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Nucleases - Deoxyribonucleases In Dna Replication And Repair

polymerase nucleotides exonuclease exonucleases

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 Hydrolysis reaction. 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.

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