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Mung bean nuclease cleavage pattern at a polypurine.polypyrimidine sequence upstream from the mouse metallothionein-I
1Department of Pharmacological Sciences, State University of New York, Stony Brook 11794.
Abstract:
Mung bean nuclease, an enzyme specific for single-stranded DNA, was used to probe a non-B DNA structure present in the mouse metallothionein-I gene. The region sensitive to the enzyme was constituted by a 128 base-pair long polypurine.polypyrimidine sequence located at 1.2-kb from the start of transcription. A detailed analysis of the mung bean nuclease cleavage pattern revealed that: (i) under conditions of supercoiling and low pH a triplex structure was formed, (ii) the triplex was flanked by a sequence with the potential of forming a Z-DNA structure, (iii) most of the enzymatic activity was localized at some of the junctions between double-stranded and triple-stranded DNA and at mismatches in the triplex, (iv) no unpaired bases were observed in the loop or outside the triplex, and (v) the triplex was present in more than one configuration.
Insights
Researchers used mung bean nuclease to identify a non-B DNA structure in the mouse metallothionein-I gene. This structure, a DNA triplex, formed under specific conditions and was adjacent to a potential Z-DNA region.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- The mouse metallothionein-I gene contains regulatory regions with complex DNA structures.
- Non-B DNA conformations, such as triplex and Z-DNA, play roles in gene regulation.
Purpose of the Study:
- To investigate the presence and nature of non-B DNA structures within the mouse metallothionein-I gene.
- To characterize the specific DNA conformation using enzymatic probing.
Main Methods:
- Utilized mung bean nuclease, an enzyme that cleaves single-stranded DNA.
- Analyzed the cleavage patterns of the enzyme on a specific polypurine.polypyrimidine sequence in the gene.
- Investigated DNA structure formation under varying conditions (supercoiling, pH).
Main Results:
- A 128 base-pair polypurine.polypyrimidine sequence formed a DNA triplex structure under supercoiling and low pH.
- The triplex region was found adjacent to a sequence capable of forming Z-DNA.
- Mung bean nuclease activity was concentrated at junctions between double-stranded and triple-stranded DNA, and at triplex mismatches.
- No unpaired bases were detected outside the triplex structure.
- Evidence suggested the triplex existed in multiple configurations.
Conclusions:
- The mouse metallothionein-I gene harbors a complex DNA triplex structure within a polypurine.polypyrimidine tract.
- This triplex structure is associated with a potential Z-DNA forming region, indicating complex DNA architecture.
- The enzymatic analysis provides insights into the stability and structural dynamics of these non-B DNA forms in a gene regulatory context.