Reading: 7.1 Mutations: Primary Tools of genetic Analysis p. 200 Suggested Problems: Solved Problem I: 12, 13. Also work the practice problems following these lecture notes. 7.1.1 Mutations are heritable changes in DA base sequences p. 200 reverse mutation vs. suppressor mutation 7.1.2 Mutations may be classified by how they change DA p. 200 DA Protein Phenotype Base Pair Substitution Silent Missense onsense eutral Alteration of Function ull Insertion/Deletion Frameshift spontaneous vs. induced [intrinsic vs. extrinsic] SPOTAEOUS vs IDUCED (Intrinsic vs Extrinsic) MUTATIOS The distinction between "spontaneous" and "induced" mutations is based on an operational definition of induced mutations as those which result from intentional exposure of an organism by an investigator to chemicals, radiation or other mutagenic treatment. Spontaneous mutations, then, are those which occur "naturally" in the environment without intervention of an investigator. By these definitions spontaneous mutations could result from the same type of molecular processes as induced mutations because there are naturally occurring environmental mutagens and naturally occurring sources of radiation. A more meaningful distinction can be drawn between those mutations which result from the interaction of DA with some external influence (such as chemicals or radiation) whether or not they were intentionally applied by an investigator and those which result from inherent properties of DA itself or from essential attributes of DA replication. These latter would be "spontaneous" in a non-trivial sense, although admittedly some mutagenic treatments act by amplifying the rate of spontaneous events or by interfering with mechanisms that repair spontaneouly occuring premutagenic lesions. BP Substitutions Base Pair Substitutions are one class of what the text calls Point Mutations. The other class of point mutation is the indel class. The term indel refers to changes by addition or deletion of one or several base pairs. Base pair substitution implies a change in the identity of a single base pair with no change in the total number of base pairs. There are 2 flavors of bp substitution Transition replacement of one purine by the other purine (or of one pyrimidine by the other) Transversion replacement of a purine by a pyrimidine (or of a pyrimidine by a purine) 1 of 9
For each of the 4 base pairs 2 transversions and one transition are possible. Example: AT GC = transition AT CG = transversion AT TA = transversion (ote that AT GC, TA CG, A G, and T C all designate the same type of bp substitution.) The effect of a base pair substitution on the phenotype may range from neutral to lethal depending on the genomic and environmental contexts. Let s consider the effect of bp substitution within a coding sequence (gene), on the amino acid sequence of the protein product of the gene. BP substitution in a coding sequence may be either silent, missense or nonsense. Silent substitutions change an amino acid codon to a synonomous codon. The sequence of the protein is unaltered. Missense substitutions alter a codon for one amino acid to a codon for a different amino acid. This changes the identity of one amino acid in the protein sequence. onsense substitutions alter an amino acid codon to one of the three termination codons. The protein product in this case is only a shortened amino terminal fragment of the original and that usually has no function. BP substitutions that convert a termination codon to an amino acid codon have no specific designation that I am aware of. 7.1.3 Spontaneous mutations occur at a very low rate p. 201 The "low" rate of spontaneous mutations is primarily a result of effecient DA repair mechanisms. 7.1.4 Spontaneous mutations arise from many kinds of random events p. 202 We will not cover the Fluctuation Test (Sub-section 7.1.4.1 and Fig. 7.4). We will not cover Unequal crossing-over and transposable elements (Sub-section 7.1.4.4). We will not cover Unstable trinucleotide repeats (Sub-section 7.1.4.5). random vs. directed (adaptive) The most important types of spontaneous processes leading to mutation are: misincorporation of bases during DA replication depurination deamination of cytosine* or (less commonly) adenine small additions and deletions (indel mutations) due to "slipped mispairing" insertional inactivation by transposable elements * I will this mechanism in detail. 2 of 9
Spontaneous Mutation by Deamination of Cytosine Cytosine is the least stable of the four bases in nucleic acids and undergoes "spontaneous" oxidative deamination at a rate of 5 x 10-13 sec - 1 in double- stranded DA. At this rate, 40 to 100 deamination events should occur in a human genome per day. H H H 2 H O C U O O R R Deamination of cytosine produces U, leading to GU abnormal base pair mismatches. The GU mismatch will be fixed as a GC - - > AT transition substitution following two subsequent rounds of DA replication. A repair enzyme, Uracil DA glycosylase, can excise U from the GU mispair by cleaving the glycoside bond. This is shown in Fig. 13.23. Again, the figure is misleading because it does not suggest GU is a mismatch. The structure resulting from base excision is called an "AP Site". The AP site is subsequently a substrate for the AP endonuclease excision and ss gap reapair pathways (strand excision; discussed in the text) which restore the original target GC. The text also briefly notes the possibility that T ocurrs in DA, rather than U, because it creates a context for efficient repair of AU lesions. (See discussion of 5-meC below.) The best explanation for the use of T rather than U in DA is that it facilitates the repair of GU mismatches. C:G to T:A transitions dominate the spectra of spontaneous base substitutions in Escherichia coli and in mammalian cells. In primates, C:G to T:A transitions are thought to account for over 40% of all base substitutions within the globin cluster. These data argue for the significance of cytosine deamination in mutagenesis. 3 of 9
H2... G C G U REPLICATIO Ạ. U REPLICATIO Ạ. T Base Excision! (Uracil DA Glycosylase) G AP Site REPLICATIO ucleotide Excision G Gap Repair! Polymerase + Ligase itrous Acid itrous acid is a well- known chemical mutagen that accelerates cytosine deamination, and also produces a significant level of adenine deamination. The deamination product of adenine is hypoxanthine, which pairs with cytosine (See Fig. 13.15 5 or 13.8 6 ). Thus, nitrous acid is a bidirectional transition mutagen. 5-meC The rate of deamination of 5-methylcytosine (5meC) in DA is 2 to 4 times higher than that for cytosine. Additionally, deamination of 5meC creates GT normal base pair mismatches that are more difficult to repair than GU mismatches. (Though at least one example of a GT* specific glycosylase has been reported.) Specific C residues are methylated to 5-meC in the DA of most, if not all, organisms. (Many bacterial restriction/modification methylases, for example, create 5-me C residues.) Several studies have shown these 5-meC residues to be hotspots for base substitution mutations in organisms as diverse as E. coli and humans. In human DA, approximately 1% of the bases are 5-meC, and the vast majority of these occur in 5' CpG sequences. An estimated 30% of germ-line point mutations associated with inherited human genetic disorders are GC to AT transitions at CpG sequences. Furthermore, there is strong circumstantial evidence that deamination of 5-meC at CpG sequences is responsible for a large fraction of somatic point mutations in the p53 tumor suppressor gene (16). It may be appropriate to consider 5-meC as an endogenous mutagen and carcinogen. The repair enzyme Uracil DA glycosylase can excise U from the GU mispair to produce an AP site subject to most of the same scenarios as the AP sites generated by depurination. 4 of 9
7.1.5 Mutagens Induce mutations p. 207 I will cover mutagenesis and repair of UV-induced lesions. I differ from the book insofar as I do not consider UV mutagenesis to be "spontaneous". UV-Induced DA Damage and Repair Bacteriologists discovered in the 19 th Century that direct sunlight exposure was lethal to bacteria and other microorganisms. Subsequent studies showed the lethal action of sunlight to be primarily attributable to the UV portion of the spectrum between 250-260 nm. This corresponds to the Amax for the DA bases (whereas the Amax for proteins is near 280 nm). UV irradiation produced by (germicidal lamps) is has been used a method for disinfection since the early 20th Century. UV absorbance (A 260 ) is a common and simple assay for [DA] in relatively pure samples (50 ug/ml = A = 1.0). A 260 increases 40% in ss relative to ds DA (hyperchromic shift). UV mutagenicity (as opposed to lethality) for bacteria was demonstrated in 1914 by V. Henri, 13 years before Muller s demonstration of X-ray mutagenesis in Drosophila. DA Damage Caused by Short Wave UV UV-induced DA lesions primarily involve covalent crosslinking of two adjacent pyrimidines in the same strand. The best known, and most common photoproducts are cyclopyrimidine dimers (CPD s) involving adjacent thymines (i.e. the so-called "thymine dimer"). Another photoproduct is a 6,4 linkage of adjacent pyrimidines. These crosslinked bases are the principal reason that exposure to short wave UV is lethal to cells. DA replication complexes stall at these damaged bases, blocking cellular reproduction. Along with other types of DA damage caused my certain chemicals, these altered bases are often referred to as "non-coding lesions". It is fascinating to contemplate the predicament of organisims on the early earth, who were potentially exposed to intense solar UV radiation flux. The atmospheric ozone layer responsible for blocking short wave UV did not exist for the first several billion years of earth history. We should not be surprised to find that organisms have evolved multiple, redundant molecular strategies to repair, or to overcome UV damage. I believe it is correct to claim these survival strategies were all discovered and first characterized by studying bacteria, particularly E. coli. 5 of 9
Direct Repair of UV-Induced DA Damage by Photolyases Photoreactivation is the term suggested by Delbrück for a phenomenon reported by Kelner (1949) in Streptomyces griseus. Following a UV exposure sufficient to reduce survivors to 10-5, a subsequent, immediate exposure to visible light raises survivors to 10-1. Photoreactivation requires the visible light exposure AFTER the UV exposure. The productivity of visible exposure decreases to background over 2 hours in E. coli. Photoreactivation is due the activity of a class of enzymes known as CPD photolyases. Properties of CPD Photolyases: Single subunit protein. Bind to CPD lesions in dark. Catalyze reversal to native structure following exposure to 370 nm light. ucleotide Excision Repair Many versions of DA nucleotide excision repair (ER) mechanisms are known, with specificity for various types of lesion. Excision repair of TT dimers in E. coli was the first such mechanism characterized. The UvrABC complex binds to DA specifically in the vicinity of pyrimidine dimers and makes 2 single strand cuts in the dimer-containing strand on either side of the lesion. With the aid 6 of 9
of UvrD (helicase II), a short fragment containing the dimer is released. The resulting gap in the DA helix is repaired by a generic scenario involving DA Polymerase I and DA ligase. Xeroderma pigmentosa (=Xerodoma pigmentosum) refers to a group of closely related human clinical conditions related to hereditary insufficiency of CPD excision repair. The incidence of XP is 1/250,00, and is inherited as an autosomal recessive. In severe manifestations, an individual's activity must be severely limited to avoid sunlight exposure, and life expectancy is reduced by an extraordinarily high rate of skin cancers. 7-9 different human XP genes have been identified, most of which contribute to the excision of CPD dimers. Introduction of a recombinant CPD photolyase gene (from bacteria?) to XP individuals has been suggested as possible candidate for human gene therapy. The "SOS Response" on-coding lesions that are not repaired directly, or removed by ER, may block progress of a normal DA replication fork complex, leading to cell death. The SOS response is a strategy to avoid this fate - "Plan C" so to speak. In the SOS response, replication forks are allowed to progress on templates containing noncoding lesions ("translesional synthesis"). This requires the activity of one or more special "error-prone" DA polymerases; PolV (UmuC,D) is the predominant one in E. coli. The replication errors introduced by the activity of these polymerases are apparently the main basis for UV mutagenesis. Errors may include transitions, transversions, and the insertion or deletion of one to several bases. (If your only options were mutation or death, which would you choose?) ote that the SOS response does not repair the DA damage; it only buys more times for other repair mechanisms. Components of the SOS pathway are not expressed under normal growth conditions, their production must be induced by damaged DA. The complex mechanism by which the damage is recognized, and the SOS response proteins (such as Pol V) induced, is beyond the scope of the present discussion. 7 of 9
7.1.6 DA Repair Mechanisms minimize mutations p. 209 7.17 Mutations have consequences for species evolution as well as individual survivalp. 215 OMIT this section 8 of 9
PRACTICE PROBLEM SET 1. For a CG base pair, write all possible base pair substitution changes and label each as transition or transversion. 2. For each of the following bp substitutions, label as transition or transversion. CG GC TA GC AT GC CG AT AT TA 3. For an AAA (Lys) codon, how many of the possible bp substitution changes are synonoumous, missense, nonsense? 4. Starting with a UUA (Leu) codon, how many different missense replacements are possible following a single bp substitution? 5. The amino acid sequence of the carboxyl terminus of a protein is: - Ala 98 Gly 99 Val 100 COOH Explain how a single bp substitution could alter the protein to - Ala 98 Gly 99 Val 100 Cys 101 COOH 9 of 9