1. Genetica per Scienze Naturali a.a. 05-06 prof S. Presciuttini 1. Mechanisms of Spontaneous Mutation The origin of spontaneous hereditary change has always been a topic of considerable interest. It is known now that spontaneous mutations arise from a variety of sources, including errors in DNA replication, spontaneous lesions, and other more complex mechanisms. The origin of spontaneous hereditary change has always been a topic of considerable interest. It is known now that spontaneous mutations arise from a variety of sources, including errors in DNA replication, spontaneous lesions, and other more complex mechanisms. Spontaneous mutations are very rare, making it difficult to determine the underlying mechanisms. How then do we have insight into the processes governing spontaneous mutation? Even though they are rare, some selective systems allow numerous spontaneous mutations to be obtained and then characterized at the molecular level for example, their DNA sequences can be determined. From the nature of the sequence changes, inferences can be made about the processes that have led to the spontaneous mutations. Spontaneous mutations are very rare, making it difficult to determine the underlying mechanisms. How then do we have insight into the processes governing spontaneous mutation? Even though they are rare, some selective systems allow numerous spontaneous mutations to be obtained and then characterized at the molecular level for example, their DNA sequences can be determined. From the nature of the sequence changes, inferences can be made about the processes that have led to the spontaneous mutations.

2. Genetica per Scienze Naturali a.a. 05-06 prof S. Presciuttini 2. Errors in DNA replication Mispairing in the course of replication is a source of spontaneous base substitution. Mispairing in the course of replication is a source of spontaneous base substitution. Tautomers of bases Tautomers of bases Each of the bases in DNA can appear in one of several forms, called tautomers, which are isomers that differ in the positions of their atoms and in the bonds between the atoms. The forms are in equilibrium. The keto form of each base is normally present in DNA, whereas the imino and enol forms of the bases are rare. Mispairs resulting from the change of one tautomer into another are termed a tautomeric shift. Each of the bases in DNA can appear in one of several forms, called tautomers, which are isomers that differ in the positions of their atoms and in the bonds between the atoms. The forms are in equilibrium. The keto form of each base is normally present in DNA, whereas the imino and enol forms of the bases are rare. Mispairs resulting from the change of one tautomer into another are termed a tautomeric shift. Most mispairing mutations are transitions. This is likely to be because an A·C or G·T mispair does not distort the DNA double helix as much as A·G or C·T base pairs do. An error in DNA replication can occur when an illegitimate nucleotide pair (say, AC) forms in DNA synthesis, leading to a base substitution. Most mispairing mutations are transitions. This is likely to be because an A·C or G·T mispair does not distort the DNA double helix as much as A·G or C·T base pairs do. An error in DNA replication can occur when an illegitimate nucleotide pair (say, AC) forms in DNA synthesis, leading to a base substitution. Mispairs can also result when one of the bases becomes ionized. This type of mispair may occur more frequently than mispairs due to imino and enol forms of bases. Mispairs can also result when one of the bases becomes ionized. This type of mispair may occur more frequently than mispairs due to imino and enol forms of bases.

3. Genetica per Scienze Naturali a.a. 05-06 prof S. Presciuttini 3. Base mismatches Mismatched bases. (a) Mispairs resulting from rare tautomeric forms of the pyrimidines; (b) mispairs resulting from rare tautomeric forms of the purines.

4. Genetica per Scienze Naturali a.a. 05-06 prof S. Presciuttini 4. From mispairs to mutations (a) A guanine undergoes a tautomeric shift to its rare enol form (G*) at the time of replication. (b) In its enol form, it pairs with thymine. (c and d) In the next replication, the guanine shifts back to its more stable keto form. The thymine incorporated opposite the enol form of guanine, seen in part b, directs the incorporation of adenine in the subsequent replication. The net result is a GC  AT mutation. If a guanine undergoes a tautomeric shift from the common keto form to the rare enol form at the time of incorporation (as a nucleoside triphosphate, rather than in the template strand diagrammed here), it will be incorporated opposite thymine in the template strand and cause an AT  GC mutation.

5. Genetica per Scienze Naturali a.a. 05-06 prof S. Presciuttini 5. 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.

6. Genetica per Scienze Naturali a.a. 05-06 prof S. Presciuttini 6. Tautomeric bases and point mutationsTransitions 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.

7. Genetica per Scienze Naturali a.a. 05-06 prof S. Presciuttini 7. 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.

8. Genetica per Scienze Naturali a.a. 05-06 prof S. Presciuttini 8. 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).

9. Genetica per Scienze Naturali a.a. 05-06 prof S. Presciuttini 9. 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.

10. Genetica per Scienze Naturali a.a. 05-06 prof S. Presciuttini 10. 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.

11. Genetica per Scienze Naturali a.a. 05-06 prof S. Presciuttini 11. 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.

12. Genetica per Scienze Naturali a.a. 05-06 prof S. Presciuttini 12. 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.