Repair of Damaged Bases

Those of us who focus on DNA repair processes in plants do so with a variety of motivations. There are those labs that have a focus on genetic engineering–we try to understand the mechanism of recombination and mutation with the ultimate goal of controlling these processes. Can we modify the frequency and location of meiotic recombinational events? Can we enhance the efficiency of gene replacement? Can we alter the nature and frequency of mutational events? There are also groups that focus on plant response to environmental stresses. Can plants withstand the toxic effects of predicted increases in environmental UV-B? Will enhanced UV-B result in enhanced mutagenesis? Do general environmental stresses also have a genotoxic effect? Is the plant genome destabilized by environmental stress, and if so, is this a programmed response to environmental change? The completion of the Arabidopsis genome project has revealed that the suite of repair proteins produced by Arabidopsis is remarkably similar to that of humans. Although there are some critical differences (discussed below), the similarities are impressive. Given this, Arabidopsis may turn out to be an excellent model system for the genetic analysis of many aspects of repair in higher eukaryotes. Gene-specific knockout mutants are a less expensive to isolate in Arabidopsis than in mice. Recent publications, and much unpublished data, also suggests that plants are far more tolerant to persisting DNA damage than mammals. Many mutations that are viable in bacteria or yeast lead to embryonic lethality in mammals. However, at least some of these lethal mutations can be rescued by a second mutation in the signal transduction pathway required for an apoptotic response to DNA damage, indicating that embryonic lethality is a suicidal response to damage, rather than an intrinsic effect of DNA damage itself (Frank et al., 2000; Sekiguchi et al., 2001). The embryonic lethality of repair defective mutants makes it difficult to assess the potential roles of repair genes in later aspects of development, including meiosis. In contrast, Arabidopsis lacks a p53 homolog and there is as of yet no evidence that plants undergo an apoptotic response to the accumulation of DNA damage. Repair defects that are lethal in mice are viable in plants. For example, knockouts of RAD50 are embryonic lethal in mice; the same knockout produces a viable plant line, enabling us to determine, for the first time in a higher eukaryote, that RAD50 plays an important role in both meiosis and telomere maintenance (Gallego et al., 2001; Gallego and White, 2001). Arabidopsis may soon emerge as a robust and inexpensive higher eukaryotic model for the genetic analysis of the roles of DNA repair pathways in the repair of various lesions, and for the effects of the persistence of these lesions on organismal development. In the review below I'll provide an update on the state of our understanding of the mechanisms for the repair of damaged bases in Arabidopsis. This review will not address the repair of double strand breaks, and I direct the reader to recent reviews on that topic (Puchta, 1998; Gorbunova and Levy, 1999). Nor will I cover the study of DNA damage tolerance pathways in plants, a field in which there is only one publication which is not entirely speculative (Garcia et al., 2000). I'll point out some interesting features revealed by the Arabidopsis genome project, the substrate-specificities defined by biochemical analysis of isolated Arabidopsis repair proteins, and the characteristics of various repair-defective mutants. The accession numbers of most of the Arabidopsis repair gene homologs described below can be obtained in Table 5 of the supplementary data to (The Arabidopsis Genome Initiative, 2000), as well as at the Plant DNA Metabolism website at the University of Arizona (http://ag.arizona.edu/dnametab/). The biological significance of damaged bases. DNA damage products can be defined as any chemical alteration to DNA that covalently alters its normal structure. Bases can be oxidized, reduced, crosslinked, and alkylated. Each lesion can often be processed via a number of different repair pathways, and different organisms carry a different suite of repair-related genes (Friedberg et al., 1995). Different lesions have different biological consequences depending on the frequency of their induction, their ability to affect or alter the activity of polymerases, and the efficiency and accuracy of their repair. Lesions that do not affect the ability of RNA polymerase to transcribe DNA, or of DNA polymerase to faithfully replicate it, are harmless in and of themselves (though many of these harmless products decay to form more dangerous ones). “Noncoding” damage products (for example, UV-induced pyrimidine dimers) act as blocks to both RNA and DNA polymerase, and their repair can be critical to the survival of the cell. Other more insidious types of “miscoding” damage products (for example, O6-methylguanine) do not block DNA polymerase but are often misinterpreted by this enzyme, directly leading to the induction of mutations. The significance of a DNA damage product also depends on its location in the genome and the developmental state of the cell in which it resides. A pyrimidine dimer in a nontranscribed region, or even a nontranscribed strand, of DNA in a nonreplicating cell has no biological significance. Given the amount of noninformational DNA in the genomes of most higher eukaryotes, and the fact that most cells are nonreplicating, this means that the majority of DNA damage products are innocuous. In contrast, the induction of a dimer on a transcribed strand of a potentially replicating genome is both a toxic and a genotoxic event, and its repair is a very high priority for the cell. The mechanisms by which eukaryotic cells set priorities for the repair of the same kinds of lesions in different types of cells or different parts of the genome have only recently been addressed in yeast and mammals (Hanawalt, 2001). Whether plants share the same mechanisms, or even the same priorities, remains to be seen.