Mung bean nuclease
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Mung bean nuclease (Nuclease MB) is a nuclease derived from sprouts of the mung bean (Vigna radiata) that removes nucleotides in a step-wise manner from single-stranded DNA molecules (ssDNA) and is used in biotechnological applications to remove such ssDNA from a mixture also containing double-stranded DNA (dsDNA). This enzyme is useful for transcript mapping, removal of single-stranded regions in DNA hybrids or single-stranded overhangs produced by restriction enzymes, etc. It has an activity similar to Nuclease S1 (both are EC 3.1.30.1), but it has higher specificity for single-stranded molecules.[1]
The enzyme degrades single-stranded DNA or RNA to nucleoside 5’-monophosphates, but does not digest double-stranded DNA, double-stranded RNA, or DNA / RNA hybrids. Mung Bean Nuclease catalyzes the specific degradation of single-stranded DNA or RNA, and produces mono and oligonucleotides carrying a 5′-P terminus. Mung bean nuclease has a stringent single-stranded specificity for DNA or RNA.
Mung bean nuclease has an estimated molecular weight of 39 kDa by SDS-PAGE. A glycoprotein, 29% of this mass is sugars.[2] As of April 2019[update], the specific gene encoding for this protein is unknown, and all production relies on a purification process on bean sprouts from 1980.[1] Some is known about its structure, with one exposed Cysteine residue and 3 pairs of disulfide bonds. Some is known about its amino acid composition.[2]
Requirements
[edit]Mung bean nuclease requires Zn2+. The addition of EDTA or SDS causes irreversible inactivation. Mung bean nuclease is not active at pH below 4.6, nor at low salt concentration.
Description
[edit]Nuclease MB is a specific DNA and RNA exo-endonuclease which will degrade single-stranded extensions from the ends of DNA and RNA molecules, leaving blunt, ligatable ends. Its higher single-strand specificity makes it the enzyme of choice for most applications requiring a single-strand-specific nuclease.
Unlike S1 Nuclease, Mung Bean Nuclease will not cleave the intact strand of nicked duplex DNA.
Its ability to recognise double-stranded nucleic acids depends on the base sequence.
It tends to cleave at ApN and at T(U) pN. It completely degrades ApA, but does not degrade G and C. Unlike S1 Nuclease, it does not cleave the strand opposite to that which has been nicked.
Mung Bean Nuclease catalyzes the specific degradation of single-stranded DNA or RNA, and produces mono- and oligonucleotides carrying a 5′-P terminus.
More than 1000- fold amount of enzyme can degrade oligomer into all mononucleotides.
An excess of the enzyme is required to degrade double-stranded DNA or RNA and DNA-RNA hybrids, and in this case, AT-rich regions are selectively degraded.
This enzyme work well at A↓pN, T ↓pN sites, and especially A↓pN sites are 100% degraded.
However, it is difficult to degrade C↓pC, C↓pG site.
Mung bean exonuclease is a nuclease derived from mung beans that removes nucleotides in a step-wise manner from single stranded DNA molecules and is used to remove such ssDNA from a mixture also containing double stranded DNA (dsDNA).
Unit Definition:
One unit of Mung Bean Nuclease converts 1 µg of heat-denatured calf thymus DNA into an acid-soluble form in 1 minute at 37 °C under standard assay conditions.
Applications in biotechnology and biochemical research
[edit]- Removal of hairpin loops during cDNA synthesis.
- High-resolution mapping of the termini and exon structures of RNA transcripts (commonly termed Berk-Sharp or S1 Mapping) using either internal-labelled or end-labelled probes.
- Restriction-site modification or removal by digestion of single-stranded protruding ends.
- Cleavage of single-basepair mismatches, as a replacement for CEL 1 Nuclease in TILLING.
- Unidirectional deletion of large DNA (in combination with Exonuclease III) to generate ordered deletions for sequencing.
- Removal of 3´ and 5´ extensions from DNA or RNA termini.
- Transcriptional mapping.
- Cleavage of hairpin loops.
- Excision of gene coding sequences from genomic DNA.
References
[edit]- ^ a b "BRENDA: 3.1.30.1".
- ^ a b Eun, HM (1996). "Nucleases". Enzymology primer for recombinant DNA technology. Academic Press. pp. 145–232. doi:10.1016/B978-012243740-3/50006-5. ISBN 978-0-12-243740-3.
Further reading
[edit]- Kowalski et al's many-article series from the 1970s: Kowalski, David; Kroeker, Warren D.; Laskowski, M. (1976). "Mung bean nuclease I. 6. Physical, chemical, and catalytic properties". Biochemistry. 15 (20): 4457–4463. doi:10.1021/bi00665a019. PMID 9973.
