Introduction to Molecular and Cell Biology, Biol. 220 Lecture 15: DNA damage and Repair
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DNA damage

Scope of the problem

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.

What are the sources of mutation?

Mutations in DNA can result from

-incorporation of incorrect bases during replication

-DNA may undergo chemical changes

-either spontaneously (Fig. 12.23)

Fig. 12.23

-a result of exposure to chemicals (Fig. 12.22)

Fig. 12.22

-radiation (Fig. 12.28).

Fig. 12.28

 Cells have evolved mechanisms to repair damaged DNA:

During replication proofreading catches many of the nucleotide misincorporations.

Fig. 12.21  Proofreading mechanism of DNA Polymerase.

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. 12.25).
  Fig. 12.25

 

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 by the enzyme photolyase, 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 (Fig. 12.26).

 Types of excision repair:
1) base excision repair
2) nucleotide excision repair
3) mismatch repair

Base Excision Repair (movie)

 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. 12.23).

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

Nucleotide excision repair removes a whole oligonucleotide that containes the damage.  Thymine-thymine dimer residues are examples of damage caused by UV radiation (Fig. 12.25).

Fig. 12.26

 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 (movie)

Recognizes mismatches resulting from replication. Scans newly
replicated DNA, identifies mismatch, excises the mismatched base specifically from new strand so error can be repaired (Fig. 12.24).

Fig. 12.24

 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.

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.
 

Recombinational repair (Repair mechanisms for gaps) (movie)
 

 

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. 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.

 

 

  SOS repair,  Error-prone repair (movie)
 

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.

 

Created 2004 by CA Rinehart• email CA Rinehart

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