DNA Damage

Mutations arise not only from the errors in the replication but also from damage to the DNA. Some damage is caused by environmental factors, such as radiation and chemicals like mutagens, which are chemical agents that increase the rate of mutation. DNA also undergoes spontaneous damage from the action of water.

Mechanisms which causes DNA Damage include:

1. Hydrolysis (Deamination and Depurination)
2. Alkylation
3. Oxidation
4. Radiation Ractions
5. Base analogue and intercalating agents.

1. Hydrolysis

The most frequent and important kind of hydrolytic damage is deamination of the base cytosin. Deamination is the removal of an amine group from a molecule. Enzymes that catalyse this reaction are called deaminases.

• The deamination of cytosin to uracil happens at a significant rate in cells. Deamination can be repaired by a specific repair process which detects uracil, not normally present in DNA; otherwise the U will cause A to be inserted opposite it and cause a C:G to T:A transition when the DNA is replicated.
• Deamination converts adenine to hypoxanthine, which hydrogen bonds to cytosine rather than to thymine; guanine is converted in to xanthine, which continues to pair with cytosin, though with only two hydrogen bonds.
• Deamination of 5-methylcytosine to thymine also occur. 5- Methylcytosine occurs in the human genome at the sequence 5’CpG3′, which normally avoided in the coding regions of genes. if the meC is deaminated to T, there is no repair system which can recognise and remove it (becase T is normal base in DNA). This means that whatever CpG occurs in genes it is a “hotspot” for mutation.

DNA also undergoes depurination by spontaneous hydrolysis of the N-glycosyl linkage, and this produces an abasic site (that is deoxyribose lacking a base) in the DNA.

Depurination in DNA is a chemical reaction of purine deoxyribonucleosides, deoxyadenosine and deoxyguanosine, in which the β-N-glycosidic bond is hydrolytically cleaved releasing a nucleic base, adenine or guanine

2. Alkylation

Alkylation is the transfer of an alkyl group from one molecule to another. In alkylation, methyl orethyl groups are transferred to reactive sites on the bases and to phosphates in DNA backbone.

Alkylations do not lead immediately to mispairing. However, they do make the bond between sugar and base more labile, or more apt to break. When this break occurs, it leaves an apurinic site, a sugar without its purine. This obviously cannot be replicated properly unless it is first repaired, but cells sometimes attempt to replicate apurinic DNA anyway. If they do, they frequently insert the wrong base across from an apurinic site, and this generates a mutation. Alkylation can also enhance the tendency of a base to form the rare (wrong) tautomer, which changes the base-pairing properties, leading to mutations. Moreover, all of the nitrogen and oxygen atoms involved in base-pairing are also subject to alkylation, which can directly disrupt base-pairing and lead to mutation.

Many environmental carcinogens, or cancer-causing agents, are electrophiles that act by attacking DNA and alkylating it. Many of the favorite mutagens used in the laboratory for the express purpose of creating mutations are also alkylating agents. One example is ethylmethane sulfonate (EMS), which transfers ethyl (CH₃-CH₂) groups to DNA. The product of this methylation, O6-rthylgaunine, often mispaires with thymine, resulting in the change of G:C base pair into an A:T base pair when the damaged DNA is repliacted.

3. Oxidation

DNA oxidation is the process of oxidative damage on Deoxyribonucleic Acid. It occurs most readily at guanine residues due to the high oxidation potential of this base relative to cytosine, thymine, and adenine. Reactive oxygen species (O2-, H2O2, and OH) are generated by ionizing radiation and by chemicals agents that generate free radicals. An important oxidation product is 8-hydroxyguanine, which mispairs with adenine, resulting in G:C to T:A transversions.

4. Radiation Ractions

Ultraviolet radiation (UV radiation) cross-links adjacent pyrimidines on the same DNA strand, forming pyrimidine dimers, usually thymine dimers. Figure shows the structure of a thymine dimer and illustrates how it interrupts base-pairing between the two DNA strands. These dimers block DNA replication because the replication machinery cannot tell which bases to insert opposite the dimer. As we will see, replication sometimes proceeds anyway, and bases are inserted at random. If these are the wrong bases, a mutation results.

Thymine dimers. (a) Ultraviolet light cross-links the two thymine bases on the top strand. This distorts the DNA so that these two bases no longer pair with their adenine partners. (b) The two bonds joining the two thymines form a four-membered cyclobutane ring (red).

Ultraviolet radiation has great biological significance; it is present in sunlight, so most forms of life are exposed to it to some extent. The mutagenicity of UV radiation explains why sunlight can cause skin cancer: Its UV component damages the DNA in skin cells, which leads to mutations that sometimes cause those cells to lose control over their division.

Damage by Gamma and X -rays:

The much more energetic gamma rays and X rays, like ultraviolet rays, can interact directly with the DNA molecule. However, they cause most of their damage by ionizing the molecules, especially water, surrounding the DNA. This forms free radicals, chemical substances with an unpaired electron. These free radicals, especially those containing oxygen, are extremely reactive, and they immediately attack neighboring molecules. When such a free radical attacks a DNA molecule, it can change a base, but it frequently causes a single- or double-stranded break. Single-stranded breaks are ordinarily not serious because they are easily repaired, just by rejoining the ends of the severed strand, but double-stranded breaks are very difficult to repair properly, so they frequently cause a lasting mutation. Because ionizing radiation can break chromosomes, it is referred to not only as a mutagen, or mutation-causing substance, but also as a clastogen, which means “breaker.”

5. Base analogue and intercalating agents.

Mutations ae al;so caused by compounds that substitue for normal bases (base analogs) or slip between the bases (intercalating agents) to cause errors in replication.

Base analogs are structurally similar to proper bases but differ in ways that make them treacherous to the cell. Thus base analogs are similar enough to the proper base to get taken by cells, converted in to nucleoside triphosphates, and incoporated into DNA during replication. But, because of the structural difference between these analogues and the proper bases, the analogues base-pair inaccurately, leading to frequent mistakes during the replication process.

one of the most mutagenic base analogs is 5-bromouracil, an analog of thymine. The presence of bromo substituent allows the base to mispair with guanine via enol tautomer.

5-bromouracil (5-BU) ia a derivative of uracil and behaves as a thymine analog, which increases the probability of a tautomeric shift from the normal keto form to the enol form, mis-pairing with guanine instead of adenine. After one round of replication, an A – T to G – C transition mutation results.

Intercalating agents are flat molecules containing several polycyclic rings that bind to the equally flat purine or pyrimidine bases of DNA. Intercalating agents are compounds, such as proflavin, acridine and ethidium,  that can bind to the major and minor grooves of DNA and cause addition or deletion of bases during replication. They may result in a frameshift mutation, which can alter the codon reading frame and result in aberrant DNA transcription and replication.