DNA Repair Mechanisms
Mismatch Repair and Base Excision
Jul 19, 2009 Alicia Mae Prater
The integrity of cell function and the health of organisms are dependent on the fidelity of DNA replication and the capability of the nucleic acid to withstand damage. Changes in the genetic material can result in disease, the best known of which is cancer. Alterations can come about due to exposure to ionizing radiation, carcinogens, and reactive oxygen species or due to problems in DNA replication. The cell possesses enzymes that can recognize DNA damage in the form of problematic nucleotide placement and correct the problem, called DNA repair.
Normal DNA Sequence
Normal DNA consists of two parallel strands consisting of a sequence made from four nucleotides, or bases: adenine, thymine, guanine, and cytosine. The parallel strands are held together via hydrogen bonds between complementary bases: adenine and thymine, and cytosine and guanine. This is called normal Watson-Crick pairing. Deviations in this pairing or chemical alterations of the bases result in altered DNA structure, which impairs cellular function and the progression of necessary processes involving the genetic material.
Mismatch Repair
The insertion of a wrong nucleotide by the replication machinery results in mismatched base pairs. Repair of the mismatch requires recognition and removal of the wrongly inserted base. The mismatch repair mechanism was first seen and studied in E. coli. If the cell does not correct mismatches, future generations of DNA will have the mutation incorporated, possibly altering the protein product and cell function.
Mismatch repair shares enzymes with the other repair pathways, discussed below. A protein encoded by the MSH genes recognizes mismatched bases. An enzyme encoded by the MLH (or PMS) genes takes the lead in removing the base. The DNA is then patched by DNA polymerase in a process similar to DNA replication. Mutations in these genes have been associated with colon cancer
Example 1: cytosine paired with adenine – the hydrogen bonding between the two nucleotides is disrupted and the altered conformation is recognized as damage.
Base Excision Repair
The enzymes DNA glycosylase, DNA polymerase beta, and DNA ligase are capable of removing, inserting, and patching the DNA strand, respectively, in the event of oxidative damage. A common point mutation is the alteration of cytosine to thymine by the addition of a methyl group followed by deamination.
DNA glycosylase is actually a family several proteins that recognize specific mutations, with a number that are specific for uracil recognition and removal. After recognizing the aberrant base, the glycosylase removes the base, leaving an apurinic/apyrimidinic site with no nucleoside attached to the backbone. AP endonuclease then nicks the backbone to create a 3′ hydroxyl (OH) terminus that can be used by DNA polymerase to add a base. If 2-4 bases are removed it is called long patch repair, which occurs in response to ionizing radiation at certain points in the cell cycle. The active glycosylases are thought to be ones that can remove dihydrouracil and 8-oxoG.
Mutations in the genes of the base excision pathway have been associated with accumulated DNA damage and the occurrence of some cancers.
Example 2: the insertion of uracil instead of thymine. Uracil is a nucleotide that occurs in RNA rather than DNA and does not allow the progression of transcription if present. The enzyme DNA glycosylase recognizes and removes the uracil, which is then replaced with thymine by the transcription machinery.
Nucleotide Excision Repair
A slightly different process from base excision is nucleotide excision repair. This repair system removes a patch of nucleotides that includes the damaged base and proceeds rapidly in cells actively undergoing transcription. An important gene in this process is XP (Xeroderma Pigmentosum), which is also the name of a rare genetic disorder caused by an impaired repair process.
Repair has been linked to RNA polymerase II, the enzyme that builds the RNA strand during transcription. This indicated that there is potential for correcting errors as the transcription machinery runs into chemically altered bases that are not binding correctly. In this context, nucleotide excision allows transcription to correct the code before mutations occur. The entire process requires approximately 20 genes.
There are five steps in nucleotide excision repair:
- Recognition of the damaged base
- Incisions are made on the damaged strand on both sides of the lesion.
- The damaged nucleotide(s) is removed
- A patch is synthesized by DNA polymerase using the other strand as a template.
- DNA ligase seals off any remaining cut via ligation.
Example 3: Exposure to UVB rays forms pyrimidine dimers. However, excess sun exposure is thought to overwhelm the process, resulting in unrepaired damage in skin cells and commonly leading to skin cancer.
Another type of DNA damage and repair mechanism due to environmental factors is chromosomal breaks, which can also occur if nucleotide excision repair is impeded.
DNA Repair Fork
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