Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.1.31.1 (micrococcal nuclease)
 2,818 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

NF-Y is a CCAAT-binding trimer with two histonic subunits, NF-YB and NF-YC, resembling H2A-H2B. We previously showed that the short conserved domains of NF-Y efficiently bind to the major histocompatibility complex class II Ea Y box in DNA nucleosomized with purified chicken histones. Using wild-type NF-Y and recombinant histones, we find that NF-Y associates with H3-H4 early during nucleosome assembly, under conditions in which binding to naked DNA is not observed. In such assays, the NF-YB-NF-YC dimer forms complexes with H3-H4, for whose formation the CCAAT box is not required. We investigated whether they represent octamer-like structures, using DNase I, micrococcal nuclease, and exonuclease III, and found a highly positioned nucleosome on Ea, whose boundaries were mapped; addition of NF-YB-NF-YC does not lead to the formation of octameric structures, but changes in the digestion patterns are observed. NF-YA can bind to such preformed DNA complexes in a CCAAT-dependent way. In the absence of DNA, NF-YB-NF-YC subunits bind to H3-H4, but not to H2A-H2B, through the NF-YB histone fold. These results indicate that (i) the NF-Y histone fold dimer can efficiently associate DNA during nucleosome formation; (ii) it has an intrinsic affinity for H3-H4 but does not form octamers; and (iii) the interactions between NF-YA, NF-YB-NF-YC, and H3-H4 or nucleosomes are not mutually exclusive. Thus, NF-Y can intervene at different steps during nucleosome formation, and this scenario might be paradigmatic for other histone fold proteins involved in gene regulation.
 ...
PMID:NF-Y associates with H3-H4 tetramers and octamers by multiple mechanisms. 1056 83

The analysis of nucleosome positions and transcription factor binding in chromatin is a central issue for understanding the mechanisms of gene expression in eukaryotes. Here, we have developed a footprinting technique, using multi-cycle primer extension with an infrared-fluorescence DNA sequencer, to analyze chromatin structure in isolated yeast nuclei and transcriptional activator binding in living yeast cells. Using this technique, the binding of the yeast activators Hap1 and Hap2/3/4/5 to their cognate sites was detectable as hypersensitive sites by in vivo UV-photofootprinting, and the locations of nucleosomes in yeast minichromosomes were determined by micrococcal nuclease mapping. We also applied this method to determine the position of the nucleosome in the 5S DNA fragment reconstituted in vitro. This technique allowed us to eliminate the use of radioactive materials and to perform experiments on common benches. Thus, the footprinting procedure established in this study will be useful to researchers studying DNA-protein interactions and chromatin structure in vivo and in vitro.
 ...
PMID:In vivo and in vitro footprinting of nucleosomes and transcriptional activators using an infrared-fluorescence DNA sequencer. 1823 71