DNA Damage and Repair

The more important UV-B-induced DNA alterations are the formation of cyclobutane pyrimidine dimers (CPDs) and pyrimidine (6-4) pyrimidine dimers (6-4 photoproducts, 6-4PPs) (Dany et al. 2001). The DNA repair mechanisms operating in plants include the following processes: (a) direct reversal (DR); (b) photoreactivation that induces photolyases; (c) dark repair (Tuteja et al. 2001; Britt 2004). The DR is a simple mechanism that involves a single-enzyme reaction for the removal of certain types of DNA damage. Alkyltransferases simply extract alkyl groups from the alkylated bases that are transferred to internal cysteine residues and thus inactivate themselves. The best example for DR is the correction of miscoding alkylation lesion 06-methylguanine, which is generated endogenously in small amounts by the reactive cellular catabolites. This reaction is catalyzed by a specific enzyme, called methylguanine methyltransferase (MGMT), which removes a methyl group from a guanine residue of the DNA molecule and transferring it to one of its own cysteine residues in a rapid and error-free repair process (Tuteja et al. 2001). The photoreactivating enzyme DNA photolyase (PRE) is a DR phenomenon performed by the combined action of one or more photolyases and the visible light (blue, violet, or long-wave UV) (Hidema et al. 2007). Photolyases specifically recognize and bind the pyrimidine dimers to form a complex molecular structure which is stable in absence of the light. After absorbing a blue light photon the pyrimi-dine dimers are reversed to pyrimidine monomers without excision of the damaged base (Tuteja et al. 2001). The repair reaction is fast and requires about 1 h for completion (Takeuchi et al. 2007). In plants, two specific types of pho-tolyases have been characterized: (a) CPD-photolyase; (b) 6-4PP-photolyase (Tuteja et al. 2001). The dark repair processes include the nucleotide excision repair (NER), base excision repair (BER), mismatch repair (MMR), and other DNA repair pathways. These mechanisms have been observed in several plant species and some of the genes required for the processes were identified (Kimura et al. 2004) . The wide class of helix-distorting lesions such as CPDs and 6-4PPs are repaired by the NER process. It is one of the most versatile DNA repair pathway operating in plants. Unlike other DNA repair pathways that are specific repair processes, the NER pathway is capable of removing various DNA damage classes, including those induced by the UV-B

radiation (pyrimidine dimers) and chemical agents (bulky DNA adducts) (Kimura et al. 2004). The NER pathway sequentially involves recognition of the DNA damage, incision on the damaged strand, excision of the damage-containing oligonucleotides, and the DNA synthesis and ligation (Liu et al. 2003). Also, the NER pathway is a slow process (about 24 h for completion) and includes several enzymes. There are two subpath-ways of the NER process that are designated as:

(a) global genomic repair (GGR); (b) transcription-coupled repair (TCR). While the GGR pathway repairs the DNA damage over the entire genome, the TCR pathway is selective for the transcribed DNA strand in expressed genes (Kimura et al. 2004). Oxidized or hydrated bases and single-strand breaks are repaired by the BER pathway that is considered an essential process for maintenance of the DNA molecule. The BER mainly removes the DNA damages that are arising spontaneously in the cell from hydrolytic events such as deamination or base loss, fragmented bases resulting from ionizing radiation (e.g., UV-B radiation), and oxidative damage or meth-ylation of the ring nitrogen by endogenous agents. The process that involves the BER mechanism is initiated by DNA glycosylases that release the damaged base by cleavage of the sugarphosphate chain followed by excision of the abasic residue or abasic residue containing oligonucleotides and then the synthesis and ligation of the DNA occurs. The BER pathway involves several enzymatic steps and depends strongly on the presence of nicotinamide adenine dinucleotide (NAD+). Also, the BER process comprises two subpath-ways that are designated as: (a) BERshort-path;

(b) BERlong-path. The BERshort-patch is a DNA polymerase beta-dependent mechanism, while the BERlong-patch is a DNA polymerase delta/ epsilon-dependent mechanism (Kimura et al. 2004) . The major difference between BER and NER pathways is the way by which the DNA damage is removed. The NER pathway cuts out the damage as a part of an oligonucleotide fragment, while the BER mechanism excises only one nucleotide (Tuteja et al. 2001). The excision repair processes (NER and BER) are very important for maintaining the genome stability and essential for the survival of plants. The mismatch repair pathway (MMR) is also important in the DNA repair processes when by errors of replication or homologous recombination can be produced mismatched bases. The MMR pathway basically discriminates between correct and incorrect bases and after DNA synthesis the error is corrected (Tuteja et al. 2001). Although most of the present understanding of the eukaryotic MMR has come from studies of the E. coli MutS and MutL proteins (Kolodner and Marsischky

1999) . recent studies carried out in Arabidopsis and rice have reported interesting findings on the MMR pathway operating in plant cells (Tuteja et al. 2001; Kimura et al. 2004). According to the E. coli model, the MutS dimer recognizes mis-pairs and then binds on it followed by the MutL binding, which activates the MutH (endonu-clease) that makes a single-strand incision (nick). The MutH incision can be done on either side of the mismatch. Subsequent to incision the excision is initiated and proceeds toward mismatch. To fill the gap (100-1,000 nucleotide gap), the original template strand can then be replicated and finally sealed by ligation. The proteins involved in the last step of eukaryotic MMR are: (a) DNA polymerase 8 . RP-A (replication protein); (b) PCNA (proliferating cell nuclear antigen); (c) RFC (replication factor) (Kolodner and Marsischky 1999).

Although the UV-A wavelengths can mediate the photoxidative damage (Turcsanyi and Vass

2000) the UV-B radiation is the most important photooxidant agent for terrestrial plants. The DNA damage can also be caused by reactive oxygen species (ROS) and free radicals produced by the UV-B radiation. This damage includes several modifications such as cross-linking, aggregation, denaturation, and degradation (Hidema et al. 2007) . The formation of 7,8-dihydro-8-oxoguanine (GO) is a common oxidative DNA lesion generated by a direct modification mediated by ROS. The GO is mutagenic and can mispair with adenine (A) during the DNA replication (Yang et al. 2001). If the resulting A/GO is not repaired before the next round of the DNA replication, a C/G ^ A/T transversion occurs and the opportunity for repair is lost. The A/GO is repaired via the BER which is initiated by the DNA repair enzyme adenine-DNA glycosylase (Yang et al. 2001). The UV-absorbing compounds (e.g., flavonoids, anthocyanins, hydroxycinnamic acid derivatives, phenolics) accumulating in epidermal and subepidermal cell layers have traditionally been thought to function as UV-B filters, but also play an important role as quenchers of the ROS and free radicals in the amelioration of the UV-B-induced DNA oxidative damage (Agati and Tattini 2010). The UV-absorbing compounds are also effective in reducing the induction of cyclobutane pyrimidine dimers (CPDs) in plants exposed to high UV-B levels (Hidema et al. 2007) . The inhibition of CDP formation seems to be high enough to compensate the DNA damage arising even from unusually strong solar irradiations (Tuteja et al. 2001). Other related UV-absorbing compounds, that is, anthocyanins through an anthocyanin-DNA complex could also provide protection against the oxidative damage. Since both anthocyanins and DNA mutually protect each other in vitro, it is likely that such protection mechanism may also operate in vivo (Sarma and Sharma 1999). In the plant cells, anthocyanins are predominantly localized inside the vacuoles and thus their putative role in the protection of DNA should be critically examined. Accepting this fact, it has been demonstrated that the excess accumulation of anthocyanins reduces the amount of blue/UV-A radiation reaching the cell and may sometimes lower the ability to photorepair the damaged DNA. For example, the purple rice is a highly UV-B sensitive species despite possessing an elevated level of anthocyanins in their leaves (Hada et al. 2003). Although significant amounts of flavonoids have been found in the chloroplasts or etioplasts isolated from a wide range of plants growing under both ambient and enhanced UV-B irradiances (Tattini et al. 2005; Agati et al. 2007), it is also likely that some amount of anthocyanins can be present in the nuclei and organelles and then may associate with the DNA molecule, offering to it a certain protection against the oxi-dative damage (Feucht et al. 2004). In this context, the UV-absorbing compounds seem to have an important protective function against the DNA

damage induced by shorter solar wavelengths (Schmitz-Hoerner and Weissenbock 2003) and so the speculations concerning a great biological risk with regard to increases in solar UV-B radiation after the depletion of the ozone layer are presumably premature.

In the natural populations, both protection and DNA repair are complementary and necessary processes for the plant development. Thereby, it is expected that the plants growing under different UV-B irradiances can exhibit different levels of the DNA protective mechanisms (Turunen and Latola 2005). Under field conditions, the observed DNA damage can often be modified by climatic conditions and then a direct extrapolation of the DNA changes obtained in controlled-environment experiments under artificially enhanced UV-B radiation to plants growing under the ambient solar UV-B is complex and unrealistic. Differences between damage, repair, and defense can be subtle and identification of a particular mechanism does not always occur as the explanation underlying a given phenomenon. For example, the UV-induced degradation of the D1 protein of the PSII can be seen either as damage or as a part of the repair mechanism leading to substitution of the damaged components of the PSII (Turcsanyi and Vass 2000) . Consequently, understanding these differences and potentially using the DNA repair mechanisms could become very important for producing UV-B-tolerant plants.

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