1.1. Basic definitions Unlike all other polymers in living cells, DNA cannot be completely broken down to monomers and rebuilt from scratch. Its synthesis always requires a pre-existing single strand of DNA as a template and a double-strand region at least several bp long, providing a 3'-OH group from which the synthesis to start. The DNA of the cell is the single blueprint that contains all the information on how to make a new copy of any other polymer molecule in the cell. Therefore, it is sensible to make sure that the genetic information coded in DNA could always be recovered even from a single strand. The preservation of the genetic information in its original state is dependent not only on the accuracy of its copying during DNA replication, but also on the timely repair of any event which may compromise the integrity of the DNA molecule and/or may change its building blocks or their arrangement in any manner. Such events constitute DNA damage. The definitions of DNA damage are numerous in the literature. One of the most succinct but, at the same time, complete definitions is the definition given by Benjamin Lewin in his famous 'Genes' textbook: "Damage to DNA consists of any change that introduces a deviation from the usual double-helical structure" [1]. One of the most commonly used and, in the authors' opinion, the most comprehensive, is the definition of DNA damage given by the US National Library of Medicine, [Unified Medical Language system (UMLS)]: "Drug- or radiation-induced injuries in DNA that introduce deviations from its normal double-helical conformation. These changes include structural distortions which interfere with replication and transcription, as well as point mutations which disrupt base pairs and exert damaging effects on future generations through changes in DNA sequence". The latter definition, however, implies that DNA damage is always a product of some external influence, that is, it is not produced by physiological means. This is not true, however, as the most part of DNA damage occurs as a result of normal cellular metabolism. Therefore, for the purposes of the present book, we may adopt the following definition of DNA damage: "Any modification in the physical and/or chemical structure of DNA resulting in an altered DNA molecule which is different from the original DNA molecule with regard to its physical, chemical and/or structural properties". To understand more fully the mechanisms of repair of DNA, one needs to be familiar with the basic types of DNA damage and, respectively, the agents which cause it. Basic types of DNA damaging (genotoxic) agents classified by their essential properties are presented below. 1.2. Classification of factors causing DNA damage 1.2.1. Factors of exogenous and endogenous origin DNA damage may occur under the influence of factors external to the cell (factors of exogenous origin, e.g. environmental factors) or potentially aggressive agents produced by normal cell metabolism (factors of endogenous origin). The consequences for the cell's DNA damage caused by the action of endogenous factors may be more serious and/or more extensive than the effect of most of the exogenous DNA damaging factors. DNA damaging events caused by endogenous factors generally occur much more frequently than damage caused by exogenous factors. For example, several thousands of nitrogenous bases are lost daily from DNA in eukaryotic cells as a result of spontaneous base hydrolysis alone. DNA damaging factors of endogenous and of exogenous origin may exert their genotoxic action on DNA using the same mechanism. For example, both ionising radiation (an agent of environmental origin) and normal oxidative phosphorylation (obviously of endogenous origin) generate reactive oxygen species which may damage the cell's DNA. DNA damage may cause appearance of structures that do not occur naturally in DNA or may cause structures that are perfectly normal for some DNA regions, to appear at ectopic sites. For example, thymine dimers are structures which normally do not exist at all in undamaged DNA. Occurrence of triple-strand DNA regions is perfectly in order in certain genome areas, but may accidentally arise de novo in other areas, where it constitutes damage. DNA damaging agents may work directly on DNA (e.g. base modifying agents) or may induce structural changes that increase the risk for further damage. For example, the occurrence of triple-strand structures in DNA increases the risk for copying errors during subsequent replication of the affected DNA region (see below). 1.2.2. Nature of DNA damaging agents (physical and chemical agents) Genotoxic agents may be broadly classified into factors of chemical or of physical nature, respectively. Some genotoxic agents are capable of damaging DNA because of both their chemical and their physical properties. The damaging agents acting on DNA by virtue of their physical properties are, for example, short-wavelength electromagnetic energy such as ultraviolet (UV) radiation and ionising radiation. Under specific conditions, low-energy electromagnetic waves, such as infrared radiation (heat) and microwave and radio wave radiation may also cause DNA damage [2, 3]. Among DNA damaging agents of chemical nature prominent are alkylating agents, oxidising agents, chemicals creating DNA-DNA or DNA-protein crosslinks, and others. DNA damaging agents may work directly on DNA to alter its structure or may exert its activity via inducing structural change which, in turn, alters its properties, or these two may occur simultaneously. For example, agents introducing bulky adducts in DNA work both ways, as they may chemically modify the DNA molecule and at the same time may physically alter (bend, distort, twist) its structure. The latter may increase the risk for additional damage (e.g. introduction of strand breaks). 1.2.3. Factors causing alterations in the information content of DNA and factors causing structural alterations Damage in DNA is often, but by no means always accompanied by alteration of the information content of DNA. Only certain damaging events, left unrepaired, can produce a lasting, heritable change in the DNA sequence, thus producing a mutation. Among these are, for example, events producing a mismatch in DNA. Mismatched bases are usually recognised and promptly repaired by a designated mismatch repair system, but if the DNA template containing the mismatches is replicated despite the presence of damage, the resulting daughter strands will carry different 'meaning' in their DNA. Since there is no system in the cells that could recognise which daughter molecule is identical to the 'maternal' molecule and which is different, the cell carrying the alteration would faithfully copy the altered sequence during subsequent cell divisions. DNA damage or erroneous copying of the damaged fragment may change one or more of the building blocks of DNA, but the resulting altered variant may carry the same coding sense. Some types of DNA damage, if they are left unrepaired, would cause replication blockage. Generally, 'nominal' DNA polymerases of replication would stop when they encounter damage in the template. This would, in turn, result in recruitment of the repair machinery to the blockage site, and the damage would be repaired without any lasting changes in the DNA sequence. Other types of DNA damage (for example, some of the events producing modified nucleotides) are sometimes managed in a manner that accommodates the modified nucleotide/s - that is, they are not exactly ignored, but during replication the fragment containing the modified nucleotide/s is copied in a manner that restores the original sequence, despite the fact that the template is damaged. DNA damage may affect structures of higher order than the nucleotide sequence level. For example, the occurrence of regions with Z-conformation or the already mentioned triple-strand DNA may constitute DNA damage. DNA damage involving higher-order structures may affect crucial cellular processes, but may not be transmitted to the cellular progeny during replication, as the nucleotide sequence remains essentially the same. Other types of alteration of higher-order structures may result in inaccurate copying during replication, which may produce a heritable genetic change (that is, a mutation). 1.2.4. Cytotoxic and genotoxic agents Factors of any origin which are injurious to living cells may be damaging to the cell as a whole (cytotoxic agents) or specifically damaging to the cellular DNA (genotoxic agents). The same agent may have predominantly cytotoxic or predominantly genotoxic action in living cells depending on the type of the agent, the type of the cells in question and the duration of the action of the damaging agent. For example, ionising radiation may be cytotoxic in large doses, that is, cells and cell populations may die directly as a result of the massive oxidative stress caused by rapid elevation of the levels of free radical species in the cell. In smaller doses, however, ionising radiation may exhibit only genotoxic properties - it produces significant amounts of DNA damage, but not all damaged cells would die, neither straight away nor as a late effect. Same is the case with many chemical damaging agents. For example, aromatic amines (e.g. 2-acetylaminofluorene) in lower concentrations exhibit predominantly genotoxic properties, as they work by introducing bulky adducts in DNA, which makes copying of the templates carrying those adducts difficult and more error-prone than copying undamaged DNA. In larger doses, however, aromatic amines may be directly cytotoxic [4]. Finally, cytotoxic effects may be exerted via genotoxic action. For example, most chemotherapeutic anticancer agents work by introduction of significant amount of damage in DNA in the hope that this may cause rerouting of the cancer cells towards the programmed cell death pathway. 1.2.5. Mutagens and teratogens Those two terms are often used synonymously or, at least, interchangeably, but this is incorrect most of the times. There is an essential difference between the intended targets and sometimes in the modes of action of mutagenic and teratogenic agents. Mutagen is a term that denotes an agent that directly or indirectly affects DNA so that may eventually cause a potentially heritable change in its sequence or structure. Mutagens usually have inherent carcinogenic properties, as the damage caused by their action may trigger neoplastic transformation. Teratogens are by definition agents that may induce abnormal prenatal development or may increase the risk for such. This, however, may or may not be related to any change or damage in DNA. For example, thalidomide is known to be a powerful teratogen (as, unfortunately, was found out the hard way in the 1950's) but is not a mutagen, as it works by interference with the vascularisation of developing body parts during the morphogenesis of the embryo. There is no DNA damage involved whatsoever. UV light is powerful somatic mutagen but is usually not considered to be a teratogen, as embryos inside the womb are very well protected from UV irradiation. It is understood, however, that the same agent may exhibit both mutagenic and teratogenic properties - for example, ionising radiation or chemical agents such as formaldehyde may very well cause both somatic mutagenesis and teratogenic effects, but the one does not automatically include the other as well. The fluorescent dye ethidium bromide, commonly used in molecular biology laboratories, is considered to be a powerful somatic mutagen (intercalating agent) but its teratogenic properties have not been definitely proven. It is possible that the usual mode and level of exposure (usually, skin coming into occasional contact with diluted solutions) minimise the potential teratogenic effects. Ethidium bromide was regularly administered in the past in veterinary medicine as an antiprotozoal drug (specifically, against Trypanosoma spp.), and had been used for over 50 years before being replaced by more modern drugs [5]. Obviously, if ethidium bromide had a pronounced teratogenic effect in mammals, it would have been noticed before. There are special circumstances, however, in which an agent that would normally demonstrate only mutagenic or only teratogenic properties may occasionally exhibit the other type of properties. This usually involves a known somatic mutagen acting as a teratogen under certain conditions. This is usually related to dose- or stage-sensitivity phenomena. For example, the already mentioned UV light, a known somatic mutagen, has been documented to actually have weak teratogenic properties. Specifically, it has been associated with a slight increase in the risk for neural tube defects for children of parents who have been exposed to therapeutic doses of UV (specifically, during phototherapy for psoriasis). The teratogenic effects are reportedly exerted via modulation of the folate metabolism and are usually restricted to very early pregnancy (the first weeks post-conception) [6, 7]. Occupational exposures to UV of the prospective father (e.g. welding) have also been found to be associated with slightly increased risk for neural tube defects (spina bifida) in the offspring [8]. When tested for mutagenic properties (e.g. using the classic Ames test [9, 10]) some teratogens may test positive for mutagenic properties. The terms 'somatic mutagen' and 'reproductive mutagen' are sometimes used instead of 'mutagen' and 'teratogen' to avoid confusion. 1.3. DNA damage does not always result in mutagenesis Undoubtedly, an event that occurs tens of thousands times per day can be described as 'common'. Therefore, DNA damage is a very common event indeed. The bulk of this damage is repaired promptly and accurately by the cellular repair machinery. This machinery, however, is not perfect (becoming even less so with ageing), and it may occasionally miss sites of damage or make mistakes - for example, it may add a non-matching nucleotide. Most mismatches in DNA result from errors in template copying during DNA replication. As DNA repair is almost always associated with DNA synthesis, it can produce mismatches as well. The error rate in repair-related DNA synthesis is generally higher (≈10^-3-10^-6 per nucleotide per cycle of synthesis for different 'repair' DNA polymerases and for different types of mispairs) than the error rate in replication (10^-5-10^-7 per nucleotide per cycle of replication) [11-15]. Repair-associated DNA synthesis, however, usually acts on much shorter templates than replication-related synthesis. For example, the length of the synthesised DNA fragment in repair by excision of nucleotides is 12-13 nucleotides in prokaryotes and 27-28 nucleotides in eukaryotes [16]. In this case, even an error rate of 10^-3 would not be too high. In any case, there is no significant difference between the net incidence of copying errors during DNA synthesis for repair purposes and in replication. Unrepaired damage in DNA may eventually bring about the death of the cell (if the damage is too extensive and/or too severe) or it may be retained, tolerated and, if the cell is capable of division, potentially transmitted to the cell's progeny. There are several DNA polymerases capable of copying damaged templates, albeit with a higher error rate, collectively known as the Y-family of polymerases. In prokaryotes, these are DNA polymerases IV and V [17]. In mammals, these are polymerases kappa, eta, zeta and iota [18-21]. "Nominal" DNA polymerases (beta, delta, and lambda) acting in repair DNA synthesis may also make mistakes, albeit at a lower rate than the error-prone polymerases (but at a higher rate than 'replication' polymerases). In the process of copying a damaged template, the DNA polymerases may add the correct nucleotide and synthesise a complementary strand which is identical in sequence with the template strand; or they may add a mismatched nucleotide, synthesising a daughter strand which carries altered nucleotide sequence at the site where the damage initially occurred. During the next cycle of replication, the altered DNA molecule will produce daughter molecules carrying the altered sequence, transmitting it on to their progeny. Eventually, a mutant clone is created. A genetic alteration which is capable of being transmitted down the generations is called a mutation. Therefore, it is prudent to assume that the main difference between DNA damage and DNA mutation is that DNA mutations are heritable, while DNA damage may be not. Generally, less than 0.1% (1 out of 1000) of the base substitutions occurring in DNA are retained and, potentially, transmitted to cell progeny. That means that over 99.9% of the damage occurrences are detected and repaired so that no trace of them is left whatsoever. The rate of spontaneous mutation varies between different organisms, but the average estimates may be summarised as follows: Mitochondrial DNA (1.66x10⁴ bp in humans) has a mutation rate of its own, which is higher than the mutation rate in nuclear DNA - about 10^-5-10^-6 per base per generation [22, 23]. It is easy to calculate that since the incidence of spontaneous mutations in higher eukaryotes is about 10^-8 per base per generation, in an average genome (about 10⁹ bp), every cell division would result in about a dozen mutations [23, 24]. The majority of these mutations, however, would affect non-coding regions; therefore, would remain 'silent' at least for some time, unless the mutation occurred in a region with control functions. Mutations in coding regions do not always result in alteration of the coded information. They may be effectively synonymous, that is, the original codon and the mutant codon would translate to the same amino acid residue (see below). Only a small proportion of all mutations in the coding regions would be non-synonymous substitutions or nonsense mutations (introducing a premature stop codon). This may, in time, together with other mutations, induce cellular senescence or trigger cancerous transformation. Spontaneous mutagenesis is generally a random process. Even among non-synonymous mutations, resulting in substitutions of amino acid residues in the resultant protein, however, there might be a mutation bias - that is, some nucleotides and some codons may be more frequently affected by mutations than others. For example, it was demonstrated that 30% of the mutations resulting in genetic disease in man affect specifically arginyl and glycyl residues [25, 26]. This cannot be explained by unique coding, as both Arg and Gly are coded by more than three codons (as are, for example, alanine, leucine, proline, serine, threonine and valine), neither is it a question of abundance, as these two amino acids are not among the most abundant in human proteins. Neither is it a matter of chemical properties, as glycine is an aliphatic amino acid while arginine has basic properties. The reason for preferential mutagenesis on some amino acid residues is still unclear. It has been proposed that the two major factors for cellular ageing are the accumulation of mutations in DNA resulting from replication errors (nucleotide substitutions, nonsense mutations); and the oxidative stress resulting from the genotoxic action of reactive oxygen species (ROS) on mitochondrial DNA [27]. 1.4. Accumulation of mutations in DNA may be responsible for 'diseases of old age' Despite the fact that the rate of occurrence of DNA damage is very high, the overall mutation rate is very low, thanks to the efficiency of the mechanisms for recognition and repair of damage in normal cells. Every cell cycle is associated with the production of new mutations in the cell's DNA, but discernible signs of ageing or age-related disease...

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