1. Genetica per Scienze Naturali a.a. 08-09 prof S. Presciuttini MUTATION MECHANISMS Questo documento è pubblicato sotto licenza Creative Commons Attribuzione – Non commerciale – Condividi allo stesso modo http://creativecommons.org/licenses/by-nc-sa/2.5/deed.it

2. Genetica per Scienze Naturali a.a. 08-09 prof S. Presciuttini Transitions vs transversions Base substitutions can be subdivided into transitions and transversions. Base substitutions can be subdivided into transitions and transversions. To describe these subtypes, we consider how a mutation alters the sequence on one DNA strand. A transition is the replacement of a base by the other base of the same chemical category (purine replaced by purine: either A to G or G to A; pyrimidine replaced by pyrimidine: either C to T or T to C). A transversion is the opposite the replacement of a base of one chemical category by a base of the other (pyrimidine replaced by purine: C to A, C to G, T to A, T to G; purine replaced by pyrimidine: A to C, A to T, G to C, G to T). In describing the same changes at the double-stranded level of DNA, we must state both members of a base pair: an example of a transition would be G·C  A·T; that of a transversion would be G·C  T·A. To describe these subtypes, we consider how a mutation alters the sequence on one DNA strand. A transition is the replacement of a base by the other base of the same chemical category (purine replaced by purine: either A to G or G to A; pyrimidine replaced by pyrimidine: either C to T or T to C). A transversion is the opposite the replacement of a base of one chemical category by a base of the other (pyrimidine replaced by purine: C to A, C to G, T to A, T to G; purine replaced by pyrimidine: A to C, A to T, G to C, G to T). In describing the same changes at the double-stranded level of DNA, we must state both members of a base pair: an example of a transition would be G·C  A·T; that of a transversion would be G·C  T·A.

3. Genetica per Scienze Naturali a.a. 08-09 prof S. Presciuttini Tautomeric bases and point mutations Transitions All the mispairs described so far lead to transitions, in which a purine substitutes for a purine or a pyrimidine for a pyrimidine. The bacterial DNA polymerase III has an editing capacity that recognizes such mismatches and excises them, thus greatly reducing the mutation rate. Other repair systems corrects many of the mismatched bases that escape correction by the polymerase editing function. All the mispairs described so far lead to transitions, in which a purine substitutes for a purine or a pyrimidine for a pyrimidine. The bacterial DNA polymerase III has an editing capacity that recognizes such mismatches and excises them, thus greatly reducing the mutation rate. Other repair systems corrects many of the mismatched bases that escape correction by the polymerase editing function. Transversions Transversions In transversions, a pyrimidine substitutes for a purine or vice versa. Transversions cannot be generated by the mismatches due to tautomeries. With bases in the DNA in the normal orientation, creation of a transversion by a replication error would require, at some point in the course of replication, mispairing of a purine with a purine or a pyrimidine with a pyrimidine. Although the dimensions of the DNA double helix render such mispairs energetically unfavorable, we now know from X-ray diffraction studies that G A pairs, as well as other purine purine pairs, can form. In transversions, a pyrimidine substitutes for a purine or vice versa. Transversions cannot be generated by the mismatches due to tautomeries. With bases in the DNA in the normal orientation, creation of a transversion by a replication error would require, at some point in the course of replication, mispairing of a purine with a purine or a pyrimidine with a pyrimidine. Although the dimensions of the DNA double helix render such mispairs energetically unfavorable, we now know from X-ray diffraction studies that G A pairs, as well as other purine purine pairs, can form.

4. Genetica per Scienze Naturali a.a. 08-09 prof S. Presciuttini Frameshift mutations A model for frameshift formation. (a-c) In DNA synthesis, the newly synthesized strand slips, looping out one or several bases. This loop is stabilized by the pairing afforded by the repetitive-sequence unit (the A bases in this case). An addition of one base pair, AT, will result at the next round of replication in this example. (d-f) If, instead of the newly synthesized strand, the template strand slips, then a deletion results. Here the repeating unit is a CT dinucleotide. After slippage, a deletion of two base pairs (CG and TA) would result at the next round of replication.

5. Genetica per Scienze Naturali a.a. 08-09 prof S. Presciuttini Spontaneous DNA lesions In addition to replication errors, spontaneous lesions, naturally occurring damage to the DNA, can generate mutations. Two of the most frequent spontaneous lesions result from depurination and deamination. In addition to replication errors, spontaneous lesions, naturally occurring damage to the DNA, can generate mutations. Two of the most frequent spontaneous lesions result from depurination and deamination. Depurination, the more common of the two, consists of the interruption of the glycosidic bond between the base and deoxyribose and the subsequent loss of a guanine or an adenine residue from the DNA. Depurination, the more common of the two, consists of the interruption of the glycosidic bond between the base and deoxyribose and the subsequent loss of a guanine or an adenine residue from the DNA.  A mammalian cell spontaneously loses about 10,000 purines from its DNA in a 20- hour cell-generation period at 37°C. If these lesions were to persist, they would result in significant genetic damage because, in replication, the resulting apurinic sites cannot specify a base complementary to the original purine. However, efficient repair systems remove apurinic sites. The deamination of cytosine yields uracil. Unrepaired uracil residues will pair with adenine in replication, resulting in the conversion of a GC pair into an AT pair (a GC  AT transition). The deamination of cytosine yields uracil. Unrepaired uracil residues will pair with adenine in replication, resulting in the conversion of a GC pair into an AT pair (a GC  AT transition).

6. Genetica per Scienze Naturali a.a. 08-09 prof S. Presciuttini Other frequent spontaneous DNA lesions Deamination of methylcytosine (meC) to thymine can also occur. meC occurs in the human genome at the sequence 5'CpG3', which is normally avoided in the coding regions of genes. If the meC is deaminated to T, there is no repair system which can recognize and remove it (because T is a normal base in DNA). This means that wherever CpG occurs in genes it is a "hot spot" for mutation. Deamination of methylcytosine (meC) to thymine can also occur. meC occurs in the human genome at the sequence 5'CpG3', which is normally avoided in the coding regions of genes. If the meC is deaminated to T, there is no repair system which can recognize and remove it (because T is a normal base in DNA). This means that wherever CpG occurs in genes it is a "hot spot" for mutation. Another type of spontaneous DNA damage that occurs frequently is damage to the bases by free radicals of oxygen. These arise in cells as a result of oxidative metabolism and also are formed by physical agents such as radiation. An important oxidation product is 8-hydroxyguanine, which mispairs with adenine, resulting in G:C to T:A transversions. Another type of spontaneous DNA damage that occurs frequently is damage to the bases by free radicals of oxygen. These arise in cells as a result of oxidative metabolism and also are formed by physical agents such as radiation. An important oxidation product is 8-hydroxyguanine, which mispairs with adenine, resulting in G:C to T:A transversions. Still another type of spontaneous DNA damage is alkylation, the addition of alkyl (methyl, ethyl, occasionally propyl) groups to the bases or backbone of DNA. Alkylation can occur through reaction of compounds such as S-adenosyl methionine with DNA. Alkylated bases may be subject to spontaneous breakdown or mispairing. Still another type of spontaneous DNA damage is alkylation, the addition of alkyl (methyl, ethyl, occasionally propyl) groups to the bases or backbone of DNA. Alkylation can occur through reaction of compounds such as S-adenosyl methionine with DNA. Alkylated bases may be subject to spontaneous breakdown or mispairing.

7. Genetica per Scienze Naturali a.a. 08-09 prof S. Presciuttini A mutational spectrum The distribution of 140 spontaneous mutations in lacI. Each occurrence of a point mutation is indicated by a box. Red boxes designate fast-reverting mutations. Deletions (gold) are represented below. The I map is given in terms of the amino acid number in the corresponding I-encoded lac repressor. Allele numbers refer to mutations that have been analyzed at the DNA sequence level. The mutations S114 and S58 (circles) result from the insertion of transposable elements. S28 (red circle) is a duplication of 88 base pairs.

8. Genetica per Scienze Naturali a.a. 08-09 prof S. Presciuttini Hot spot mutation sites In the 1970s, Jeffrey Miller and his co-workers examined mutational hot spots in the lacI gene of E. coli. Hot spots are sites in a gene that are much more mutable than other sites. The lacI work showed that certain hot spots result from repeated sequences. In lacI, a four-base-pair sequence repeated three times in tandem in the wild type is the cause of the hot spots (for simplicity, only one strand of the double strand of DNA is indicated): In the 1970s, Jeffrey Miller and his co-workers examined mutational hot spots in the lacI gene of E. coli. Hot spots are sites in a gene that are much more mutable than other sites. The lacI work showed that certain hot spots result from repeated sequences. In lacI, a four-base-pair sequence repeated three times in tandem in the wild type is the cause of the hot spots (for simplicity, only one strand of the double strand of DNA is indicated): The major hot spot, represented here by the mutations FS5, FS25, FS45, and FS65, results from the addition of one extra set of the four bases CTGG to one strand of the DNA. This hot spot reverts at a high rate, losing the extra set of four bases. The minor hot spot, represented here by the mutations FS2 and FS84, results from the loss of one set of the four bases CTGG. This mutant does not readily regain the lost set of four base pairs. The major hot spot, represented here by the mutations FS5, FS25, FS45, and FS65, results from the addition of one extra set of the four bases CTGG to one strand of the DNA. This hot spot reverts at a high rate, losing the extra set of four bases. The minor hot spot, represented here by the mutations FS2 and FS84, results from the loss of one set of the four bases CTGG. This mutant does not readily regain the lost set of four base pairs.

9. Genetica per Scienze Naturali a.a. 08-09 prof S. Presciuttini Deletions and duplications Large deletions (more than a few base pairs) constitute a sizable fraction of sponta- neous mutations, as already shown. The majority, although not all, of the deletions occur at repeated sequences. The figure below shows the results for the first 12 deletions analyzed at the DNA sequence level, presented by Miller and his co-workers in 1978. Further studies showed that hot spots for deletions are in the longest repeated sequences. Duplications of segments of DNA have been observed in many organisms. Like deletions, they often occur at sequence repeats. Large deletions (more than a few base pairs) constitute a sizable fraction of sponta- neous mutations, as already shown. The majority, although not all, of the deletions occur at repeated sequences. The figure below shows the results for the first 12 deletions analyzed at the DNA sequence level, presented by Miller and his co-workers in 1978. Further studies showed that hot spots for deletions are in the longest repeated sequences. Duplications of segments of DNA have been observed in many organisms. Like deletions, they often occur at sequence repeats. How do deletions and duplications form? Deletions may be generated as replication errors. For example, an extension of the model of slipped mispairing could explain why deletions predominate at short repeated sequences. Alternatively, deletions and duplications could be generated by recombinational mechanisms.

10. Genetica per Scienze Naturali a.a. 08-09 prof S. Presciuttini Do mutations arise spontaneously? Since the beginning of the past century it was widely known that when a population of bacteria is exposed to a toxic environment, some rare cells may acquire the ability to grow much better than most of the other cells in the population (resistance). Since the beginning of the past century it was widely known that when a population of bacteria is exposed to a toxic environment, some rare cells may acquire the ability to grow much better than most of the other cells in the population (resistance). In addition, the resistant phenotype was often stable and so appeared to be the consequence of true genetic mutations. In addition, the resistant phenotype was often stable and so appeared to be the consequence of true genetic mutations. However, it was not known if these mutants were produced spontaneously or if they were induced by the presence of the toxic agent. For many years, most microbiologists believed that mutations in bacteria were induced by exposure to a particular environment. (Salvador Luria once said that "bacteriology is the last stronghold of Lamarckism".) However, it was not known if these mutants were produced spontaneously or if they were induced by the presence of the toxic agent. For many years, most microbiologists believed that mutations in bacteria were induced by exposure to a particular environment. (Salvador Luria once said that "bacteriology is the last stronghold of Lamarckism".) The first rigorous evidence that mutations in bacteria followed the Darwinian principle of random variation and selection came from a study by Luria and Delbruck (1943). The first rigorous evidence that mutations in bacteria followed the Darwinian principle of random variation and selection came from a study by Luria and Delbruck (1943).  They studied mutations that made E. coli resistant to phage T1. Phage T1 interacts with specific receptors on the surface of E. coli, enters the cell, and subsequently kills the cell. Thus, when E. coli is spread on a plate with 10 10 phage T1, most of the cells are killed. However, rare T1 resistant (Ton R ) colonies can arise due to mutations in E. coli that alter the T1 receptor in the cell wall.

11. Genetica per Scienze Naturali a.a. 08-09 prof S. Presciuttini Spontaneous vs induced mutations Luria noted that the two theories of mutation (spontaneous vs induced) made different statistical predictions. Luria noted that the two theories of mutation (spontaneous vs induced) made different statistical predictions. If the Ton R mutations were induced by exposure to phage T1, then every population of cells would be expected to have an equal probability of developing resistance and hence a nearly equal number of Ton R colonies would be produced from different cultures. If the Ton R mutations were induced by exposure to phage T1, then every population of cells would be expected to have an equal probability of developing resistance and hence a nearly equal number of Ton R colonies would be produced from different cultures.  For example, if there was a 10 -8 probability that exposure to phage T1 would induce a Ton R mutant, then approximately 10 colonies would arise on each plate spread with 10 9 bacteria. In contrast, if Ton R mutations were due to random, spontaneous mutations that occured sometime during the growth of the culture prior to exposure to phage T1, then the number of Ton R colonies would vary widely between each different culture. In contrast, if Ton R mutations were due to random, spontaneous mutations that occured sometime during the growth of the culture prior to exposure to phage T1, then the number of Ton R colonies would vary widely between each different culture.  For example, although there is an equal probability (say 10 -8 ) that a Ton R mutant would arise per cell division, the number of resistant bacteria in each culture would depend upon whether the mutation occurred during one of the first cell divisions or one of the last cell divisions.

12. Genetica per Scienze Naturali a.a. 08-09 prof S. Presciuttini Different predictions The figure shows a cartoon of the two alternative predictions. Ton S cells are indicated in white and Ton R cells are indicated in black. The shaded area indicates when the cells were exposed to phage T1. The figure shows a cartoon of the two alternative predictions. Ton S cells are indicated in white and Ton R cells are indicated in black. The shaded area indicates when the cells were exposed to phage T1. In either of these two cases, if multiple samples from a single culture of bacteria were plated on phage T1, each of the resulting plates should yield approximately the same number of colonies. However, the two possibilities can be distinguished mathematically by comparing the mean and variance of the number of the number of mutants in each culture. In either of these two cases, if multiple samples from a single culture of bacteria were plated on phage T1, each of the resulting plates should yield approximately the same number of colonies. However, the two possibilities can be distinguished mathematically by comparing the mean and variance of the number of the number of mutants in each culture.

13. Genetica per Scienze Naturali a.a. 08-09 prof S. Presciuttini The “fluctuation test” Luria and Delbruck designed their "fluctuation test" as follows. Luria and Delbruck designed their "fluctuation test" as follows. They inoculated 20 small cultures (0.2 ml), each with a few cells, and incubated them until there were 10 8 cells per ml. At the same time, a much larger culture also was inoculated and incubated until there were 10 8 cells per milliliter. The 20 individual cultures and 10 aliquots of the same size from the large culture were plated in the presence of phage. They inoculated 20 small cultures (0.2 ml), each with a few cells, and incubated them until there were 10 8 cells per ml. At the same time, a much larger culture also was inoculated and incubated until there were 10 8 cells per milliliter. The 20 individual cultures and 10 aliquots of the same size from the large culture were plated in the presence of phage. If resistance were due to random mutation during the incubation period, each culture tube would produce a different number of resistant cells; the number would vary depending on how early in the cascade of growing cells the mutation occurred. These resistant cells would each produce a separate colony when plated with the T1 phage. If resistance were due to random mutation during the incubation period, each culture tube would produce a different number of resistant cells; the number would vary depending on how early in the cascade of growing cells the mutation occurred. These resistant cells would each produce a separate colony when plated with the T1 phage. If resistance were due to a physiological adaptation occurring after exposure of the cells on the T1 phage, all cultures would be expected to generate resistant cells at roughly the same rate and little variation from culture to culture would be expected. If resistance were due to a physiological adaptation occurring after exposure of the cells on the T1 phage, all cultures would be expected to generate resistant cells at roughly the same rate and little variation from culture to culture would be expected.

14. Genetica per Scienze Naturali a.a. 08-09 prof S. Presciuttini Results of the fluctuation test Variation from plate to plate was indeed observed in the individual 0.2-ml cultures but not in the samples from the bulk culture (which represent a kind of control experiment). This situation cannot be explained by physiological change, because all the samples spread had the same approximate number of cells. The simplest explanation is random mutation, occurring early in the incubation of the 0.2-ml cultures (producing a large number of resistant cells and, therefore, a large number of colonies) or late (producing few resistant cells and colonies) or not at all (producing no resistant cells).