Gene Mutations: Changes at the Molecular Level

A mutation can be defined as


Major changes can take place with only a small rearrangement of base pairs on the DNA strand itself, either due to errors in DNA replication or from outside damage to the DNA.

Recall:

  • adaptation is short-term change (via gene expression) in response to environmental factors; it does not (usually) involve genetic change.
  • organic evolution is a shift in allele frequencies in a population (microevolution) which can ultimately lead to speciation (macroevolution) under certain circumstances.

    The raw material of evolution is mutation.

    Evolution, not adaptation, results from changes in gene frequencies and genetic composition of the main unit of evolution: the population.

    A cistron is the smallest unit of genetic material which can undergo mutation and still produce a single phenotypic effect. (It's the functional mRNA equivalent of a gene.)
    Key ideas:

    Classes of Mutations At the DNA level:

    At the protein level, point mutations can be:

    Here's an overview.

    Mutations in coding regions of DNA
    Point mutations can alter protein structure simply be coding a different amino acid. But is it not uncommon for a point mutation to affect the exact splicing sites of mRNA processing. If this happens, the mutant protein translated from the mis-spliced mRNA is usually non-functional.

    Mutations in non-coding regions of DNA
    Mutations at non-coding regions such as

    are less predictable in their effects. Usually, the effect of such a mutation will depend on whether it creates a new or disrupts an existing binding site. These can change gene expression in many different ways (degree of expression, timing of expression, you name it).

    But mutations that occur at "spacer" DNA sequences that are neither coding nor regulatory are usually phenotypically neutral, and of no immediate consequence.

    At the level of phenotypic expression, mutations can be classified as:

    What Causes Mutations?

    Mutations can be

  • For example, errors can occur during DNA replication

    Mutations can occur naturally in the body via

    Mispairing Caused by Base Tautomers
    DNA bases exist in one of several forms called tautomers--isomers differing in the position of their atoms/bonds.

    Normal DNA - most bases are in keto form:

    More rare are the enol or imino forms, which tend to cause mispairing. The result: a tautomeric shift mutation:

    Trinucleotide Repeat Mutations In some genes, three-nucleotide repeats are normal and expected. But mutations causing expansion of these normal repeats can result in serious disorders. Several heritable diseases are known to be due to such repeats.

    Fragile X Syndrome

    Huntington's Disease
    The exact mechanism by which these repeats cause disease is not known, but it is notable that most of them affect the nervous system, causing nerve cell death. It is possible that the repeats affect chromatin structure responsible for silencing transcription of the mutant form of the gene. (Huntington's disease may be caused by a loss-of-function mutation.)

    The Wonderful World of Mutagens Induced mutations can be caused by a variety of mutagens, and many cause specific mutations. Some regularly mutate DNA "hot spots." These are useful in the laboratory.

    Chemical Mutagens

    Some fungi produce mycotoxins that can be either acutely toxic, or have a more delayed toxicity and be carcinogenic. One mycotoxin in particular, known as aflatoxin B1 is a powerful depurinating agent that is known to promote carcinogenesis via apurinic mutations. (Vindictive fungi!)

    Aflatoxin B1 binds directly to DNA at guanine residues (at the N7 position), breaking them off and creating an apurinic site.

    Short-wavelength Ionizing Radiation Ionizing electromagnetic radiation such as x rays and gamma rays easily pass through the plasma membrane and into the cell where they are absorbed by intracellular molecules such as water.

    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 plenty of free radical scavengers Can't hurt. Might help.

    Ultraviolet Radiation Short wavelength radiation just beyond the visible spectrum can be damaging to DNA. Wear your sunscreen.

    Repair Mechanisms

    UV damage repair
  • Damage reversal - The pyrimidine dimers mentioned previously can be excised (at least in E. coli) by an enzyme known as DNA photolyase. 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. Fortunately for us (but not for the dermatologists), the cell has UV repair systems. Enzymes can either: - 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:

    Postreplication Repair
  • 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:

    Interesting 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

    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.

    Various types of mutation can be studied because of their effect on. A conditional mutation is one which produces different phenotypes in its bearers, depending upon environmental conditions.

    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.

    The Genetics of Cancer
    Regulation of cell number and division

    Key Ideas

  • The cells of more derived eukaryotes contain mechanisms that control their survival and ability to proliferate.
  • These cells constantly evaluate their own condition via continuous communication among neighboring cells and tissues. Survival and proliferation controls are highly integrated and dependent on these inter-cellular communications.
  • A normal cell's proliferation is regulated at the level of the cell cycle (mitosis).
  • Apoptosis (from the Greek apo meaning "from, or away" and pto meaning "fall"), or programmed cell death, (The word is pronounced ah poh toh' sis, NOT a pop toh' sis; the second "p" is silent.) is a normal process by which cells are destroyed by intra- and extra-cellular mechanisms.
  • Cells may be triggered into an apoptotic cycle if they are damaged, dangerously abnormal, or needed only transiently during development.
  • Intercellular signaling systems allow organized cell proliferation and apotosis to proceed within any given population of cells.
  • In cancer cells, proliferation and apoptosis mechanisms have failed due to mutations in normal tumor-suppressing genes, preventing self-destruct mechanisms from operating. Cancer cells are immortal and highly proliferative.
  • Many of the genes in which mutations cause cancer are those which contribute either directly or indirectly to the normal control of growth and differentiation mechanisms in the cell.
  • Early detection and treatment of cancer is becoming more sophisticated with the application of functional genomics (i.e., discovering not only what genes are in the genome, but what they code for).

    To understand what happens when a Cell Goes Bad, we must first understand the behavior of a Nice, Normal Cell....

    Normal cell proliferation is necessary for Cell death is necessary for Cell proliferation and cell death balance one another, and when mechanisms controlling either or both go awry, neoplasia (from the Greek neo meaning "new" and plas meaning "form" or "shape") can result.

    Cell Proliferation Recall that mitosis consists of

    Of these, only the G1 phase is variable in length, mainly because of the variation in an optional resting phase known as G0. The cell must pass through checkpoints--fail-safe mechanisms that won't allow the cell to proceed to one phase until all the parts of the previous phase are complete--during the cell cycle.

    Enzymes involved in the proliferation process are

    Cell Death

  • In multicellular organisms, programmed cell death occurs primarily in somatic cells.
  • cell proliferation replaces somatic cells lost to cell death.
  • mechanisms have evolved to eliminate certain cells, and the process of such programmed cell death is known as apoptosis. Enzymes & cells involved in apoptosis are

    The cell must respond to internal and external environmental cues to know when to proliferate and when to die. These consist of

  • intercellular chemical signals
  • receptors of those signals
  • transduction systems that relay the signal from receptor to other parts of the cell

    Cyclins

  • exist in families of related enzymes, each of which is present only during a specific phase of the cell cycle.
  • are generated by the previous phase's specific cyclin-CDK complex, which acts as a transcription factor for its gene activation
  • don't last long in the cell. One type is rapidly degraded and replaced by the next via

    CDK

    Variations in cyclin-CDK follow the cell cycle...

    One example of a well-known transcription factor activation is the Rb/E2F protein regulation of the G1 to S phase transition in mammals cells...

    Apoptosis This is sometimes referred to as "programmed cell death" and it is

    Proliferation vs. Apoptosis Controls These are interrelated, and may induce apoptosis in cells that fail to successfully complete some phase of cell cycle.

    Intracellular signals

  • cell cycle negative controls: inhibition of CDK-cyclin (see illustration below)
  • cell cycle positive controls: activation of CDK-cyclin

  • apoptosis positive controls: leakage of cytochrome c from defective mitochondria acts as a trigger for apoptosis
  • apoptosis negative controls: proteins such as Bcl-2 and Bcl-x block the release of cytochrome c from mitochondria, possibly stabilizing the mitochondrial membrane and preventing its rupture). This maintains the apoptosis system in "off" mode

    Extracellular signals

  • based on cell-cell communication
  • secreted molecules (paracrine signals act locally, are not sent via circulatory system)
  • direct cell-cell contact

    An example of inhibitory control:

    1. DNA is damaged by some mutagen during G1

    2. This inhibits the activity of CDK-cyclin complexes

    3. The protein responsible for this inhibition is named p53 (and the gene that encodes it, p53); it senses mismatches in the DNA strand.

    4. In the presence of such mismatches, p53 protein activates another protein named p21 (encoded by a gene named p21).

    5. When p21 is present in high concentration, it binds to CDK-cyclin complexes in the cell, inhibiting their kinase activity and preventing phosphorylation of proteins.

    6. Without proper protein phosphorylation, the cell cycle cannot continue until DNA mismatches have been repaired.

    7. Inhibitory processes are reversed once the DNA mismatches are repaired, as p53 levels drop in response to lowered levels of DNA mismatches.

    8. As p53 levels drop, the binding capacity of p21 also drops.

    9. As p21 levels drop, these proteins diffuse off the cyclin-CDK proteins, which then can resume their normal activity.

    Mutations in the genes encoding any of these highly specific "tumor suppressor" genes can result in cancer.

    Cancer cells are

    Many different cell types can be altered to become cancerous. What are the common threads uniting them?

  • A cancer cell can be considered an aberrent cell with an accumulation of mutations that cause it to lose its proliferation and apoptotic controls. (In other words, a single mutation in a cell is not likely to cause it to become cancerous.) A cell that has a mutation preventing apoptosis will have more time to accumulate proliferation-promoting mutations that will cause it to become cancerous.
  • Some such mutations can be inherited via the germline (as in familial/heritable cancers)
  • Others can arise de novoin the somatic cell lineage of a particular cell due to mutagenesis.
    Two major types of mutations are associated with carcinogenesis. Mutations in

    Oncogenes An oncogene is a dominant mutant gene that contributes to the formation of (animal) cancer.

  • The non-mutant form of an oncogene is known as a proto-oncogene.

    Oncogenes have been isolated from certain viruses known to have carcinogenic activity.

  • About 100 different oncogenes have thus far been identified.
  • Oncogenes can change due to Tumor Suppressor Genes These are genes that encode an active form of protein that ordinarily functions to maintain normal proliferation (e.g., rb; a mutant form of this gene encodes a mutant, non-functional RB protein, and cells containing this mutation proliferate out of control.

    Other tumor suppressor mutations involve problems with positive regulation of apoptosis (e.g., p53).

    Mutations of any of these genes result in inactive forms of the protein, allowing uncontrolled proliferation and/or lack of apoptosis.

    Tumor suppressor mutations are generally recessive.

    Tumor-promoting mutations were first identified by study of cells from cancer patients in single families showing the same type of cancer. These shared cancers variously showed As you can see, a wide variety of mutations can potentially cause cancer, and cancer cannot be considered a single "disease." It is a failure of normal gene function.

    As geneticists continue to work in the realm of FUNCTIONAL GENOMICS, determining not only the sequence of our genes, but their function, greater headway will be made in finally finding the answer to controlling this genetic disorder.

    (Will you be the one to find the key?)