Nucleases

DNA and RNA are polymers made by linking together smaller units called nucleotides . Nucleases are enzymes that break the chemical bonds, called phosphodiester bonds , that hold the nucleotides of DNA or RNA polymers together. Enzymes that cleave the phosphodiester bonds of DNA are called deoxyribonucleases, and enzymes that cleave the phosphodiester bonds of RNA are called ribonucleases.

Nucleases can be divided into two classes, exonucleases and endonucleases, based on the positions of the cleaved bonds within the DNA or RNA polymers. The exonucleases are involved in trimming the ends of RNA and DNA polymers, cleaving the last phosphodiester bond in a chain. This cleavage results in the removal of a single nucleotide from the polymer. If the enzyme removes nucleotides from the 3′ ("three prime") end, it is referred to as a 3′ exonuclease. If cleavage is at the 5′ end, the enzyme is called a 5′ exonuclease. The endonucleases cleave phosphodiester bonds of DNA or RNA at positions other than at the end of the polymer. The cutting reactions of the endonucleases produce fragments of DNA or RNA.

Individual nucleases frequently show preferences for various structures of DNA and RNA. Some nucleases prefer single-stranded polymers, while others prefer double-stranded polymers. Some nucleases cleave at specific nucleotide sequences, and others cleave at positions in the polymers independent of nucleotide sequence. Exonucleases can show preference for DNA ends that are correctly base-paired or for ends that are mispaired. Some endonucleases involved in DNA repair recognize damaged nucleotides and cleave phosphodiester bonds at these sites. The preferences exhibited by the nucleases reflect the wide biological functions for these enzymes .

The Nuclease Mechanism

There are two different mechanisms used by various nucleases to cleave the chemical bonds of the DNA or RNA polymer. The most common mechanism is one in which a water molecule is used to break the phosphodiester bond . This is called a hydrolysis reaction. Under most conditions the P-O (phosphorous and oxygen) bond of the DNA or RNA polymer is very stable, and the H₂O molecule is not usually very reactive. However, nucleases that use the hydrolysis mechanism make the H₂O reactive by removing one of the hydrogens to generate a highly reactive OH^- (hydroxyl). The negatively charged OH^- can then attack the P-O bond to cleave the polymer. An alternative mechanism used by some DNA repair endonucleases involves the initial cleavage of a C-O (carbon and oxygen) bond and subsequent P-O bond cleavage. This is called a lyase reaction and does not involve water.

Deoxyribonucleases in DNA Replication and Repair

During DNA synthesis the 3′ and 5′ exonucleases function to remove unwanted nucleotides from the DNA. Occasionally, a DNA polymerase will add an incorrect nucleotide to the growing DNA polymer. A 3′ exonuclease removes nucleotides that have been incorrectly polymerized into DNA chains. These exonucleases are referred to as "proofreading" exonucleases. The proofreading exonucleases work in close association with the DNA polymerases to increase the overall accuracy of DNA synthesis.

In many cases the exonuclease activity is contained in the same protein as the DNA polymerase activity. For example, the Escherichia coli DNA polymerase I is a single polypeptide with three separate domains, or regions of function. Each of these three domains contains an enzymatic activity. The DNA polymerase activity is in one domain, and the two other domains contain 3′ and 5′ exonuclease activities. The 3′ exonuclease proofreads for the DNA polymerase, and the 5′ exonuclease removes unwanted nucleotides in advance of the DNA polymerase. In contrast, the proofreading exonuclease of E. coli DNA polymerase III is located in a separate protein called the ε subunit, while the polymerase activity is contained on the α subunit. These two separate proteins, encoded by different genes, associate and interact in a complex to assure a high level of accuracy during DNA replication.

Multiple DNA repair pathways also use nucleases to restore the correct nucleotide base-pairing if it becomes altered during the life of the cell. Reactive molecules originating from inside the cell during normal metabolism or brought into the cell during exposure to external sources can damage the nucleotides of DNA. A damaged nucleotide opposite a normal nucleotide creates a distortion in the shape of the DNA double helix that is recognized by DNA repair proteins. A DNA helix distortion is also generated when normal but mismatched nucleotides are generated during DNA replication—for example, if a nucleotide is paired with C rather than A. Mismatches occur when DNA polymerases misinsert nucleotides and fail to proofread the misinserted base. These DNA helix distortions are repaired to minimize introduction of mutations into the genome. The steps in these DNA repair pathways include recognition of the distorted DNA, incision of the DNA by endonucleases on the 5′ or 3′ side of the damage, excision (removal) of nucleotides by exonucleases from the damaged region, and synthesis of a new DNA strand by a DNA polymerase. Some of the genes encoding the repair endonucleases and exonucleases have been identified in E. coli and in human cells, and the precise functions of these enzymes in cells are an active area of research.

The topoisomerases are a specialized class of nucleases functioning in cells to alter the topological structure of DNA. During replication the DNA becomes twisted, creating a barrier to progression of the DNA replication apparatus. The topoisomerases recognize these twisted regions of DNA and restore them to their untwisted state. This is accomplished by incising the DNA, removing the topological strain by unwinding, then resealing the DNA to regenerate the intact polymer. The Type I topoisomerases function by cutting one of the DNA strands. The Type II topoisomerases cut both DNA strands. The incision of DNA is transient, and both classes of topoisomerases reseal the DNA strands.

The restriction endonucleases are involved in the DNA restriction-modification systems of bacteria, which protect these cells from invading viruses. These enzymes have become powerful tools for DNA manipulation by molecular biologists. They recognize specific sequences in DNA and cut the DNA at these sites. The recognition sequences are usually between four and six nucleotides in length in duplex DNA. Each restriction enzyme has a different recognition sequence, making it possible to cut DNA in a variety of very predictable patterns.

Ribonucleases in RNA Maturation and Degradation

The expression of genes into protein products requires the generation of a messenger RNA (mRNA) by transcription and the subsequent translation of the mRNA into protein. In bacteria, the mRNA is transcribed, translated, and then degraded by ribonucleases in rapid succession. Thus, the ribonucleases are primarily responsible for mRNA degradation in bacteria. In animal cells, RNA molecules are transcribed as precursors that require processing by ribonucleases to generate functional RNAs. This RNA maturation process requires cleavage by endonucleases and trimming by exonucleases. After the mRNA is translated into protein it is degraded by additional ribonucleases.

see also Carcinogens; DNA Polymerases; DNA Repair; Mutation; Nucleotide; Restriction Enzymes.

Fred Perrino

Bibliography

Gerlt, John A. "Mechanistic Principles of Enzyme-Catalyzed Cleavage of Phosphodiester Bonds." In Nucleases, 2nd ed., Stuart M. Linn, R. Stephen Lloyd, and Richard J. Roberts, eds. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1993.

Inhalation of recombinant human DNA endonuclease breaks up sticky airway secretions in cystic fibrosis.