Most of the time nucleases are the enemy of the molecular biologist who is trying to preserve the integrity of RNA or DNA samples. However, deoxyribonucleases (DNases) and ribonucleases (RNases) have certain indispensable roles in molecular biology laboratories.

Numerous types of DNase and RNase have been isolated and characterized. They differ among other things in substrate specificity, cofactor requirements, and whether they cleave nucleic acids internally (endonucleases), chew in from the ends (exonucleases) or attack in both of these modes. In many cases, the substrate specificity of a nuclease depends upon the concentration of enzyme used in the reaction, with high concentrations promoting less specific cleavages.

The most widely used nucleases are DNase I and RNase A, both of which are purified from bovine pancreas.

Deoxyribonuclease I cleaves double-stranded or single stranded DNA. Cleavage preferentially occurs adjacent to pyrimidine (C or T) residues, and the enzyme is therefore an endonuclease. Major products are 5'-phosphorylated di, tri and tetranucleotides.

In the presence of magnesium ions, DNase I hydrolyzes each strand of duplex DNA independently, generating random cleavages. In the presence of manganese ions, the enzyme cleaves both strands of DNA at approximately the same site, producing blunt ends or fragments with 1-2 base overhangs. DNase I does not cleave RNA, but crude preparations of the enzyme are contaminated with RNase A; RNase-free DNase I is readily available.

Some of the common applications of DNase I are:

• Eliminating DNA (e.g. plasmid) from preparations of RNA.
• Analyzing DNA-protein interactions via DNase foot printing.
• Nicking DNA prior to radio labeling by nick translation.
  

Ribonuclease A is an endoribonuclease that cleaves single-stranded RNA at the 3' end of pyrimidine residues. It degrades the RNA into 3'-phosphorylated mononucleotides and oligonucleotides.

Some of the major use of RNase A are:

• Eliminating or reducing RNA contamination in preparations of plasmid DNA.
• Mapping mutations in DNA or RNA by mismatch cleavage. RNase will cleave the RNA in RNA:DNA hybrids at sites of single base mismatches, and the cleavage products can be analyzed.

In the alpha-helix the polypeptide folds by twisting into a right handed screw so that all the amino acids can form hydrogen bonds with each other. This high amount of hydrogen bonding stabilizes the structure so that it forms a very strong rod-like structure. The amino group of each AA residue is hydrogen bonded to the carboxyl group of the 4th following AA residue, which is on an adjacent turn of the helix.

Along the axis of the helix, it rises 0.15 nm per AA residue, and there are 3.6 residues/turn of the helix. This means that AA residues spaced 4 apart in the linear chain are quite close to one another in the alpha-helix. The screw-sense of any helix can be RH or LH, but the alpha-helix found in proteins is always RH.

A sequence of asp and or glu residues together can also destabilize the helix because they are highly -vely charged, and repel each other. The forces of repulsion are stronger than the H-bonding. Also a cluster of ile residues with their large bulky R groups tends to disrupt the alpha-helix structure by disrupting the H-bonding.

After discovery of helix, Pauling & Corey discovered that polypeptide chains could fold in another way, which they named beta-pleated sheet (beta is second, alpha was first).

In this case more H-bonding is achieved by stretching out the polypeptide chain, and laying it side by side to form H-bonds between lengths of polypeptide chain. Thus providing both inter and intra-H bonds.

Called a beta-pleated sheet because of zig zag appearance when viewed from the side. Substantially different from the alpha-helix in that it's a sheet rather than a rod and polypeptide chain is fully stretched rather than tightly coiled as in helix. The H-bonds are formed from amino and carboxyl groups as for alpha-helix, but bonding also occurs between different stands of a polypeptide.

The stands can run in opposite directions to give antiparallel beta-pleated sheet or they can run in same direction to give parallel beta-pleated sheets. Beta sheets occur in variable amounts in the polypeptide chains of globular proteins e.g. lysozyme and carboxypeptidase, but more commonly associated with fibrous proteins such as silk and keratin.

Twisting of beta-sheets

It is found that extended polypeptide chain beta-sheets tend to be twisted for greater stability and rigidity (e.g. take a sheet of paper, if straight very flexible, but introduce a twist and introduce stability and rigidity). So twisting is a common feature in beta-sheets and twisted beta-sheets frequently form the backbone of many protein structures.

We see in globular proteins that a high degree of folding and close packing is required to reduce the exposure of protein chains to solvent and create a "dry" inside. So vital for sharp turns or bends in polypeptide chains and these super secondary structures are a common feature in many globular proteins.

Normally it involves formation of hydrogen bonds between one AA and another 3 residues on to form a hairpin loop. Found that in many bends gly and or pro are present in the bend to enable the turn to occur. So in the pre-determined amino acid sequence of a protein the occurrence of these AA in particular places can promote the formation of a bend or turn. The gly is small and provides no steric resistance to a tight turn.

The tertiary structure of a polypeptide chain is the next level of conformation or shape adopted by the alpha-helices or beta-pleated sheets of the chain. Most proteins tend to fold into shapes that are broadly classified as globular in arrangement, and some, particularly structural proteins form long fibers. These are the main forms of gross tertiary structure. A term often used is domain, which refers to a compact unit of globular structure in a polypeptide chain.

Globular is a all-encompassing term like house, but many different shapes and sizes of house, and many types of globular structure. Like secondary structure the tertiary structure of a protein is stabilized by mostly non-covalent forces although tertiary structure can also be stabilized by covalent bonds. 

(a) electrostatic interactions non-covalent

(b) H-bonds non-covalent

(c) hydrophobic interactions non-covalent

(d) disulfide bridges covalent bond

(e) isopeptide linkage covalent bond

Insight into the relationship between AA sequence i.e. primary structure and final conformation in tertiary structure came from work of Anfinsen studying enzyme ribonuclease (RNase), which hydrolyzes RNA.

Ribonuclease is a single polypeptide chain 124 AA, and 4 disulfide bridges with an overall globular structure. Anfinsen decided to see what would happen if he unfolded the polypeptide chain by disrupting the stabilization forces. Used either urea or guanidine-HCl to disrupt the forces that stabilize the secondary and tertiary structure. They do not affect the covalent peptide bonds in primary structure.

These chemicals cause the polypeptide chain to unfold to form a random coil structure. The concentrations of these chemicals required are quite high 6M Gdn-HCl or 8M urea. They only disrupt the non-covalent interactions so it was necessary to add beta-mercaptoethanol, which reduces and breaks disulfide bonds.

-S-S- -----> -SH + SH-

When ribonuclease was treated with urea + beta-mercaptoethanol, it unfolded to form a randomly coiled polypeptide chain with no enzyme activity i.e. RNAse was denatured. This experiment proved that proteins need the 3D conformation (shape) to perform the given function, which in this case was enzymic hydrolysis of RNA.

In 1960 Standford Moore and William Stein determined the amino acid sequence of bovine RNase (pancreatic ribonuclease), which was the first enzyme and only the second protein to be sequenced in full. It is a small protein of 124 amino acids and it is extremely stable. You can autoclave this enzyme and its activity will return after it cools. The enzyme cuts (hydrolyzes) RNA by cleaving the phosphate-oxygen ester bond on the 3' side of the phosphate of a pyrimidine base. When a synthetic copy of the enzyme was made it was found to be active. If the enzyme was cut with a protease into two parts, the parts by themselves were not enzymatically active. However, if the two parts were mixed, they would associate into an active enzyme again. This occurred in spite of the fact that the initial single polypeptide chain (α-carbon backbone) had been broken in two and remained so. With this information at hand, it was possible to determine the arrangement of the active site of the protein, how the RNA substrate fits into the active site, what amino acids were present at the active site, and which amino acids at the active site were necessary for ribonuclease activity. The important amino acids at the active site are:

Two His residues -- His12 and His119 -- are implicated in the catalytic mechanism of this enzyme. The imidazole ring of His12 has an anonymously low pK value (pK < 6.0) suggesting it must be deprotonated for catalysis. Conversely, the imidazole ring of His119 has an anonymously high pK value (pK > 6.0) suggesting it must be protonated for catalysis.

A two-step reaction is proposed for hydrolysis of RNA.

1. His12 acts as a general base accepting 2'-OH proton of the sessile ribonucleic sugar ring thus promoting a nucleophilic attack by the 2'-O on the more positively-charged phosphorus (P) atom thereby creating a 2’,3’-cyclic ribonucleotide phosphate intermediate. The creation of this intermediate is facilitated by the imidazole of His119 which acts as a general acid donating its proton to the oxygen atom in the susceptible P-O-R' bond.