Course: SAT 170 Introductory Biotechnology

Unit 3 Reading and Multimedia Assignment

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Page 12 of 13
Attributions
Excerpt from “Biology: Boundless Biology Open Textbook”, CC BY SA 3.0

https://www.boundless.com/biology/dna/
Introduction to DNA Repair and Proofreading
While DNA replication errors are rare, they must be corrected by various DNA repair pathways.
In general, DNA replication is extremely accurate because cellular proofreading and error correcting mechanisms ensure the near perfect outcome for DNA replication. Replicated DNA molecules have an error rate of one nucleotide for every 10 billion (1010) nucleotides added. However, the initial addition of nucleotides by DNA polymerase has an error rate of one per 105 (100,000) nucleotides added.

Error correction is a property of some DNA polymerases. This process corrects mistakes in newly-synthesized DNA. When an incorrect base pair is recognized, DNA polymerase reverses its direction by one base pair of DNA and excises or clips off the incorrect base. Following base excision, the polymerase can reinsert the correct base and replication can continue. This activity is known as proofreading.

Proofreading occurs during DNA replication because some DNA polymerases have 3'->5' exonuclease activity that allows an incorrect base to be excised. A nuclease is an enzyme capable of cleaving the phosphodiester bonds between the nucleotide subunits. Nucleases are usually further divided into endonucleases and exonucleases, although some of the enzymes may fall into both categories. Exonucleases are enzymes that work by cleaving nucleotides one at a time from the end (exo) of a polynucleotide chain.

Nucleotide errors not caught and corrected by DNA polymerase proofreading need alternative methods of correction. DNA mismatch repair is a system for recognizing and repairing erroneous insertion, deletion, and misincorporation of bases that can arise during DNA replication and recombination, as well as repairing some forms of DNA damage.

DNA damage, due to environmental factors and normal metabolic processes inside the cell, occurs at a rate of 1,000 to 1,000,000 molecular lesions per cell per day. While this constitutes only 0.000165% of the human genome's approximately 6 billion bases (3 billion base pairs), unrepaired lesions in critical genes (such as tumor suppressor genes) can impede a cell's ability to carry out its function and increase the likelihood of tumor formation. The vast majority of DNA damage affects the primary structure of the double helix; the bases themselves are chemically modified. These modifications can in turn disrupt the molecules' regular helical structure by introducing non-native chemical bonds or bulky adducts that do not fit in the standard double helix.

Agents known to cause DNA damage include:
• Certain wavelengths of ionizing radiation such as gamma rays and X-rays
• Ultraviolet rays, especially the UV-C rays (~260 nm) that are absorbed strongly by DNA but also the longer-wavelength UV-B that penetrates the ozone shield
• Highly-reactive oxygen radicals produced during normal cellular respiration, as well as by other biochemical pathways
• Many hydrocarbons, including some found in cigarette smoke
• Some plant and microbial products, e.g., the aflatoxins produced in moldy peanuts
• Chemicals used in chemotherapy, especially chemotherapy of cancers

DNA in the living cell is subject to many chemical alterations, a fact often forgotten in the excitement of being able to do DNA sequencing on dried and/or frozen specimens. If the genetic information encoded in the DNA is to remain uncorrupted, any chemical changes must be corrected. A failure to repair DNA produces a mutation. Defects in mismatch repair can allow DNA errors to accumulate, sometimes leading to tumors and cancers. Nearly 100 DNA repair enzymes have been identified in bacteria (E. coli). The recent publication of the human genome has already revealed 130 genes whose products participate in DNA repair. More will probably be identified soon.

Some of these repair enzymes are involved in the mismatch repair system. Others are associated with nucleotide excision repair (NER), a particularly important mechanism by which the cell can prevent unwanted mutations by removing the vast majority of UV-induced DNA damage (mostly in the form of thymine dimersand 6-4-photoproducts). The importance of this repair mechanism is evidenced by the severe human diseases that result from in-born genetic mutations of NER proteins, including Xeroderma pigmentosum and Cockayne's syndrome. While the base excision repair (BER) machinery can recognize specific lesions in the DNA, it can correct only damaged bases that can be removed by a specific enzyme; in contrast, the nucleotide excision repair enzymes recognize bulky distortions in the shape of the DNA double helix. Recognition of these distortions leads to the removal of a short single-stranded DNA segment that includes the lesion, creating a single-strand gap in the DNA. This is subsequently filled in by DNA polymerase, which uses the undamaged strand as a template. Ligase finishes the repair by sealing the newly repaired section to the original DNA strand.