DNA REPAIR
Damage can occur to all cellular molecules. If RNAs or proteins are damaged, they can be degraded and newly synthesized via transcription and translation using DNA as the template (later).
However, because DNA is the genetic material, changes in its structure can result in mutations (in the change of the base sequence). Although mutations can sometimes be beneficial, the overwhelming majority is not.
Mutations in DNA can result from incorporation of incorrect bases during replication or DNA may undergo chemical changes either spontaneously or as a result of exposure to chemicals or radiation.
As we will see, some chemical changes occur with an extraordinary frequency. The resulting huge frequency of mutations would be devastating for the survival of the cell and overwhelm any beneficial mutation that might have ocurred among them.
Cells had to evolve mechanisms to repair damaged DNA:
Classes of repair mechanisms:
1) direct reversal of damage reaction
2) removal of damaged bases and replacement with newly synthesized DNA
Also, mechanisms to cope with damage if it cannot be repaired.
Direct reversal:
Only a few types of damage are repaired in this way although it is probably the most energy efficient. Especially the formation of pyrimidine dimers, which is the major type of damage induced by UV light. Pyrimidine dimers are formed between adjacent pyrimidines (particularly thymines) on the same strand of DNA by the formation of a cyclobutane ring resulting from saturation of the double bonds in their ring structure (Fig. 5.20 A).
Pyrimidine dimers distort the double helical structure of DNA and block transcription or replication past the damaged site. Recognition of distortions in the double helix is the major way that DNA damage is generally recognized in the cell.
One mechanism of repair (there are several others) is through direct reversal of the dimerization reaction. The process is called photoreactivation because the energy to break the cyclobutane ring is derived from visible light. Therefore, in this kind of repair mechanism the original pyrimidine bases are restored and remain in the DNA.
The repair of pyrimidine dimers by photoreactivation is common to many prokaryotes and eukaryotes (E. coli, yeast, and several species of plants and animals). However, photoreactivation is not universal. Many species (including humans) lack this kind of repair mechanism. But humans have other kinds of repair mechanisms that directly reverse certain damages.
Excision repair:
Most important repair mechanism. Damaged DNA is recognized, removed either as free bases or as nucleotides, and the gap is filled by synthesis of new DNA using the complementary strand as a template.
Types of excision repair:
1) base excision repair
2) nucleotide excision repair
3) mismatch repair
One example for base excision repair is the removal of uracil from DNA. Most uracil in DNA arises from the deamination of cytosine which directly leads to the structure of uracil (Fig. 5.19 A).
In humans it occurs at a frequency of about 100 times a day in each cell.
If uracil is not repaired, it will base pair with adenine during the next round of replication and thus cause a mutation. The general use of thymine instead of uracil in DNA allows the repair system to recognize deaminated cytosine as incorrect.
In general, the excision of a base is catalyzed by DNA glycosylase, which cleaves the bond between the base and the deoxyribose (called glycosidic bond). The result is an apyrimidinic or apurinic (AP) site: a sugar with no base attached.
AP sites also form through spontaneous loss of a base. This occurs especially often with purines, for example several thousand times a day in a human cell.
Repair of AP sites by AP endonuclease: cleaves adjacent to AP site.
The deoxyribose is removed and the single base gap filled by DNA polymerase and ligase.
Nucleotide excision repair removes a whole oligonucleotide that containes the damage.
In E. coli three genes involved (uvrA, B and C). The protein UvrA recognizes the damage, recruits UvrB and UvrC which cleave at 3' and 5' site of damage, respectively, producing an oligonucleotide of 12 or 13 bases.
Helicase necessary to remove oligonucleotide (disruption of hydrogen bonds from base pairing), DNA polymerase fills gap, ligase seals.
Nucleotide excision repair also in eukaryotes. Similar mechanism.
Mismatch repair system.
Recognizes mismatches resulting from replication. Scans newly replicated DNA, identifies mismatch, excises the mismatched base specifically from new strand so error can be repaired.
How can the old strand of DNA be distinguished from the new strand after replication?
In E. coli, because new strand not yet methylated at GATC sequences as is normally the case (A of GATC methylated).
In E. coli mismatch repair is initiated by the protein MutS, which recognizes mismatch, and forms complex with MutL and MutH. Then MutH (an endonuclease) cleaves the unmethylated DNA strand at a GATC sequence (Fig. 5.25).
Eukaryotes have a similar mismatch repair system, but the mechanism by which they identify the newly replicated DNA strand is not known.
If DNA contains damaged bases (like a pyrimidine dimer) that cannot be repaired, replication and transcription are blocked at this site. However, there are mechanisms to circumvent the damaged site. For example, replication can be initiated downstream of the damaged site by an Okazaki fragment. The result is a gap in the new daughter strand opposite the damage of the parental strand.
Repair mechanisms for gaps:
Recombinational repair
{SOS repair} Error-prone repair
Recombinational repair makes use of the undamaged parental strand to undergo recombination shifting the gap to the other newly synthesized DNA molecule, the one that does not contain the damage (Fig. 5.26). Because the gap is now opposite an undamaged strand, it can be filled by DNA polymerase. And the damage lies now opposite a normal strand and can be dealt with later.
In error-prone repair, the gap opposite the site of DNA damage is directly filled by newly synthesized DNA. But because of damage to the template the repair is very inacurate and leads to frequent mutations. Error-prone repair is used only in bacteria that have been subjected to potentially lethal conditions (such as extensive UV irradiation), where damage is so enormous that cell death is probably the only alternative.