Cells have mechanisms for minimizing the amount of mutation that takes place. As stated previously, these are not perfect, but they do reduce greatly the frequency of mutation. The two mechanisms we'll consider are proofreading and mismatch repair.

• Proofreading - This occurs during DNA replication. As DNA polymerase III adds nucleotides to the growing chain, it checks each one for correct base pairing. If the correct nucleotide has not been inserted, the polymerase uses its 3' to 5' exonuclease activity to remove the incorrect nucleotide. The polymerase can then carry on and insert the correct nucleotide. This is very much like a word processor: if you type in the wrong letter, you just hit the delete key to remove it, allowing you to type in the correct letter. (For more on 3' to 5' exonuclease activity, see the module on DNA replication.) In bacteria, a wrong nucleotide gets inserted for every 10⁵ nucleotides added during replication. Proofreading corrects most of these, so that the overall error ratein replication is one mistake for every 10⁷ nucleotides added.
• Mismatch Repair - This mechanism is used soon after replication, to correct errors that escaped proofreading. Because mismatched bases don't hydrogen bond, they create a distortion in the double helix, which can be recognized and repaired by excision repair. The question in this case is how does the repair system recognize which strand to repair? There are two nucleotides (one on each DNA strand) that won't base pair - which one is the wrong nucleotide? The answer comes from DNA methylation. DNA under normal circumstances is methylated; these methyl groups do not interfere with the function of the DNA in any way. Newly replicated DNA is not methylated however; the methyl groups are added enzymatically after replication. If mismatch repair is done immediately after replication (before methylation occurs), the original DNA strand will be methylated, and the newly-synthesized strand (the one containing the error) will be unmethylated. The mismatch repair system therefore repairs the unmethylated strand.

Excision repair can be used to correct other problems as well. For example, if a deoxynucleotide containing uracil is ever inserted into a DNA molecule, the base is detected and removed by an enzyme called uracil DNA glycosylase. This enzyme removes the base, but leaves the sugar and phosphate in the DNA molecule. This base-less site is then recognized by specific endonucleases, which initiate excision repair.

Recombination

There is a mechanism of genetic variation other than mutation, and it involves exchange between DNA molecules with significant sequence similarity. Because the molecules must be homologous in order for recombination to occur, the process is often called homologous recombination. This process occurs in viruses, bacteria, and eukaryotes. In eukaryotes, recombination is the molecular basis of crossing over between homologous chromosomes. (For a review of crossing over, see the module on meiosis.)

Recombination begins with paired homologues, lined up so that the homologous sequences are adjacent.
A single-stranded nick is introduced at the same point on each molecule.
The nicked ends are displaced to the other molecule, and base pair with the complementary sequence on that molecule. (This is why the two molecules must be homologous - without homologous sequences, this base pairing can't occur.)
DNA ligase seals the nick on each translplanted strand, creating a heteroduplex molecule from the two homologues.
After the initial base pairing, more of each strand is displaced across to the other molecule in a zipper-like action. This process is known as branch migration, because the branch point between the two molecules (the crossover point) moves along the heteroduplex.
To understand how recombination is resolved, and how the heteroduplex is returned to two separate DNA molecules, we need to mentally manipulate the heteroduplex. First, we bend the 'arms' of each DNA molecule around the point of crossover. Then, the two molecules are rotated 180 degrees with respect to each other.
The result of the manipulations is the following structure. (Realize that these manipulations would not likely occur in a cell; they are sinply done to aid our understanding.) To return the heteroduplex to two separate molecules, nucleases cut the DNA in two places . However, there is a choice of the two locations used: the cuts can occur at positions 1 and 2, or at positions 3 and 4. (Each possibility is used with equal frequency.)
If sites 1 and 2 are cut, the molecules produced would be as depicted at right. There has been substantial recombination between the homologues.
If sites 3 and 4 are cut, then the molecules at right would be produced. In this case, there has been little recombination (only one small segment of one strand from each molecule has been exchanged). Even this small amount of recombination can have an effect, however - this is what occurs during recombination repair of DNA.