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In this report we will be looking at various mechanisms which have evolved in mammalian cells to limit the damaging effects of the oxidised base 8-oxogaunine to genetic integrity. DNA damage is caused by reactive oxygen species and is present in all living organisms. Oxygen is required for human existence and is essential for many different processes in the body. Oxygen is often viewed as having no harmful effects however the by products caused during metabolic processes, such as respiration, are usually referred to as reactive oxygen species (ROS), which can also be formed by inflammatory response and the metabolism of xenobiotics, and can pose a major threat to the genetic integrity of cells which can then lead to cellular damage which, if unrepaired can promote diseases such as cancer, neurodegenerative disorders, atherosclerosis and likely becoming a contributing factor to the ageing process .
8-oxogaunine is an important pre-mutagenic base in oxidatively damaged DNA which is produced during metabolic processes in the body. In normal circumstances phagocytic cells, specifically neutrophills and macrophages, are activated and a burst of reactive oxygen and nitrogen chemicals form a cytoxic armoury against invading organisms. These reactive by products have the ability to attack most cellular components which consist of proteins, lipids, carbohydrates and DNA which will be our main focus. If these go unrepaired as mentioned earlier it could result in cellular damage. Modified proteins and lipids can go through the process of being degraded and resynthasised where as DNA has to be repaired before replication and cell division takes place.
The most important reactive oxygen species have been categorised as super oxide, hydrogen peroxide and hydroxyl radical. These chemicals reacting with DNA can lead to the formation of many intermediates of oxidative damage such as modified bases and typical single strand breaks. Persistence of these damages can assist unwanted genetic change which, in turn, is another cause of cell death and also the formation of cancer. This is because ROS are genotoxic and therefore generates strand breaks and an excess of damaged bases in DNA. As a result organisms have evolved multiple repair systems so that DNA can return back to it unmodified state. The interactions of ROS with DNA have been researched and a pattern that has been noticed is that G-C T-A transversions are the most commonly found mutations resulting from oxidative damage to DNA.
DNA has the potential of being either cytotoxic or mutagenic however damage is dependant on the repair capacity of the cell. This means that cells that have insufficient repair facilities or are not able to undergo the repair mechanisms will result in persistent DNA damage. As mentioned earlier this will promote genetic mutation or cell death. Cells that regulate growth or differentiation can affect the organism's natural biology and therefore promote disease by damage induced mutations at loci.
7,8-dihydro-8-oxogaunine (8-oxoG) is a result of an oxidative DNA modification
which as mentioned earlier is pre- mutagenic and gives rise to G-C T-A transversions. 8-oxoG is generated in high quantities involving the reaction of all oxidants with DNA. Guanine has the lowest oxidation potential out of the four nucleobases therefore it the most easily oxidised. 8-oxoG is a result of undergoing hydroxyl radical attack of the 8' position imidazoyl ring on guanine. The 8-oxoG residue exists mainly in its keto form at physiological PH. This results in the normal anti- conformation around the N-glycosylic bond which forms a Watson-crick base pair with cytosine. When paired with adenine 8-oxoG forms a syn conformation around the N-glycolsylic bond which forms a stable hoogsteen mispair which contains two hydrogen bonds. This provides the structural basis G-C T-A transversion which is associated with base modifications. After undergoing infa-red spectroscopy it has been revealed that 8-oxoG promotes unique local dynamics in duplex DNA which is an important feature in terms of biological handling of this lesion. 8-oxoG is more receptive to oxidative attack than guanine alone. After analysing Nuclear magnetic resonance (NMR) it showed that 8-oxoG was able to form a thermodynamically stable hoogsteen mispair with guanine which was a big indication to the ability of G-C T-A transversions being induced.
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8-oxoG glycosylase (OGG1) is categorised into the helix-harpin-helix (HhH) super family of DNA binding proteins. This is the enzyme that has a major role in humans and is responsible for the repair of 8-oxoG in non-replicating DNA for example when the lesion is positioned opposite a cytosine. OGG1 can exist primarily in two iso forms which arise from alternative joining. The first being a smaller alpha (~39kDa) form which is localised mainly in the nucleus and the second being a beta form (~47kDa) which is found mainly in the inner membrane of the mitochondria. Nuclear OGG1, which has the same active core site found in the mitochondrial protein but has a different C terminal end, is a bifunctional DNA glycosylase. This means that not only does it excise the substrate base but incises the DNA backbone 3' to the resulting AP site via AP lyase activity that involves a beta elimination reaction.
There are several repair mechanisms which have evolved in mammalian cells to limit the damaging effects such as the Base Excision Repair (BER) pathway, nucleotide excision repair (NER) and DNA mismatch repair (MMR).
BER, up to now, has been the simplest and most defined of all repair processes. However, deficiencies in this repair process have been linked to cancer and other diseases. BER is a cellular pathway that has the role of removing damaged nucleobases from the genome. BER appears to be the simplest repair process in comparison to others. It requires five distinct enzymatic activities in the basic reaction steps. The most important step being the excision of damaged bases from the DNA by a class of enzymes called DNA glycoslylases these are enzymes that organize irregular bases in DNA and catalyze their removal through glycosidic-bond cleavage.
Glycosylases are small proteins that do not require ATP for activity to take place. Research involving X-ray crystallography has shown that many glycosylases have a base flipping process. This averts the target nucleobase from the helix into an active site pocket on the enzyme. It enables the damaged nucleobase to be more accessible to the various side-chain functions in the enzyme that contributes to catalysis and substrate discrimination.
BER pathway involves several proteins that act to remove a single damaged nucleobase from DNA and it replaces it with the correct undamaged nucleotide. In the BER pathway the role of glycosylases is to recognise the damaged nucleobase and as mentioned before catalyze the hydrolysis of the glycosydic bond in order to release the adduct.
Studies have shown that BER in mammalian cells have revealed the presence of two pathways that are distinctive. The pathways are according to the proteins required and the intermediate products.
The repair is completed in several subsequent steps starting with cleavage of the DNA backbone by AP endonuclease (APE), or by the intrinsic AP lyase activity which is associated with oxidized base-specific DNA glycosylases. The strand cleavage by APE generates 3'-OH and 5' deoxyribose phosphate (dRP) termini, while the AP lyases produce 3' blocking phosphoribose or phosphate termini together with 5' phosphates. The later step involves DNA polymerase β which has intrinsic dRP lyase activity, this carries out both 5' cleaning and DNA repair synthesis incorporating the suitable nucleotide at the site of the base damage. The closing step results in the nick being sealed by DNA ligase which in turn completes the repair.
As mentioned earlier there are two distinct pathways involved in BER, which are the short patch repair, and long patch repair pathway.
The short patch can take place by either a monofunctional or bio functional DNA glycosylase initiated event. The repair takes place after the initial step that is performed by the DNA glycosylase which has been discussed in the earlier paragraph; the resynthesis of DNA polymerase β followed by ligation by ligase iii. The protein XRCC1 protein is also involved in this pathway even though the function of this protein is not known.
The long patch repair differs to the short patch as it involves different polymerases and ligase. It also requires a structure specific nuclease (FEN1) as well as PCNA dependent DNA polymerase such as POLÎ or POLδ these execute strand displacement synthesis. FEN1 then excises the 5' flap DNA intermediate. It generates a resynthesis patch of 3 to 6 nucleotides.