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DNA damage
DNA damage, due to normal metabolic processes inside the cell, occurs at a rate of 50,000 to 500,000 molecular lesions per cell per day. However, many more sources of damage can drive this number even higher. Whilst this constitutes only 0.0002% of the human genome of 3,000,000,000 (3 billion) bases, a single unrepaired lesion to a critical cancer related gene (such as a tumor suppressor gene) could have catastrophic consequences for the cell.
Nuclear versus mitochondrial DNA damage
In human, and eukaryotic cells in general, DNA is found in two cellular locations - inside the nucleus and inside the mitochondria (mitochondrial genetics). Nuclear DNA (nDNA) exists in large scale aggregate structures known as chromosomes which are composed of DNA wound up around bead-like proteins called histones. Whenever the cell needs to access the genetic information encoded in nDNA it will unravel the required section, read it, and then allow it to wind up once more in its protected conformation. In contrast, mitochondrial DNA (mtDNA) which is located inside mitochondria organelles, exists in single or multiple copies of a circular loop without any histone association. Consequently, mtDNA is far more prone to damage than nDNA because it lacks the structural protection afforded by histone proteins. In addition, the highly oxidative environment inside mitochondria that exists due to the constant production of adenosine triphosphate (ATP) makes mtDNA even more prone to damage. Even though human mtDNA encodes only 13 proteins, a malfunctioning mitochondrion can activate apoptosis.
Sources of damage
DNA damage can be subdivided into two main types:
- • endogenous processes such as attack by reactive oxygen radicals produced from normal metabolic byproducts (spontaneous mutation);
- • agents of environmental origin, such as
- - ultraviolet (UV) radiation from the sun
- - other radiation frequencies, including x-rays and gamma rays
- - certain plant toxins
- - human-made mutagenic chemicals, such as hydrocarbons from cigarette smoke
- - cancer chemotherapy and radiotherapy
Replication of damaged DNA can lead to the incorporation of the wrong base opposite the damaged one. This "incorrect" base is now fixed in the next generation cell, permanently changing the DNA sequence. This change in sequence is a mutation.
Types of damage
Endogenous damage affects the primary rather than secondary structure of the double helix. It can be subdivided into four classes:
- • oxidation of bases [e.g. 8-oxo-7,8-dihydroguanine (8-oxoG)] and generation of DNA strand interruptions from reactive oxygen species,
- • methylation of bases, such as formation of 7-methylguanine
- • hydrolysis of bases, such as depurination and depyrimidination.
- • mismatch of bases, due to DNA replication in which the wrong DNA base is stitched into place in a newly forming DNA strand.
DNA repair mechanisms
The cell cannot tolerate DNA damage as it interferes with the integrity and accessibility of the information encoded in its genome. Depending on the type of damage inflicted on the DNA's double helical structure, a variety of repair strategies have evolved to replace the lost information. The information to be replaced must be made available either by an intact version from the complementary strand of DNA or from the sister chromosome. Without access to this information repair cannot take place.
Damaged DNA results in an altered configuration of the molecule which can be rapidly detected by the cell. Specific DNA repair related molecules are attracted to and bind at or near the site of damage inducing other molecules also to bind and form a complex that enables repair to take place. The types of molecules involved and the mechanism of repair that takes place is based on:
- • the type of damage on the DNA molecule
- • whether the cell has entered into a state of senescence
- • the phase of the cell cycle that the cell is in
Single strand damage
In order to repair of damage to one of the two helical domains of DNA, there are numerous mechanisms by which DNA repair can take place. These include:
- • direct reversal of damage are specialized mechanisms for reversing of one specific type of damage. Example include methyl guanine methyl transferase (MGMT) which specifically removes methyl groups from guanine or photolyase in bacteria, which break the chemical bond created by UV light between adjacent thymidine bases.
- • Excision repair mechanisms in which the damaged nucleotide is removed and an undamaged nucleotide put back in by using the information from the undamaged copy. These include:
- - base excision repair (BER), which repairs damage due to alkylation or deamination;
- - nucleotide excision repair (NER), which largely repairs bulky, helix distorting damage, including damage caused by UV light; and
- - mismatch repair (MMR), which corrects errors of DNA replication and recombination
- • single strand break repair , which rejoins interruptions in a single strand of the DNA chain caused by oxidation.
Double strand breaks
A particularly hazardous type of DNA damage to dividing cells is a break to both strands in the double helix. There are two mechanisms that exist to repair this damage. They are generally known as homologous recombination and Non-Homologous End-Joining.
Homologous recombination utilizes a source of identical or nearly identical sequence in the genome as a template for repair of the break. This mechanism is believed to be predominantly used during the phases of the cell cycle when the DNA is replicating or has completed replicating it's DNA. This allows a damaged chromosome to be repaired using the newly created sister chromatid which is an identical copy. The human genome is highly repetitive and contains many possible sources of identical sequences. Recombination with these other sequences can be very harmful since the crossing over which may result, can cause a chromosomal translocation or other chromosome rearrangements. The enzymatic machinery responsible for this repair process is nearly identical to the machinery responsible for chromosomal crossover in germ cells during meiosis.
Non-Homologous End-Joining (NHEJ) essentially rejoins the two ends of the break, however there is often DNA sequence lost during this process and so this repair can be mutagenic. NHEJ can occur at all stages of the cell cycle but is predominant before DNA replication when homologous recombination with the sister chromatid is not yet available. Since the vast majority of the genome in humans and other multicellular organisms is made up of DNA which are not genes, the so-called "junk DNA", this mutagenic repair is less likely to be harmful to a cell than homologous recombination with sequences other than the sister chromatid. The enzymatic machinery used for NHEJ is also utilized in B-cells to rejoin breaks created by the RAG proteins during VDJ recombination in the generation of antibodies in the immune system.
DNA repair in disease and aging
Poor DNA repair induces pathology
As cells get older the amount of DNA damage accumulates overtaking the rate of repair and resulting in a reduction of protein synthesis. As proteins in the cell are used for numerous vital functions the cell becomes slowly impaired and eventually dies. When enough cells in an organ reach such a state the organ itself will become compromised and the symptoms of disease begin to manifest. Experimental studies in animals, where genes associated with DNA repair were silenced, resulted in accelerated aging, early manifestation of age related diseases and increased susceptibility to cancer. In studies where the expression of certain DNA repair genes was increased resulted in extended lifespan and resistance to carcinogenic agents in cultured cells.
DNA repair rate is variable
If the rate of DNA damage exceeds the capacity of the cell to repair it, the accumulation of errors can overwhelm the cell and result in senescence, apoptosis or cancer. Inherited diseases associated with faulty DNA repair functioning result in premature aging (e.g. Werner's syndrome) and increased sensitivity to carcinogens (e.g Xeroderma Pigmentosum). Studies in animals, where DNA repair genes are prevented from functioning, show similar disease profiles.
On the other hand, organisms with enhanced DNA repair systems, such as Deinococcus radiodurans (also known as "Conan the bacterium", listed in the Guinness Book of World Records as "the world's toughest bacterium"), exhibit remarkable resistance to lethal dosages of radioactivity, because their DNA repair enzymes are able to perform at unusually fast rates to keep up with radiation induced-damage, and because it carries 4–10 copies of the genome. In human studies, Japanese centenarians have been found to have a common mitochondrial genotype, which predisposes them to reduced DNA damage in their mitochondria.
Studies in smokers have found that, for people with a mutation that causes them to express less of the powerful DNA repair gene hOGG1, their vulnerability to lung and other smoking related cancers are increased. Single nucleotide polymorphisms (SNP) associated with this mutation can be clinically detected.
Hereditary DNA repair disorders
Defects in the NER mechanism are responsible for several genetic disorders, including:
- • xeroderma pigmentosum: hypersensitivity to sunlight/UV, resulting in increased skin cancer, incidence and premature aging
- • Cockayne syndrome: hypersensitivity to UV and chemical agents
- • trichothiodystrophy: sensitive skin, brittle hair and nails
Mental retardation often accompanies the latter two disorders, suggesting increased vulnerability of developmental neurons.
Other DNA repair disorders include:
- • Werner's syndrome: premature aging and retarded growth
- • Bloom's syndrome: sunlight hypersensitivity, high incidence of malignancies
- • ataxia telangiectasia: sensitivity to ionizing radiation and some chemical agents
Other diseases associated with reduced DNA repair function include, hereditary breast cancer and hereditary colon cancer.
Chronic DNA repair disorders
Chronic disease can be associated with increased DNA damage. For example, smoking cigarettes causes oxidative damage to the DNA and other components of heart and lung cells, resulting in the formation of DNA adducts (molecules that disrupt DNA). DNA damage has now been shown to be a causative factor in diseases from atherosclerosis to Alzheimer's, where patients have a lesser capacity for DNA repair in their brain cells. Mitochondrial DNA damage has also been implicated in numerous disorders.
Longevity genes and DNA repair
Certain genes are known to influence variation in lifespan within a population of organisms. Studies in model organisms such as yeast, worms, flies and mice have identified single genes, which when modified, can double lifespan (eg. a mutation in the age-1 gene of the nematode Caenorhabditis elegans). These genes are known to be associated specifically with cell functions other than DNA repair, but when the pathways that they influence are followed to their final destination, it was observed that they mediate one of three functions:
- • increasing the rate of DNA repair,
- • increasing the rate of antioxidant production, or
- • decreasing the rate of oxidant production.
Therefore, the common pattern across most lifespan influencing genes is in their downstream effect of altering the rate of DNA damage.
Caloric restriction increases DNA repair
Caloric restriction (CR) has been shown to increase lifespan and decrease age related disease in all organisms where it has been studied, from single celled life such as yeast, to multicellular organisms such as worms, flies, mice and primates. The mechanism by which CR works is associated with a number of genes related to nutrient sensing which signal the cell to alter metabolic activity when there is a shortage of nutrients, particularly carbohydrates. When the cell senses a decrease in carbohydrate availability, activation of the lifespan influencing genes DAF-2, AGE-1 and SIR-2 (see accompanying illustration "Most lifespan influencing genes affect the rate of DNA damage") is triggered.
The reason why a shortage of nutrients, will induce in a cell a state of increased DNA repair and an increase in lifespan is suggested to be associated with an evolutionarily conserved mechanism of cellular hibernation. Essentially this permits a cell to maintain a dormant state until more favorable conditions are met. During this period, the cell must decrease its normal rate of metabolism and one of the ways it can accomplish this is by reducing genomic instability. Thus the cellular rate of aging is mutable and can be influenced by environmental factors such as nutrient availability which mediate their effect by altering the rate of DNA repair.