DNA Proofreading and Repair
DNA replication is a highly accurate process, but mistakes can occasionally occur, such as a DNA polymerase inserting a wrong base. Uncorrected mistakes may sometimes lead to serious consequences, such as cancer. Repair mechanisms correct the mistakes. In rare cases, mistakes are not corrected, leading to mutations, in other cases, repair enzymes are themselves mutated or defective.
Fortunately, cells have evolved highly sophisticated means of fixing most, but not all, of those mistakes. Some of the mistakes are corrected immediately during replication through a process known as proofreading, and some are corrected after replication in a process called mismatch repair. When an incorrect nucleotide is added to the growing strand, replication is stalled by the fact that the nucleotide’s exposed 3′-OH group is in the “wrong” position. During proofreading, DNA polymerase enzymes recognize this and replace the incorrectly inserted nucleotide so that replication can continue. Proofreading fixes about 99% of these types of errors, but that’s still not good enough for normal cell functioning.
Some errors are not corrected during replication, but are instead corrected after replication is completed; this type of repair is known as mismatch repair. The enzymes recognize the incorrectly added nucleotide and excise it. This is then replaced by the correct base. If this remains uncorrected, it may lead to more permanent damage. In E. coli, after replication, the nitrogenous base adenine acquires a methyl group; the parental DNA strand will have methyl groups, whereas the newly synthesized strand lacks them. Thus, DNA polymerase is able to remove the wrongly incorporated bases from the newly synthesized, non-methylated strand. In eukaryotes, the mechanism is not very well understood, but it is believed to involve recognition of unsealed nicks in the new strand, as well as a short-term continuing association of some of the replication proteins with the new daughter strand after replication has completed.
The polymerase checks whether the newly added base has paired correctly with the base in the template strand. If it is the right base, the next nucleotide is added. If an incorrect base has been added, the enzyme makes a cut at the phosphodiester bond and releases the wrong nucleotide. This is performed by the exonuclease action of DNA pol III. Once the incorrect nucleotide has been removed, a new one will be added again.
In another type of repair mechanism, nucleotide excision repair, enzymes replace incorrect bases by making a cut on both the 3′ and 5′ ends of the incorrect base. Some DNA-damaging chemical reactions can be directly “undone” by enzymes in the cell. It is called a Direct reversal.
Another repair is the Double-stranded break repair in which two major pathways, non-homologous end joining and homologous recombination, are used to repair double stranded breaks in DNA.
There are many events that contribute to replication stress, including;
- Disincorporation of ribonucleotides
- Unusual DNA structures
- Conflicts between replication and transcription
- Insufficiency of essential replication factors
- Common fragile sites
- Overexpression or constitutive activation of oncogenes
- Chromatin inaccessibility