WHAT CAUSES MUTATIONS?

Mutations can be INDUCED, or intentionally produced by treating organisms with mutagens (e.g., ionizing radiation or chemical mutagens), or SPONTANEOUS, which arise in the absence of *known* mutagens.

Spontaneous mutations are obviously caused by something, but they are what could also be termed "natural" mutations, which occur at a relatively constant rate in natural situations due to naturally occuring mutagenesis.

1. A-C bond accidentally forms
2. repair mechanisms accidentally remove the original base instead of the wrong base on the new strand, and replace it with the complementary base.
3. result: base pair substitution.

Various types of mutations include...

1. depurination: base-deoxyribose bond (a glycosidic bond) is broken or disrupted; loss of a G or A is the most common result.

(note, at normal body temperature, a mammal cell loses about 10,000 purines per 20-hour cell cycle; repair mechanisms are very busy! Most of these apurinic sites are repaired.) Many fungi produce MYCOTOXINS that are either highly toxic almost immediately, or highly CARCINOGENIC down the line. One mycotoxin in particular, known as AFLATOXIN B₁ is a powerful depurinating agent that can cause cancer due to apurinic mutations.

2. deamination (of cytosine, causing C --> U)

result: G-C pair --> A-T pair.

3. oxidatively damaged bases: free radicals in the cell change bases and cause mispairing.

Normal DNA - most bases are in keto form.
More rare are the imino or enol forms.

The imino forms tend to cause mispairing. Result: mutation known as a tautomeric shift.

INDUCED MUTATIONS can be caused by a variety of different mutagens, and many mutagens cause specific mutations, and may regularly cause mutations at DNA "hot spots." These are useful in the laboratory, and may also sometimes occur spontaneously.

CHEMICAL MUTAGENS

• Base modifying agents can actually change the chemical structure of the nitrogenous bases, resulting in mispairing and other problems.

• Base analogs can resemble nucleotides so closely that they replace them in the DNA molecule, but do not pair w/ normal bases. Hence: replication, transcription and translation are disrupted. Two examples are 5-bromouracil and 2-aminopurine.

  

• some chemical mutagens selectively remove NH₃ group from A or C. Result: mispairing.

• some can add hydrocarbon groups to the bases, also causing mispairing.

• some chemicals act as intercalating agents, causing the insertion or deletion of an entire base pair (and hence, frameshift errors, insertions and deletions, since the fit is not exact).

SHORT WAVELENGTH IONIZING RADIATION

• ionizing rays easily pass through the plasma membrane and into the cell where they are absorbed by intracellular molecules such as water.

• electrons of the absorbing molecules are boosted to such a high energy state that they spin out of their orbitals and are lost.

• the remaining particle (often a proton) is a positively charged FREE RADICAL which is highly unstable and highly reactive.

• free radicals react energetically with anything nearby, from enzymes to RNA to DNA. Obviously, reactions with DNA are the most long-lasting (other reactions usually being more ephemeral in their effect).
• ionizing radiation events such as these can also break the DNA across both backbones, effectively snapping it in half.
  • Eukaryotes can repair damage like this during synapsis, when the homologs lie in close apposition and the DNA can be re-linked by repair mechanisms.

  • Is meiosis the evolutionary result of a mechanism whose original benefit was to serve as a repair mechanism?)

    Who knows? But just to be on the safe side: EAT MORE FREE RADICAL SCAVENGERS!

ULTRAVIOLET RADIATION damage

Fortunately for us (but not for the dermatologists), the cell has UV repair systems. Enzymes can either:

1. break the covalent bond between the two pyrimidines OR

2. remove the faulty piece and use the complementary DNA strand as a template to fix the piece.

xeroderma pigmentosum: recessive disorder in which victims lack the normal UV repair enzymes. Result: even the slightest exposure to UV results in copious skin tumors.

REPAIR MECHANISMS

• Damage prevention - The cell has various enzymes that de-tox potential mutagens before they can act. SUPEROXIDE DISMUTASE can change superoxide free radicals into hydrogen peroxide, which is then broken down by CATALASE. (Hey! Remember that wonderful lab in BIL 150?)
• Damage reversal - the pyrimidine dimers mentioned previously can be excised (at least in E. coli) by an enzyme known as DNA photoylase. In the dark, the enzyme binds to dimerized thymines. In the presence of light, the enzyme breaks the dimer bonds with light energy, and then falls off the broken DNA strand. This has not been found in other organisms, though different repair enzymes are probablye there.
• Excision repair - a variety of enzymes can sense distortion of the strand, and cooperate to excise the mistake and replace it with a correct DNA sequence. Three basic types:
  
• UV damage repair - endonuclease detects a dimer, and makes a "nick" in the DNA strand on either side of the dimer, 8 phosphodiester bonds upstream of the dimer, and five phosphodiester bonds downstream from the dimer. DNA helicase II releases the whole segment, which is degraded. DNA polymerase I and ligase then fill in the gap.

  In prokaryotes and some simple eukaryotes, an enzyme known as photolyase splits photodimers, restoring original base configuration.

• AP repair - this is the repair of apurinic and apyrimidinic sites on the DNA, where a base has been removed (either by a mutagen or by DNA glycosylase, which senses a wrong base). AP endonucleases then initiate repair at the site of excision: Class I endonucleases nick the 3' side of the site; Class II endonucleases nick the 5' side of the site. Exonucleases removes the nicked out section, which is then repaired/replaced by DNA polymerase I and ligase. Have a look:

  

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  POSTREPLICATION REPAIR

  • If DNA polymerase III encounters a mutation that it cannot polymerize through (such as a thymine dimer), it skips that area, leaving a broken piece.

  • A group of enzymes repairs this gap in a process known as postreplicative repair.

  • Mismatch repair - This is responsible for about 90% of all DNA repair, usually due to mismatching during replication.
    A mismatch repair system, enzymes encoded by a series of genes, zip along behind the replication fork, removing incorrect bases, and able to distinguish between the new and old strands because only the OLD strand is methylated.

    The genes encoding these enzymes are called mut genes, which is short for "mutator" genes because when these genes mutate, it results in an unusually high level of mutation in the cell (due to faulty repair!).

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• One model--the SOS System--has been studied in E. coli. (It apparently does not exist in more recently evolved eukaryotes, such as mammals.)

  In this model, of the most important enzymes in this group is one encoded by a gene called recA. The enzyme product of the gene (RecA) is instrumental in the SOS response of E. coli, which is a "stop gap" measure taken by the cell to survive a lethal mutation while repair takes place, or to accept a non-lethal level of mutation instead of just succumbing.

• The other player in this drama is a gene called lexA, which encodes a protein known as LexA.

• Under normal conditions, LexA squats on the promoter region of a polycistronic region of about 18 genes (including its own), all of which are involved in the repair of DNA damage of various types.

• In the presence of single stranded DNA (caused by the deletion of bases or series of bases), RecA somehow becomes activated, and binds to LexA, causing it to release its hold on the 18 repair genes.

• The genes on this area all have a consensus sequence known as the SOS box: 5'-CTG--ten bases--CAG-3'.

  All genes having an SOS box are transcribed, once LexA releases the promoter.

• Over time, the damage is repaired.

• In the absence of single stranded DNA, RecA loses its activity, and stops reacting with LexA.

• This means that newly manufactured LexA can now diffuse back onto the operator region of the 18-gene area, and stop transcription of the emergency repair enzymes.
• Here's the scenario:

  

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  AMAZING SIDE NOTE: the lambda prophage (a virus) also exhibits the SOS response: it enters a vegetative state when exposed to UV. When this happens, RecA in the HOST cell also inactivates the prophage's repressor protein, which is normally preventing transcription of repair enzymes.
  The "parasitic" phage utilizes the host's repair enzymes to activate its own system!

SOMATIC VERSUS GERMLINE MUTATIONS

• In animals, only germline mutations can be passed on to future generations. Somatic mutations can result in phenotypes from differing color patches to cancer.

• In plants, protists and fungi, somatic mutations *can* be passed on, since these organisms exhibit INDETERMINATE GROWTH, and have tissues that can potentially develop into germinal cell lines throughout the life cycle.

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  What's the deal with mutations? Judging from what we see in the lab and in the field, the vast majority of unrepaired mutations are recessive. In homozygous condition, they are often maladaptive or lethal.

Some non-lethal mutations can be dominant over the wild type allele. These changes, known as GAIN-OF-FUNCTION MUTATIONS, can produce new phenotypes that are then subject to the same natural selection as any other allele.

• Morphology (morphological mutations)

• Viability (lethal mutations)
  Note: This feather mutation

  

  is labeled in your text as "lethal if it had occurred in nature". This is not exactly correct. It is more precise to say that it would have conferred a decided selective DISADVANTAGE on the birds that expressed it. Since the mutation does not cause developmental death, and can be artificially maintained, it is not lethal in the most precise sense of the word.

• Biochemical pathways (biochemical mutations)

• Phenotype in combination with environmental conditions (conditional mutations)

CONDITIONAL MUTATIONS

• Restrictive conditions - defined as those which result in mutant phenotype in an organism having the particular conditional mutation.
• Permissive conditions - defined as those which result in wild-type phenotype in an organism having the particular conditional mutation.

The organism that has this mutation is known as a CONDITIONAL MUTANT.

Example: heat sensitive lethal mutations in Drosophila. These types of mutants are useful for study, since they can be raised in permissive conditions, then switched to restrictive conditions in order to study the actual gene expression/protein consequences of the mutation when the environment changes.

MUTATIONS AND HUMAN DISEASE

1. mitochondrial encephalomyopathy

Disorders of the CNS or muscles may be caused by dysfunction of oxidative phosphorylation in the mitochondria.
Common cause: deletions between normal, repeated sequences in mtDNA.

2. Fragile X sydrome

This is caused by expansion of a 3-base pair repeat

(1/1500 males and 1/2500 females exhibit this disorder, which is characterized by some degree of mental retardation). Normal repeat of the FMR-1 gene is about 29. Some individuals have repeats of this gene numbering about 50 -200, but are not affected. However, their offspring ARE affected, and show even more repeats. It appears that the presence of the repeats themselves fosters additional repeats during DNA replication, causing huge expansion of repeats in the offspring that inherit them. Result: hundreds of repeats in affected individuals.)

3. Fabry disease

This is characterized by anemias and thalassemias (salt balance problems),and is caused by deletions/duplications in short, direct repeats.