Although DNA synthesis happens under strict regulations (Proofreading system), however; faults can still take place and have to be corrected. Otherwise; they will be converted to permanent mutations and when inherited will end with a number of serious drawbacks mainly cancers.
Types of DNA Repairs
A. Methyl-directed mismatch repair (nucleotide excision repair): Sometimes replication errors escape the proofreading function of DNA polymerases during DNA synthesis, causing a mismatch of one to several bases. In E. coli, mismatch repair is mediated by a group of proteins known as the Mut proteins, analogous proteins are present in humans.Note: Mutation to the proteins involved in mismatch repair in humans is associated with hereditary nonpolyposis colorectal cancer (HNPCC), also known as Lynch syndrome. Only about 5% of all colon cancers is the result of mutations in mismatch repair.
An adenocarcinoma involving the cecum and ileo-caecal valve in HNPCC patient on surgery
B. Repair of damage caused by ultraviolet (UV) lightExposure of a cell to UV light can result in the covalent joining of two adjacent pyrimidines (usually thymines), producing a dimer. These thymine dimers prevent DNA polymerase from replicating the DNA strand beyond the site of dimer. Such dimers will be excised in bacteria and a related pathway occurs in humans.
Pyrimidine dimers can be formed in the skin cells of humans exposed to unfiltered sunlight and normally corrected, but in a rare genetic disease xeroderma pigmentosum, there will be hereditary defects in the genes responsible for producing enzymes that correct the UV- produced dimers mainly (UV specific endonuclease or UV rABC exonuclease) leading to accumulation of mutations and subsequently cancers.
C. Correction of base alterations (base excision repair)
The bases of DNA can be altered, either spontaneously, as is the case with cytosine, which slowly undergoes deamination to form uracil, or by the action of deaminating or alkylating compounds. For example, nitrous acid, which is formed in the cell from precursors, such as the nitrosamines, nitrites, and nitrates, is a potent compound that deaminates cytosine, adenine, and guanine. Moreover, bases can also be lost spontaneously. For example, approximately 10,000 purine bases are lost this way per cell per day. Lesions involving base alterations or loss can be corrected by base excision repair.Abnormal bases, such as uracil, which can occur either by deamination of cytosine or improper use of dUTP instead of dTTP during DNA synthesis, are recognized by base specific glycosylases that hydrolytically cleave them from the deoxyribose–phosphate backbone of the strand e.g (Uracil-N-glucosylase). This leaves an AP site (apyrimidinic site or apurinic).
Later on, Specific AP-endonucleases recognize that there is a missing base and initiate the process of excision and gap-filling by making an endonucleolytic cut just to the 5′-side of the AP site. Soon after this, a “deoxyribose phosphate lyase” removes the single, empty, sugar phosphate residue. Finally, a DNA polymerase and DNA ligase complex complete the repair process.
D. Repair of double-strand breaks
High-energy radiation and oxidative free radicals can cause potentially cell lethal double-stranded DNA breaks which lead to produce 2 separate fragments of DNA double stranded helix. Such cut is corrected by two systems, either: nonhomologous end-joining repair, this mechanism of repair is error prone and mutagenic and associated with a predisposition to cancer and immunodeficiency syndromes. homologous recombination repair, uses the enzymes that normally perform genetic recombination between homologous chromosomes during meiosis. This system is much less error prone than nonhomologous end-joining.Biotechnology and Human Diseases
Recombinant DNA Technologyالهندسة الوراثيةIn the past, efforts to understand genes and their expression have failed due to the large size and extreme complexity of human genomic DNA. The Human Genome Project has enabled us to determine the nucleotide sequence of long DNA stretches and even the entire human genome sequence. Thus it has greatly contributed to our understanding of many genetic diseases and it produced several methods for prenatal diagnosis of genetic diseases and lead to initial successes in the treatment of patients using “Gene Therapy”. Several techniques are used in recombinant DNA technology, including at least 3 right now.
I. Restriction Endonucleases The discovery of a special group of bacterial enzymes, called restriction endonucleases (restriction enzymes), which cleave double-stranded (ds) DNA into smaller, more manageable fragments, has opened the way for DNA analysis. Because each enzyme cleaves DNA at a specific nucleotide sequence, restriction enzymes are used experimentally to obtain precisely defined DNA segments called restriction fragments.
Restriction endonucleases recognize short (4-6 base sequences) stretches of DNA that contain specific nucleotide sequences-known as the “recognition sites”. These sequences which differ for each restriction endonuclease, are palindromes, that is, they exhibit twofold rotational symmetry. This means that, within a short region of the double helix, the nucleotide sequence on the “top” strand, read 5′→3′, is identical to that of the “bottom” strand, also read in the 5′→3′ direction.
There are hundreds of restriction enzymes having different cleavage specificities. They are commercially available as valuable analytical reagents.Generally, there are four classes of Res based on their composition, restriction sites, sequence specificity& co-factors needed.Example: HgaI belongs to Class II. 5ˊ- G A C G C (N) 5 …3ˊ 3ˊ- C T G C G (N) 10 …5ˊ
https://www.id-press.eu/mjms/article/view/oamjms.2018.156
II. DNA Cloning
It implies the insertion or the introduction of a foreign DNA molecule into a replicating cell leading to its amplification i.e. production of too many copies of that foreign molecule. The DNA segment of interest is first cleaved with a specific restriction enzyme, and then joined to a DNA molecule called as cloning vector to form a hybrid or a recombinant DNA molecule. This process leads to create hundreds of thousands of fragments. Each recombinant DNA molecule conveys its inserted DNA fragment into a single host cell, for example, a bacterium, where it is replicated (or “amplified” or “cloned”).The process of introducing foreign DNA into a cell is called transformation for bacteria and transfection for eukaryotes. As the host cell multiplies, it forms a clone in which every bacterium carries copies of the same inserted DNA fragment, hence the name “cloning.” The cloned DNA is eventually released from its vector by cleavage using the appropriate restriction endonuclease and is isolated. By this mechanism, many identical copies of the DNA of interest can be produced.
Vectors It is a molecule of DNA to which the fragment of DNA to be cloned is joined. Essential properties of a vector include: 1) It must be capable of autonomous replication within a host cell. 2) it must contain at least one specific nucleotide sequence recognized by a restriction endonuclease.3) it must carry at least one gene that confers the ability to select for the vector, e.g. an antibiotic resistance gene& β- galactosidase gene.
Commonly used vectors include plasmids and bacterial and animal viruses(eg. Bacteriophages) Prokaryotic plasmids: In addition to the single circular large chromosome of prokaryotes, there are normally other small circular extrachromosomal DNA molecules called plasmids. Plasmid DNA undergoes replication that may or may not be synchronized to chromosomal division. Plasmids may carry genes that convey antibiotic resistance to the host bacterium, and may facilitate the transfer of genetic information from one bacterium to another.
Bacteria are grown in the presence of antibiotics (most commonly Ampicillin and Tetracyclin), thus selecting for cells containing the hybrid plasmids, which provide antibiotic resistance. Plasmids can be readily isolated from bacterial cells, their circular DNA cleaved at specific sites by restriction endonucleases, and foreign DNA inserted. The hybrid plasmid can be reintroduced into a bacterium, and large numbers of copies of the plasmid containing the foreign DNA produced.
2. Other vectors
In addition to prokaryotic plasmids, new improved vectors have been developed that more efficiently accommodate larger DNA fragments or express the passenger gene in different cell types like: naturally occurring viruses that infect bacteria (e.g. bacteriophage) or infect mammalian tissues (e.g. retroviruses) artificial constructs like cosmids and bacterial or yeast artificial chromosomes that are all in wide use nowadays as cloning vectors.Example: Human insulin is nowadys used for treating type 1 diabetes mellitus. It was the first commercial product created by recombinant DNA technology developed by Genentech- the first American biotechnology company in 1982. Prior to its development, diabetics used insulin isolated from pigs and cows.