13.2: DNA Repair

Learning Objectives

• Define DNA repair and why it is essential for cell survival
• Compare and contrast dark repair with light repair
• Explain how cells recognize and fix thymine-dimers caused by UV radiation

What happens when you DNA is damaged?

 

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The DNA in just one of your cells gets damaged tens of thousands of times per day. Because DNA provides the blueprint for the proteins your cells need to function, this damage can cause serious issues—including cancer. Fortunately, your cells have ways of fixing most of these problems, most of the time. Monica Menesini details the processes of DNA damage and repair.

 

DNA Repair

The process of DNA replication is highly accurate, but mistakes can occur spontaneously or be induced by mutagens. Uncorrected mistakes can lead to serious consequences for the phenotype. Cells have developed several repair mechanisms to minimize the number of mutations that persist.

Proofreading

Most of the mistakes introduced during DNA replication are promptly corrected by most DNA polymerases through a function called proofreading. In proofreading, the DNA polymerase reads the newly added base, ensuring that it is complementary to the corresponding base in the template strand before adding the next one. If an incorrect base has been added, the enzyme makes a cut to release the wrong nucleotide and a new base is added.

Mismatch Repair

Some errors introduced during replication are corrected shortly after the replication machinery has moved. This mechanism is called mismatch repair. The enzymes involved in this mechanism recognize the incorrectly added nucleotide, excise it, and replace it with the correct base. One example is the methyl-directed mismatch repair in E. coli. The DNA is hemimethylated. This means that the parental strand is methylated while the newly synthesized daughter strand is not. It takes several minutes before the new strand is methylated. Proteins MutS, MutL, and MutH bind to the hemimethylated site where the incorrect nucleotide is found. MutH cuts the nonmethylated strand (the new strand). An exonuclease removes a portion of the strand (including the incorrect nucleotide). The gap formed is then filled in by DNA pol III and ligase.

Repair of Thymine Dimers

Because the production of thymine dimers is common (many organisms cannot avoid ultraviolet light), mechanisms have evolved to repair these lesions. In nucleotide excision repair (also called dark repair), enzymes remove the pyrimidine dimer and replace it with the correct nucleotides (Figure \(\PageIndex{6}\)). In E. coli, the DNA is scanned by an enzyme complex. If a distortion in the double helix is found that was introduced by the pyrimidine dimer, the enzyme complex cuts the sugar-phosphate backbone several bases upstream and downstream of the dimer, and the segment of DNA between these two cuts is then enzymatically removed. DNA pol I replaces the missing nucleotides with the correct ones and DNA ligase seals the gap in the sugar-phosphate backbone.

The direct repair (also called light repair) of thymine dimers occurs through the process of photoreactivation in the presence of visible light. An enzyme called photolyase recognizes the distortion in the DNA helix caused by the thymine dimer and binds to the dimer. Then, in the presence of visible light, the photolyase enzyme changes conformation and breaks apart the thymine dimer, allowing the thymines to again correctly base pair with the adenines on the complementary strand. Photoreactivation appears to be present in all organisms, with the exception of placental mammals, including humans. Photoreactivation is particularly important for organisms chronically exposed to ultraviolet radiation, like plants, photosynthetic bacteria, algae, and corals, to prevent the accumulation of mutations caused by thymine dimer formation.

Figure \(\PageIndex{6}\): Bacteria have two mechanisms for repairing thymine dimers. (a) In nucleotide excision repair, an enzyme complex recognizes the distortion in the DNA complex around the thymine dimer and cuts and removes the damaged DNA strand. The correct nucleotides are replaced by DNA pol I and the nucleotide strand is sealed by DNA ligase. (b) In photoreactivation, the enzyme photolyase binds to the thymine dimer and, in the presence of visible light, breaks apart the dimer, restoring the base pairing of the thymines with complementary adenines on the opposite DNA strand.

Query \(\PageIndex{1}\)

Query \(\PageIndex{1}\)

Identifying Bacterial Mutants

One common technique used to identify bacterial mutants is called replica plating. This technique is used to detect nutritional mutants, called auxotrophs, which have a mutation in a gene encoding an enzyme in the biosynthesis pathway of a specific nutrient, such as an amino acid. As a result, whereas wild-type cells retain the ability to grow normally on a medium lacking the specific nutrient, auxotrophs are unable to grow on such a medium. During replica plating (Figure \(\PageIndex{7}\)), a population of bacterial cells is mutagenized and then plated as individual cells on a complex nutritionally complete plate and allowed to grow into colonies. Cells from these colonies are removed from this master plate, often using sterile velvet. This velvet, containing cells, is then pressed in the same orientation onto plates of various media. At least one plate should also be nutritionally complete to ensure that cells are being properly transferred between the plates. The other plates lack specific nutrients, allowing the researcher to discover various auxotrophic mutants unable to produce specific nutrients. Cells from the corresponding colony on the nutritionally complete plate can be used to recover the mutant for further study.

The Ames Test

The Ames test, developed by Bruce Ames (1928–) in the 1970s, is a method that uses bacteria for rapid, inexpensive screening of the carcinogenic potential of new chemical compounds. The test measures the mutation rate associated with exposure to the compound, which, if elevated, may indicate that exposure to this compound is associated with greater cancer risk. The Ames test uses as the test organism a strain of Salmonella typhimurium that is a histidine auxotroph, unable to synthesize its own histidine because of a mutation in an essential gene required for its synthesis. After exposure to a potential mutagen, these bacteria are plated onto a medium lacking histidine, and the number of mutants regaining the ability to synthesize histidine is recorded and compared with the number of such mutants that arise in the absence of the potential mutagen (Figure \(\PageIndex{8}\)). Chemicals that are more mutagenic will bring about more mutants with restored histidine synthesis in the Ames test. Because many chemicals are not directly mutagenic but are metabolized to mutagenic forms by liver enzymes, rat liver extract is commonly included at the start of this experiment to mimic liver metabolism. After the Ames test is conducted, compounds identified as mutagenic are further tested for their potential carcinogenic properties by using other models, including animal models like mice and rats.

Figure \(\PageIndex{7}\): Identification of auxotrophic mutants, like histidine auxotrophs, is done using replica plating. After mutagenesis, colonies that grow on nutritionally complete medium but not on medium lacking histidine are identified as histidine auxotrophs.

Figure \(\PageIndex{8}\): The Ames test is used to identify mutagenic, potentially carcinogenic chemicals. A Salmonella histidine auxotroph is used as the test strain, exposed to a potential mutagen/carcinogen. The number of reversion mutants capable of growing in the absence of supplied histidine is counted and compared with the number of natural reversion mutants that arise in the absence of the potential mutagen.

Key Concepts and Summary

• Cells have mechanisms to repair naturally occurring mutations. DNA polymerase has proofreading activity. Mismatch repair is a process to repair incorrectly incorporated bases after DNA replication has been completed.
• Pyrimidine dimers can also be repaired. In nucleotide excision repair (dark repair), enzymes recognize the distortion introduced by the pyrimidine dimer and replace the damaged strand with the correct bases, using the undamaged DNA strand as a template. Bacteria and other organisms may also use direct repair, in which the photolyase enzyme, in the presence of visible light, breaks apart the pyrimidines.
• Through comparison of growth on the complete plate and lack of growth on media lacking specific nutrients, specific loss-of-function mutants called auxotrophs can be identified.
• The Ames test is an inexpensive method that uses auxotrophic bacteria to measure mutagenicity of a chemical compound. Mutagenicity is an indicator of carcinogenic potential.