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This lecture covers
- What is DNA damage?
- How it is happened?
- How it is different from mutation?
- How does the DNA damage get repaired?
DNA damage
DNA damage
DNA damage refers the modification of base pairs (especially the nitrogenous bases) and affects the DNA double helix as well as the gene’s expressions. The damage can be either due to irradiation and harmful chemicals (exogenous damages) or due to metabolic byproducts of their own cellular activities (endogenous damages).
Endogenous damages
The cellular metabolic activities produce several reactive oxygen species (includes singlet oxygen, peroide; super oxides etc) which attack the DNA and causes oxidative deamination (amine groups reacts with these species and removed from the base pairs in the form of ammonia). This internal damage is referred as endogenous damage.
The replication errors made by PolI enzyme during DNA replication (change of 1/million) also a type of endogenous DNA damage. The replication of damaged DNA before cell division can lead to the incorporation of wrong bases opposite damaged ones. Daughter cells that inherit these wrong bases carry mutations from which the original DNA sequence is unrecoverable.
Exogenous damages
- The external sources that cause damage to the DNA include:
- ultraviolet (200-400 nm) radiation from the sun
- Other radiation frequencies, including x-rays and gamma rays
- Hydrolysis or thermal disruption
- Certain plant toxins
- Human-made mutagenic chemicals, especially aromatic compounds that act as DNA intercalating agents
- Viruses
Types of damage
Types of damage
There are five main types of damage to DNA due to endogenous cellular processes:
1. Oxidation of bases [e.g. 8-oxo-7,8-dihydroguanine (8-oxoG)] and generation of DNA strand interruptions from reactive oxygen species.
2. Alkylation of bases (usually methylation), such as formation of 7-methylguanine, 1-methyladenine, 6-O-Methylguanine
3. Hydrolysis of bases, such as deamination, depurination, and depyrimidination.
4. Bulky adduct formation refers to the DNA covalently bound to a chemical. Examples: benzo[a]pyrenediol epoxide-dG adduct, aristolactam I-dA adduct.
5. Mismatch of bases, due to errors in DNA replication, in which the wrong DNA base is stitched into place in a newly forming DNA strand, or a DNA base is skipped over or mistakenly inserted.
Some example exogenous DNA damages are:
a. UV-B light causes crosslinking between adjacent cytosine and thymine bases creating pyrimidine dimers. This is called direct DNA damage.
b. UV-A light creates mostly free radicals. The damage caused by free radicals is called indirect DNA damage.
c. Ionizing radiation such as that created by radioactive decay or in cosmic rays causes breaks in DNA strands. Low-level ionizing radiation may induce irreparable DNA damage (leading to replicational and transcriptional errors needed for neoplasia or may trigger viral interactions) leading to pre-mature aging and cancer.
d. Thermal disruption at elevated temperature increases the rate of depurination (loss of purine bases from the DNA backbone) and single-strand breaks. For example, hydrolytic depurination is seen in thethermophilic bacteria, which grow in hot springs at 40-80 °C.
e. Industrial chemicals such as vinyl chloride and hydrogen peroxide, and environmental chemicals such as polycyclic aromatic hydrocarbons found in smoke, soot and tar create a huge diversity of DNA adducts- ethenobases, oxidized bases, alkylated phosphotriesters and Cross-linking of DNA.
UV damage, alkylation/methylation, X-ray damage and oxidative damage are examples of induced damage. Spontaneous damage can include the loss of a base, deamination, sugar ring puckering and tautomeric shift.
DNA damage and mutation
DNA damage and mutation
DNA damage and mutation are two types of errors in DNA which are fundamentally different. Hence, it is important to distinguish between damage and repair.
Damages are physical abnormalities in the DNA, such as single- and double-strand breaks, 8-hydroxydeoxyguanosine residues, and polycyclic aromatic hydrocarbon adducts. DNA damages can be recognized by enzymes, and, thus, they can be correctly repaired if redundant information, such as the undamaged sequence in the complementary DNA strand or in a homologous chromosome, is available for copying. If a cell retains DNA damage, transcription of a gene can be prevented, and, thus, translation into a protein will also be blocked. Replication may also be blocked and/or the cell may die.
In contrast to DNA damage, a mutation is a change in the base sequence of the DNA. A mutation cannot be recognized by enzymes once the base change is present in both DNA strands, and, thus, a mutation cannot be repaired. At the cellular level, mutations can cause alterations in protein function and regulation. Mutations are replicated when the cell replicates. In a population of cells, mutant cells will increase or decrease in frequency according to the effects of the mutation on the ability of the cell to survive and reproduce. Although distinctly different from each other, DNA damages and mutations are related because DNA damages often cause errors of DNA synthesis during replication or repair; these errors are a major source of mutation.
Hence, DNA damages are a special problem in non-dividing or slowly dividing cells, where unrepaired damages will tend to accumulate over time. On the other hand, in the rapidly dividing cells, unrepaired DNA damages that do not kill the cell by blocking replication will tend to cause replication errors and thus mutation.
Lecture about DNA damage (Youtube)
Repair mechanisms
Repair mechanisms
DNA repair refers to a collection of processes by which a cell identifies and corrects damage to the DNA molecules that encode its genome. The rate of DNA repair is dependent on many factors, including the cell type, the age of the cell, and the extracellular environment. The DNA repair ability of a cell is vital to the integrity of its genome and thus to its normal functioning and that of the organism.
Methylation as protective mechanism for DNA
N base methylation is part of the restriction modification system of many bacteria, in which specific DNA sequences are methylated periodically throughout the genome. A methylase is the enzyme that recognizes a specific sequence and methylates one of the bases in or near that sequence. Foreign DNAs (which are not methylated in this manner) that are introduced into the cell are degraded by sequence-specific restriction enzymes and cleaved. Bacterial genomic DNA is not recognized by these restriction enzymes. The methylation of native DNA acts as a sort of primitive immune system, allowing the bacteria to protect themselves from infection by bacteriophage.
E. coli DNA adenine methyl transferase (Dam) is an enzyme of ~32 kDa that does not belong to a restriction/modification system. The target recognition sequence for E. coli Dam is GATC, as the methylation occurs at the N6 position of the adenine in this sequence (G meATC). Dam plays several key roles in bacterial processes, including mismatch repair, the timing of DNA replication, and gene expression. As a result of DNA replication, the status of GATC sites in the E. coli genome changes from fully methylated to hemimethylated. This is because adenine introduced into the new DNA strand is unmethylated. Re-methylation occurs within two to four seconds, during which time replication errors in the new strand are repaired. Methylation, or its absence, is the marker that allows the repair apparatus of the cell to differentiate between the template and nascent strands. It has been shown that altering Dam activity in bacteria results in increased spontaneous mutation rate. Bacterial viability is compromised in dam mutants that also lack certain other DNA repair enzymes, providing further evidence for the role of Dam in DNA repair.
Introduction to DNA repair mechanisms (Youtube)
Types of repair mechanisms found in prokaryotes:
In the presence of sun light, the photoreactivation repair mechanism is common in E. coli. In dark, excision repair, recombination repair and SOS repair are the three types of repairs found in E. coli. In this chapter, a brief mechanism of each repair system is depicted.
1. Photoreactivation
1. Photoreactivation
Photolyasesare DNA repair enzymes that repair damage caused by exposure to ultraviolet light. This enzyme mechanism requires visible light, preferentially from the violet/blue end of the spectrum, and is known as photoreactivation.
Photolyase is a phylogenetically old enzyme which is present and functional in many species, from the bacteria to the fungi to the animals.However it is no longer working in humans and other placental mammals who instead rely on the less efficient nucleotide excision repair mechanism.
Photolyases bind complementary DNA strands and break certain types of pyrimidine dimers that arise when a pair of thymine or cytosine bases on the same strand of DNA become covalently linked. These dimers result in a 'bulge' of the DNA structure, referred to as a lesion. The more common covalent linkage involves the formation of a cyclobutane bridge. Photolyases have a high affinity for these lesions and reversibly bind and convert them back to the original bases.
Watch a detailed lecture on Photoreactivation (Youtube)
2. Excision repair
2. Excision repair
When only one of the two strands of a double helix has a defect, the other strand can be used as a template to guide the correction of the damaged strand. In order to repair damage to one of the two paired molecules of DNA, there exist a number of excision repair mechanisms that remove the damaged nucleotide and replace it with an undamaged nucleotide complementary to that found in the undamaged DNA strand.
The excision repair, can repair the damages to a single base caused by oxidation, alkylation, hydrolysis, or deamination. The damaged base is removed by a DNA glycosylase. The "missing tooth" is then recognized by an enzyme called AP endonuclease, which cuts the Phosphodiester bond. The missing part is then resynthesized by a DNA polymerase, and a DNA ligase performs the final nick-sealing step.
How excision repair takes place
3. Recombinational repair
3. Recombinational repair
This type of repair is common in UV damaged E. coli. The UV induced thymine dimers blocks the DNA replication. However, the replication fork bypass the dimer block and initiate the chain growth, known as postdimer initiation. During this process, restarting DNA synthesis occur in one strand and from the daughter strand, by the process of recombination, the excision of dimers and thereby the repair occur. The essential idea in sister strand exchange is that a single segment free from any defects is excised from the homologous DNA segment and inserted into the gap produced opposite the thymine dimers. This genetic recominational repair requires RecA protein. In contrast to excision repair, the recombinational repair occurs after the DNA replication, hence it has been called as post-replicational repair.
View animation about the repair mechanism (Youtube)
4. SOS repair
4. SOS repair
Some types of DNA damage, especially large-scale damage from highly mutagenic chemicals or large doses of radiation, may interfere with replication if such lesions are not removed before replication occurs. Lesions on the template DNA may lead to stalling of DNA replication, which is a lethal event. Stalled replication, as well as certain types of major DNA damage, activate the SOS repair system. The SOS system initiates a number of DNA repair processes, some of which are error-free. However, the SOS system also allows DNA repair to occur without a template, that is, without base pairing; as expected, this results in many errors and hence many mutations. This permits cell survival under conditions that are otherwise lethal.
In E. coli, the SOS repair system regulates the transcription of approximately 40 genes located throughout the chromosome that are involved in DNA damage tolerance and DNA repair. The SOS system is a regulon, (means, a set of genes that are coordinately regulated although they are transcribed separately). The SOS system is regulated by two proteins, LexA and RecA. LexA is a repressor that normally prevents expression of the SOS regulon. The RecA protein, which normally functions in genetic recombination, is activated by the presence of DNA damage, in particular by the single-stranded DNA that results when replication stalls. The activated form of RecA stimulates LexA to inactivate itself by self-cleavage. This leads to derepression of the SOS system and results in the coordinate expression of a number of proteins that take part in DNA repair. Because some of the DNA repair mechanisms of the SOS system are inherently error-prone, many mutations arise. Once the DNA damage has been repaired, the SOS regulon is repressed and further mutagenesis ceases.
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