 and PDC1 and on the induced heat shock gene HSP82. Loss of promoter melting can be observed in as little as 30 s after a shift to the nonpermissive temperature and is accompanied by a dramatic reduction in transcription. These effects on the promoter are specific, since the mutation does not affect TATA box occupancy or, in the case of HSP82, recruitment of TATA-binding protein to the TATA element or that of heat shock factor to heat shock elements. Additionally, using the technique of formaldehyde cross-linking coupled with restriction endonuclease cleavage and ligation-mediated PCR, we were able to map the polymerase density on the promoter of HSP82. This high-resolution mapping allowed us to determine that the polymerase II (Pol II) density on the promoter is also dramatically reduced after inactivation of TFIIH. These data provide strong support for the hypothesis that TFIIH functions with Pol II in the transcriptionally required step of promoter melting and show, surprisingly, that the extent of TFIIH-dependent promoter melting observed in vivo is several times larger than that seen in vitro.
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PMID:Transcription factor TFIIH is required for promoter melting in vivo. 1040 54

Here the effect on DNA-mediated charge transport of binding by a variety of proteins is examined. DNA assemblies were constructed that contain a tethered rhodium intercalator, as photooxidant, as well as two 5'-GG-3' sites flanking the DNA-binding site for the different proteins. By monitoring the ratio of oxidative damage promoted at the guanine doublet situated distal to the protein-binding site versus that at the proximal site as a function of protein binding, the effects of binding the proteins on DNA-mediated charge transport were determined. Proteins examined included both the wild-type and mutant methyltransferase, M.HhaI, which are base-flipping enzymes, the restriction endonuclease R.PvuII, a TATA-binding protein, which kinks the DNA, and the transcription factor Antennapedia homeodomain protein, which binds DNA through a helix-turn-helix motif. In general, it was observed that yields of long-range oxidative damage correlate with protein-dependent alterations in DNA base stacking. Interactions that disturb the DNA pi-stack inhibit DNA charge transport. Alternatively, interactions that promote no helix distortion but, as a result of tight packing, may rigidify the pi-stack, serve instead to enhance the ability of the DNA base pairs to serve as a conduit for charge transport. Thus, protein binding to DNA modulates long-range charge transport both negatively and positively, depending upon the specific protein/DNA interactions in play. Long-range DNA charge transport and this modulation by protein binding may be important to consider physiologically.
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PMID:How different DNA-binding proteins affect long-range oxidative damage to DNA. 1133 Oct 21

DNA charge transport chemistry is found to provide a sensitive method for probing protein-dependent changes in DNA structure and enzymatic reactions. Here we describe the development of an electrochemical assay of protein binding to DNA-modified electrodes based upon the detection of associated perturbations in DNA base stacking. Gold electrode surfaces that were modified with loosely packed DNA duplexes, covalently crosslinked to a redox-active intercalator and containing the binding site of the test protein, were constructed. Charge transport through DNA as a function of protein binding was then assayed. Substantial attenuation in current is seen in the presence of the base-flipping enzymes HhaI methylase and uracil DNA glycosylase, as well as with TATA-binding protein. When restriction endonuclease PvuII (R.PvuII) binds to its methylated target, little base-stacking perturbation occurs and little diminution in current flow is observed. Importantly, the kinetics of restriction by R.PvuII of its nonmethylated target is also easily monitored electrochemically. This approach should be generally applicable to assaying protein--DNA interactions and reactions on surfaces.
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PMID:An electrical probe of protein-DNA interactions on DNA-modified surfaces. 1187 30

Repair of DNA double-strand breaks (DSBs) by recombination pathways is essential for plant growth and fertility. The recombination endonuclease MRE11 plays important roles in sensing and repair of DNA DSBs. Here we demonstrate protein interaction between Arabidopsis MRE11 and the histone acetyltransferase TAF1, a TATA-binding protein Associated Factor (TAF) of the RNA polymerase II transcription initiation factor complex TFIID. Arabidopsis has two TAF1 homologues termed TAF1 and TAF1b and mutant taf1b lines are viable and fertile. In contrast, taf1 null mutations are lethal, demonstrating that TAF1 is an essential gene. Heterozygous taf1+/- plants display abnormal segregation of the mutant allele resulting from defects in pollen tube development, indicating that TAF1 is important for gamete viability. Characterization of an allelic series of taf1 lines revealed that hypomorphic mutants are viable but display developmental defects and reduced plant fertility. Hypersensitivity of taf1 mutants lacking the C-terminal bromodomain to X-rays and mitomycin C, but not to other forms of abiotic stress, established a specific role for TAF1 in plant DNA repair processes. Collectively these studies reveal a function for TAF1 in plant resistance to genotoxic stress, providing further insight into the molecular mechanisms of the DNA damage response in plants.
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PMID:Arabidopsis TAF1 is an MRE11-interacting protein required for resistance to genotoxic stress and viability of the male gametophyte. 2635 8