MNase

Related Reading

Introductions

Micrococcal Nuclease (Mnase) is a nuclease that degrades only DNA in the nucleosomal junction region and is not degraded because the DNA at the nucleosome is protected by histone 1. Micrococcal nuclease is derived from Staphylococcus aureus and is a relatively non-specific endo-exonuclease. It is purified from a recombinant E. coli strain that digests double-stranded, single-stranded, circular and linear nucleic acids. The enzyme is active in the pH range of 7.0 - 10.0, with optimal activity at pH 9.2 for both RNA and DNA substrates. Cleavage preferences have been observed at sites rich in adenylate, deoxyadenylate or thymidylate. Both DNA and RNA are degraded to 3' phosphomononucleotides and dinucleotides.

Source

This enzyme is an extracellular nuclease of Staphylococcus aureus. A common source is E. coli cells carrying a cloned nuclear gene encoding a S. aureus extracellular nuclease (micrococcal nuclease).

Structure

The 3-dimensional structure of micrococcal nuclease (then known as staphylococcal nuclease) was solved early in the history of protein crystallography, and higher resolution, newer crystal structures are available for apo forms, such as Protein Data Bank file 1SNO: and thymidine diphosphate repressed forms, such as Protein Data Bank file 3H6M and 1SNC. nuclease molecules have 3 long alpha helices and a 5-linked barrel-shaped beta lamellae, in an arrangement known as an OB fold (oligonucleotide-binding fold).

Micrococcal Nuclease Procedure

• Obtain the target gene

1. By PCR method: Using the cloned plasmid containing the target gene as the template, a pair of primers are designed according to the gene sequence (different enzymatic sites are introduced in the upstream and downstream primers) and the PCR cycle is performed to obtain the desired gene fragment.
2. By RT-PCR method: extract total RNA from cells or tissues by TRIzol method, use mRNA as template, reverse transcribe to form the first strand of cDNA, and use the reverse transcription product as template for PCR cycle to obtain the product.

• Construct recombinant expression vector

1. Micrococcal Nuclease vector digestion: The expression plasmid was double digested with restriction endonuclease (same primer site), and the digested product was recovered by agarose electrophoresis, and the vector fragment was recovered by gel recovery Kit or freeze-thaw method.
2. The PCR products were recovered after double digestion and ligated into the vector by T4 DNA ligase.

• Obtain the expression strain containing recombinant expression plasmid

1. Transform the ligated product into E. coli DH5α, screen the recombinant vector according to its marker (anti-Amp or blue-white spot), pick a single spot, extract a small amount of plasmid by alkaline lysis, and double digestion for preliminary identification.
2. Sequencing to verify the target gene insertion direction and reading frame are correct, enter the next step. Otherwise, more clones should be screened, repeat subcloning or subcloning to different enzymatic sites.
3. Use this recombinant plasmid DNA to transform the receptor cells of the host bacteria.

• Induction of expression

Applications

• Hydrolysis of nucleic acids in cell-free crude extracts.
• RNA sequencing.
• Preparation of double-stranded DNA fragments with 5'-OH and 3'-phosphate.
• Studies of chromatin structure.
• Degradation of nucleic acids in crude cell-free extracts.
• Preparation of rabbit reticulocyte lysates.
• Chromatin structure studies.
• Removal of nucleic acids from laboratory protein preparations for protein folding and structure-function studies.
• Protein folding mechanism studies.