The low overall rate of mutation during DNA replication (1 base pair change in one billion base pairs per replication cycle) does not reflect the true number of errors that take place during the replication process. The number is kept so low by a proof-reading system that checks newly synthesized DNA for errors and corrects them when they are found. Errors in DNA replication can take different forms, but usually revolve around the addition of a nucleotide with the incorrect base, meaning the pairing between the parent and daughter strand bases is not complementary. The addition of an incorrect base can take place by a process called tautomerization. A tautomer of a base group is a slight rearrangement of its electrons that allows for different bonding patterns between bases. This can lead to the incorrect pairing of C with A instead of G, for example.
DNA retains its high level of accuracy is with its proof-reading function.
The 3' to 5' proof-reading exonuclease works by scanning along directly behind as the DNA polymerase adds new nucleotides to the growing strand. If the last nucleotide added is mismatched, then the entire replication holoenzyme backs up, removes the last incorrect base, and attempts to add the correct base again. The enzyme is "3' to 5'" because it scans in the opposite direction of DNA replication, which we learned must always be 5' to 3'. The mechanism of the proof-reading system offers an explanation as to why DNA replication must occur in this direction.
Keeping in mind the chemical mechanism we learned for the addition of nucleotides to the growing DNA strand, imagine what happens when the proof- reading system removes an incorrectly paired base. The exonuclease removes the base by cleaving the phosphodiester bond that had just been formed. In 5' to 3' synthesis, this leaves the 3' -OH still attached to the terminal end of the growing strand ready to attack another nucleotide.
After DNA has been completely replicated, the daughter strand is often not a perfect copy of the parent strand it came from. Mutations during replication and damage after replication make it necessary for there to be a repair system to fix any errors in newly synthesized DNA. There are three main sources of damage to DNA.
These different sources of damage lead to different categories of DNA damage. The damage that is caused by water attack can lead to unnatural bases. Chemical and radiation damage leads to the formation of bulky adducts to, or breaks in, the growing DNA strand. In the previous section we discussed the 3' to 5' proof-reading exonuclease that is responsible for fixing mismatches. Because it is not a perfect system, it can miss mismatched bases. As a result, a third category of DNA damage is mismatched bases.
Because these categories of DNA damage are different, there is a need for multiple repair systems.
The first type of repair system we will discuss is the excision repair system. To excise simply means to remove, so this repair systems works by removing the area of damage. Special enzymes recognize damaged DNA. This repair system comes in two forms: Base-excision repair and short-patch nucleotide excision.
Base-pair Excision Repair
In base-pair excision, single base-pairs are identified and removed. The resultant gap is then filled with a DNA polymerase and the nick is sealed by a DNA ligase.
Short-patch Excision Repair
Short-patch excision varies from base-pair excision in that its enzymes will recognize and remove "short patches" of DNA that are damaged. These short patches of damage arise from bulky lesions such as thymine dimers. This form of damage is radiation-induced and leads to the formation of a bond between adjacent thymine bases on the same strand of DNA. This bond leads to a distortion in the DNA that makes a short stretch around the thymine dimer unable to base pair correctly. The short-patch excision repair system recognizes such distortions and cuts the damaged strand on both sides of the damaged region leaving a 12 base pair gap in the strand. A helicase then unwinds the stretch of the helix with the damage that can then be filled and sealed with DNA polymerase and ligase. The short-patch excision repair can also be used to correct damage resulting from unnatural bases.
The other major type of repair systems corrects damage resulting from mismatched bases, called the mismatch repair system. The mismatch repair system is able to identify mismatch errors because such damage leads to a small distortion in the DNA backbone. Once it has identified a mismatched base pair, it marks the spot with a cut and then uses an exonuclease to digest or "eat up" the DNA at the marker. A DNA polymerase can then fill in the gap with the appropriate base.
There is one major question that remains: How does the mismatch repair system know the difference between strand contains the correct base and the one on which it should make the incision? The way that it tells the two strands apart is by a marker that is added to the parent strand during replication. An additional methyl (-CH3) added to the adenine base groups of the parent strand and acts as a flag for the mismatch repair system so that it knows to make its cuts on the opposite strand.