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 availability of DNA structural probes that can be applied to living cells is essential for the analysis of biological functions of unusual DNA structures adopted in vivo. We have developed a chemical probe assay to detect and quantitate left-handed Z-DNA structures in recombinant plasmids in growing E. coli cells. Potassium permanganate selectively reacts with B-Z or Z-Z junction regions in supercoiled plasmids harbored in the cells. Restriction enzyme recognition sites located at these junctions are not cleaved by the corresponding endonuclease after modification with KMnO4. This inhibition of cleavage allows the determination of the relative amounts of B- and Z-forms of the cloned inserts inside the cell. We have successfully applied this method to monitor the extent of Z-DNA formation in E. coli as a function of the growth phase and mutated topoisomerase or gyrase activities. The assay can in principle be used for any unusual DNA structure that contains a restriction recognition site inside or near the structural alteration. It can be a useful tool to analyze in vivo correlations between DNA structure and gene regulatory events.
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PMID:Potassium permanganate as an in situ probe for B-Z and Z-Z junctions. 166 68

Ligation-mediated polymerase chain reaction (LMPCR) provides adequate sensitivity for nucleotide-level analysis of single-copy genes. Here, we report that chromatin structure can be studied by enzyme treatment of permeabilized cells followed by LMPCR. DNase I treatment of lysolecithin-permeabilized cells was found to give very clear footprints and to show differences between active and inactive X chromosomes (Xa and Xi, respectively) at the human X-linked phosphoglycerate kinase (PGK-1) locus. Beginning 380 bp upstream and continuing 70 bp downstream of the major transcription start site of PGK-1, we analyzed both strands of this promoter and CpG island and discovered the following: (1) The transcriptionally active Xa in permeabilized cells has several upstream regions that are almost completely protected on both strands from DNase I nicking. (2) Nuclei isolated in polyamine-containing buffers lack these footprints, suggesting that data from isolated nuclei can be flawed; other buffers are less disruptive. (3) The Xa has no detectable footprints at the transcription start and HIP1 consensus sequence. (4) The heterochromatic and transcriptionally inactive Xi has no footprints but has two regions showing increased DNase I sensitivity at 10-bp intervals, suggesting that the DNA is wrapped on the surface of a particle; one nucleosome-sized particle seems to be positioned over the transcription start site and another is centered approximately 260 bp upstream. (5) Potassium permanganate and micrococcal nuclease (MNase) studies indicate no melted or otherwise unusual DNA structures in the region analyzed, and MNase, unlike restriction endonuclease MspI, does cut within the positioned particles on the Xi. Results are discussed in the context of X chromosome inactivation and the maintenance of protein and DNA methylation differences between euchromatin and facultative heterochromatin at CpG islands.
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PMID:Chromatin differences between active and inactive X chromosomes revealed by genomic footprinting of permeabilized cells using DNase I and ligation-mediated PCR. 204 57