SECTION E - DNA DAMAGE, REPAIR, AND RECOMBINATION E2 Mutagenesis Key Notes Mutation Physical mutagens Chemical Direct mutagenesis Indirect mutagenesis and transiesion DNA Related topics ‘Mutations are heritable permanent changes in the base sequence of DNA. Point mutations may be transitions (e.g. G-C-+A-T) or transversions (eg. G-C+T-A). Deletions and insertions involve the loss or addition of bases and can cause frameshifts in the genetic code. Silent mutations have no phenotypic effect, while missense and nonsense mutations change the amino acid sequence of the encoded protein. Replication fidelity The high accuracy of DNA replication (one error per 10"° bases incorporated) depends on a combination of (1) proper base pairing of template strand and incoming nucleotide in the active site of the DNA polymerase, (ii) proofreading of the incorporated base by 3'—95' exonuclease and (ii) mismatch. ir. The use of RNA primers ensures high fidelity of Tonizing (eg. X- and y-rays) and nonionizing (e.g. UV) radiation produce a variety of DNA lesions. Pyrimidine dimers are the commonest product of UV irradiation. Base analogs can mispair during DNA replication to cause mutations. Nitrous acid deaminates cytosine and adenine. Alkylating and arylating agents generate a variety of adducts that can block transcription and replication and cause mutations by direct or, more commonly, indirect mutagenesis. Most chemical mutagens are carcinogenic. If abase analog or modified base whose base pairing properties are different from the parent base is not removed bya DNA repair mechanism before passage of a replication. fork, then an incorrect base will be incorporated. A second round of replication fixes the mutation permanently in the DNA. ‘Most lesions in DNA are repaired by error-free direct, reversal or excision repair mechanisms before passage of a replication fork. If this is not possible, an error-prone form of translesion DNA synthesis may take place involving specialized DNA polymerases and one or more incorrect bases become incorporated opposite the lesion. (A2) Nucleic acid structure (E3) DNA repair and function (£4) Recombination and (Section D) DNA replication transposition (El) DNA damageE2~ MUTAGENESIS 83 Mutation Mutations are permanent, heritable alterations in the base sequence of the DNA. They arise either through spontaneous errors in DNA replication, or meiotic recombination (Section E4), or as a consequence of the damaging effects of physical or chemical agents on the DNA. The simplest mutation is a point mutation — a single base change. This can be either a transition, in which one purine (or pyrimidine) is replaced by the other, or a transversion, where a purine replaces a pyrimidine or vice versa. The phenotypic effects ‘ofsuch a mutation can be various. Ifitis in a noncoding or nonregulatory piece of DNA or inthe third position ofa codon, which often has no effecton the amino acid incorporated into a protein (Section K1), then it may be silent. If itresults in an altered amino acid ina .gene product then itis called a missense mutation, and its effect can vary from nothing to lethality, depending on the aminoacid affected. Mutations that generate newstop codons are nonsense mutations and give rise to truncated (shortened) protein products. Inser- ions or deletions involve the addition or loss of one or more bases. These can produce frameshift mutations in genes, where the translated protein sequence to the C-terminal ide of the mutation is completely changed. Mutations that affect the processes of cell ‘growth and cell death can result in carcinogenesis (Sections N2 and N3). The accumu- lation of many silent and other nonlethal mutations in populations produces genetic polymorphisms - acceptable variations in the ‘normal’ DNA and protein sequences {Section C4). Replication fidelity ‘The error rate of DNA replication is much lower than that of transcription because of the need to preserve the meaning of the genetic message from one generation to the next. For example, the spontaneous mutation rate in £. coli is about one error per 10! bases incorporated during replication. One reason for replication errors is the presence of the minor tautomeric forms and conformational isomers of the bases, which have altered base pairing properties (Figure 1b, Section B1). For example, the (minor) enol form of Gcan form a base pair with the (major) keto form of T that has the same dimensions as normal ‘Watson-Crick’ A-T and G-C base pairs and so appears normal. Small additions of extra bases or deletions can also occur spontaneously during replication. The error rate is minimized by avariety of mechanisms. First, DNA polymerases will only efficiently incorporate an incoming nucleotide if it forms the correct base pair with the template nucleotide in its active site (base selection). Secondly, the occasional error (Figure 1a) is detected by the 3'-95’ proofreading exonuclease associated with the polymerase (Section D2) because it has the wrong shape. This removes the incorrect nucleotide from the 3’-end before any further incorporation, allowing the polymerase then to insert the correct base (Figure 1b). In order for the proofreading exonuclease to work properly, @ SHATGCATGCS S-ATGCATGCS SLTACGTg. -TACGT PPI xa acte omP. Figure 1. Proofreading of newly syrthesized DNA. Ki, for example, a’G' residue is wrongly incorporated oppesite a template T’ (2, itis removed by the 325’ proofreading exonuclease (b).84 SECTION E - DNA DAMAGE, REPAIR, AND RECOMBINATION it must be able to distinguish a correct base pair from an incorrect one. The increased. ‘mobility of tunanchored’ base pairs at the very 5’-end of newiy initiated lagging strand fragments of DNA (Section D1) means that they can never appear correct and so cannot be proofread. Hence, ribonucleotides are used for the first few nucleotides (the RNA primer) so that they subsequently can be identified as low fidelity material and replaced later with DNA elongated (and proofread) from the adjacent fragment. Finally, errors that escape proofreading are corrected by mismatch repair (Section E3). Physical mutagens Absorption of high-energy ionizing radiation such as X-rays and y-rays causes the target ‘molecules to lose electrons. These electrons can cause extensive chemical alterations to DNA, including strand breaks, both single and double, and base and sugar destruction. Nonionizing radiation causes molecular vibrations or promotion of electrons to higher energy levels within the target molecules. This can lead to the formation of new chemi- cal bonds. The most important form causing DNA damage is UV light, which produces pyrimidine dimers from adjacent pyrimidine bases (Section El). Chemical mutagens Base analogs are derivatives of the normal bases with altered base pairing properties and. can cause direct mutagenesis. A wide range of other natural and synthetic organic and inorganic chemicals can react with DNA and alter its properties. Nitrous acid promotes, the deamination of C to U (Figure 1, Section E1), which base-pairs with A and causes G— (C-+A-T transitions upon subsequent replication. Deamination of Ato the guanine analog. hypoxanthine results in A~T-sG-C transitions. Alkylating agents, suchas MMS and ENU, and arylating agents (Section E1) produce lesions that usually have to be repaired to prevent serious disruption to the processes of transcription and replication. Processing of these lesions by the cell may giverise to mutations by indirect mutagenesis. Intercala- ‘ors (Section B3) generate insertionand deletion mutations. Most chemical mutagens are carcinogens and cause cancer. Direct mutagenesis Direct mutagenesis results from the presence of a stable, modified base with altered base pairing properties in the DNA. All that is required for this lesion (nonpermanent) to be fixed as a mutation (permanent and heritable) is DNA replication. For example, the keto tautomer of the thymine analog 5-bromouracil base-pairs with adenine as expected, but the frequently occurring enol form (due to electronegativity of Br) pairs with guanine (Figure 2a). After one round of replication, a guanine may be inserted opposite the 5-bromouracil. After a second round of replication, a cytosine will be incorporated oppo- site this guanine and so the mutation is fixed (Pigure 2b). The net effect is an A-T-»G-C transition. 8-Oxo-G produced by intracellular oxidation (Section E1) can base-pair with Aand, ifleft unrepaired, can give rise to A-T-+C-G transversions. Indirect mutagenesis and translesion DNA synthesis The great majority of lesions introduced by chemical and physical mutagens are substrates for one or more of the DNA repair mechanisms that attempt to restore the original structure to the DNA in an error-free fashion before the damage is encountered. by a replication fork (Section F3). Occasionally this is not possible, for example when a bulky ornoncodinglesion is created immediately ahead of an advancing fork. This could ead to a potentially lethal stalling of the replication machinery so in such cases the cellE2~ MUTAGENESIS 85 @ ® Hydrogen bone AGcTItccTA @ AGCTBCCTA TCCAAGCAT TCGAAGGAT AGCTBCCTAMIAGCTTCCTA TCGAGGGAT TCGAAGGAT AGCTRCCTAMMAGCTECCTA TCGAAGGAT TCGAGGGAT Figure 2. (a) Base-pairing of the enol form of 5-bromouracil with guanine; (b) direct mutagenesis by 5-bromouracil(B): () the ‘T’ analog ‘B' can be readily incorporated in place ‘of Ti) after one round of replication ‘G' may be incorporated opposite the ‘Bi after a second round, a ‘C’ will be incorporated opposite the ‘G'. Bru may resort to translesion DNA synthesis (TLS). This involves the temporary replacement ofthe normal replicative DNA polymerase by one of several specialized polymerases that are able to insert bases opposite the unrepaired lesion. After this, normal replication resumes. Thus, the lesion is not removed or repaired but tolerated and so the integrity of the replicated DNA is maintained. The lesion in the template strand can then be removed later, before the next round of replication. AILS polymerases lack proofreading activity and have relaxed active sites. Some are of relatively high fidelity and manage to incorporate the correct base opposite the lesion; ‘however, others are oflow fidelity and of necessity insert incorrect bases simply to ensure chromosomal integrity, leading to indirect mutagenesis. Indirect mutagenesis may be targeted, if the mutation occurs onlyat the site of the lesion, or untargeted, if mutations are also generated elsewhere in the genome. In E. coli, translesion DNA synthesis is part ofthe SOS response to DNA damage andis sometimes called error-prone repair, though it is not strictly a repair mechanism. The SOS response involves the induction of many genes whose functions are to increase survival after DNA damage. The damage-inducible DNA polymerases IV and V are essential for mutation induction by indirect mutagen- esis. For example, pol can insert a base opposite a noncoding lesion such as an AP site (Section El) leading to targetted mutagenesis while, once induced, the error-prone pol IV can cause untargetted mutagenesis even where there is no lesion. In bacteria, untar- _getted mutagenesis may be a useful and deliberate response to ahostile, DNA-damaging, environment in order to increase the mutation rate with the hope of producing a more resistant strain that could be selected for survival. Pol IV also creates many of the adap- tive mutations that arise under the stressful conditions of stationary growth phase. ‘Mammalian cells have at least seven TLS polymerases, specialized for different types of lesion. Pol-n (eta) correctly inserts adenines opposite the linked thymines in a cyclobu- ‘tane thymine dimer. Pol-t (iota) is good atinserting the correct bases oppositea 6,4-photo- product but inserts wrong bases opposite a cyclobutane dimer. These and pol-x (kappa) can also bypass other noncoding lesions with varying degrees of fidelity. When some TLS polymerases insert a wrong base, they have difficulty extending the resulting mismatch at the 3’-end of the growing strand. In this case, pol-{ (zeta) may continue polymeriza- tion before the normal replicative polymerase resumes. TLS is responsible for many of ‘the point mutations occurring in cells, particularly the high level found in cancer cells.