Tuesday, March 16, 2010

DNA Repair 1

Causes of DNA Damage
Constantly, day-in and day-out, the genome is assaulted by a number of forces that lead to damage and mistakes that can lead to severe problems for the organism under the onslaught.  Included in the forces that cause the DNA damage are chemicals, radiation, and especially reactive metabolites (reactive oxygen species, for example).  Such mutagenic agents can lead to damage in the form of thymidine dimers, single-strand breaks, creation of abasic sites, or the formation of double-stranded breaks.  Specifically, UV radiation induces pyrimidine dimers, while ionizing radiation can cause double-stranded breaks.  Toxic chemicals can be mutagenic in themselves, or they can be activated by cellular metabolism into mutagenic chemicals.  In addition, DNA replication errors and the inherent mobile genetic elements can lead to genetic mutation.

Some of the most common mutations in DNA occur via hydrolysis a nucleotide base.  Due to the high concentration of water within the cell, the DNA is constantly exposed to the harmful effects of water.  Depurination results in a nucleotide without its guanine or adenine base.  Deamination is a base conversion event in which a methylated cytosine residue to converted to thymine.  Such an event can be especially dangerous to the cell because it can induce base changes if the mutation is not fixed immediately. 

Chemicals that induce mutations come in several forms: base analogs, intercalating agents, and base modifiers.  In the case of base analogs, the chemicals resemble that of the natural bases found in the genome but have altered binding potential, leading to the misincorporation of bases that fit improperly.  Examples of base analogs include 5-bromouracil (5-BU) and 2-aminopurine (2-AP).  Intercalating agents do not pair with the DNA bases, but they affect the structure of the DNA helix during DNA synthesis, leading to improper pairing and the induction of mutations.  Examples of intercalating agents include proflavin and acridine orange.  When a base is modified, it can also affect the DNA structure, leading to altered pairing and the induction of mutations.  Chemicals that modify bases as alkylating agents include ethyl methanesulfonate (EMS) and nitrosoguanidine (NG). 

Testing for mutagenic activity
In order to examine whether a chemical is mutagenic in a living system, using the model organism Salmonella, the Ames test is employed.  First, mice are exposed to the mutagen and injected with arochlor to induce hepatic function, where most chemical mutagens are activated.  Then, liver extract is homogenized and added to Salmonella.  The Salmonella strain used contains a His- marker, making it unable to grow in the absence of histidine.  The ability for the strain to grow on media lacking histidine works as a readout for the mutagenic capability of the chemical:  the most colonies that grow on the selective media, the more mutants were able to convert to His+ and the higher the mutagenic capacity of the tested chemical.  Extensions of the Ames test have been developed to specifically look at base substitutions and insertions or deletions.

The Ames Test Simplified:
  1. Expose mouse to potential mutagen
  2. Inject mouse with arochlor to induce hepatic function
  3. Isolate and homogenize liver
  4. Combine homogenized liver with Salmonella typhimurium (His-)
  5. Plate Salmonella on agar lacking histidine
  6. Count number of colonies that have grown on the plate as a measure of mutagenicity.
More information about the Ames test can be found here and here.

Note: Links provided in this post are only a sampling of the vast amount of information available about these chemicals and processes.

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