Endogenous sources of DNA damage

Normal metabolic processes also generate ROS, which, in addition to single-strand break formations, can modify DNA bases by oxidation (De Bont and van Larebeke, 2004). Both purine and pyrimidine bases are subject to oxidation. One of the most prevalent lesions in DNA is guanine oxidized to 8-oxo-7,8-dihydroguanine, which is capable of base pairing with adenine, resulting in a G→T transversion mutation following the replication (Ruiz-Laguna et al., 2000). Products of unsaturated lipids oxydation can react with bases in DNA resulting in exocyclic etheno adducts such as etheno-dC or etheno-dA. Generally, base alterations produced by oxidizing agents are substrates of the base excision repair (BER).

Intracellular S-adenosylmethionine (SAM), which is a methyl group donor in enzymatic methylation reactions, is known as a weak non-enzymatic DNA-methylating agent (Lindahl, 1993). One of the products of its reaction with DNA bases is O⁶-methylgunine (O⁶-MeG) which base pairs with thymine rather than with cytosine. This point mutation is repaired by

damage reversal system by recruiting O⁶-MeG DNA methyltransferase, which removes the methyl group (E. C. Friedberg et al., 2006).

Another principal source of DNA damage is spontaneous hydrolysis reaction (Figure 1.1).

Especially susceptible is the N-glycosidic bond of the purines. Depyrimidination also occurs, but about 30 times slower. The resulting apurinic/apyrimidinic (AP) sites, if not repaired, can lead to DNA chain rupture. It was estimated that 10000 depurination events occur daily in a diploid mammalian cell (Friedberg, 2006). Not only glycosidic bonds, but also DNA bases suffer from hydrolytic attacks. The exocyclic amino groups of the bases are labile and readily undergo reactions of hydrolytic deamination (Lindahl, 1993). Formation and repair of these DNA damages, especially of cytosine deamination, will be described in more details as it has direct relation to this work.

Figure 1.1: DNA primary structure with four principal DNA bases and major sites of spontaneous hydrolytic attack. Green arrows indicate N-glycosidic bonds; red arrows: phosphodiester bonds; blue arrows: bonds with exocyclic amino groups. Adapted from T. Lindahl, 1993.

1.1.4.1. Hydrolytic DNA deamination

In the course of the hydrolytic deamination, purines adenine and guanine are converted into the hypoxanthine and xanthine residues, respectively. Xanthine is unable to pair stably with either cytosine or thymine and thus may result in arrested DNA synthesis, whereas hypoxanthine generates a pre-mutagenic lesion as it preferentially base pairs with cytosine (Friedberg, 2006). But as rates of purines deamination are low (for instance, conversion of adenine into hypoxanthine in single-stranded DNA occurs at about 2% of the rate of the conversion of cytosine to uracil (Lindahl, 1979)) and the resulting products are repaired efficiently, no real threat to the integrity of the genetic information is considered.

Hydrolytic deamination occurs most rapidly at 5-methylcytosine (5-meC) sites (Lindahl, 1993). 5-meC is produced by site-specific DNA (cytosine-5)-methyltransferase which transfer methyl group from S-adenosylmethionine to the C-5 position of cytosine in double-stranded DNA (Chen et al. 1994). Cytosine methylation has important functions such as modification of DNA as a defense against the invasion of the foreign DNA species in prokaryotes (Palmer and Marinus, 1994) and involvement in the regulation of gene expression, embryogenesis, genomic imprinting, aging, and some other processes in eukaryotic cells (Jaenisch and Bird, 2003). Deamination of 5-meC in DNA results in the formation of thymine and hence of T/G mispair. The subsequent replication rounds will generate a GC→AT transition mutation. Base excision repair initiated by several highly specialized enzymes, and a specific repair process in some bacteria termed very short patch repair (VSP) mechanism are responsible for the repair of T/G mismatches (Bhagwat and Lieb, 2002).

Hydrolytic cytosine deamination occurs about 50 times quicker than deamination of the purines (Lindahl and Nyberg, 1972). Resulting uracil is formed at high rates especially in the single stranded DNA during transcription, replication or recombination (Lindahl and Barnes, 2000). Although uracil is normally confined to RNA, the formation of uracil in DNA is mutagenic due to its preferential pairing with adenine residue. If not repaired, this will lead to GC→AT transition mutation in 50% of progeny when replication proceeds. E. coli strains that are defective in the removal of uracil from DNA have an increased spontaneous mutation rate, and GC→AT base pair transitions are observed at selected sites in such mutants (Duncan and Miller, 1980). Deamination of cytosine can be enhanced by a number of chemical alterations and steric factors, by the formation of UV-radiation induced cyclobutane pyrimidine dimers, by certain intercalating agents or by the positioning of a mismatched or alkylated base

opposite cytosine. Deamination can also be promoted by reaction with nitrous acid or sodium bisulfate (Friedberg et al., 2006). Generation of uracil by gamma radiation-induced deamination of cytosine and sensitivity of E. coli cells deficient in Ung and Smug1 DNA glycosylases to gamma-radiation was reported (An et al., 2005). In eukaryotic cells, uracil can arise in DNA due to the enzymatic deamination of cytosine (Harris et al., 2002), by drug treatment or folate deficiency (Kavil et al., 2007). In addition, uracil can be incorporated into DNA during semiconservative replication and the extent of this incorporation is directly related to the size of intracellular dUTP pool. Presence of U/A base pairs rather than T/A base pairs, in general, does not change the coding information, but uracil-containing DNA possesses the altered binding affinities for the transcription factors or other regulatory proteins (Verri et al., 1990).

Uracil residues in DNA exist transiently since they are subject to removal by the multi-step uracil initiated DNA base excision repair (BER) process in most organisms and by nucleotide incision repair pathway (NIR) described to date only in M. thermautotrophicus (Georg et al., 2006; Schomacher et al., 2009).