Review

Blackwell Publishing, Ltd. Tansley review DNA repair mechanisms in plants: crucial sensors and effectors for the maintenance of genome integrity

Author for correspondence: Clifford M. Bray1 and Christopher E. West2 Cliff Bray 1Faculty of Life Sciences, University of Manchester, Manchester M13 9PT, UK; 2Centre for Plant Tel: +44 161 275 5099 Fax: +44 161 275 3938 Sciences, University of Leeds, Leeds LS2 9JT, UK Email: [email protected] Received: 26 April 2005 Accepted: 9 July 2005

Contents

Summary 511 V. Molecular responses to genotoxic stress 523

I. Introduction 511 VI. Conclusions and future prospects 524

II. Photoreactivation 514 Acknowledgements 525

III. Excision repair pathways: BER, NER, mismatch repair 517 References 525

IV. DNA double-strand break repair 520

Summary

Key words: DNA repair, excision repair, As obligate phototrophs, plants harness energy from sunlight to split water, producing genotoxic stress, photoreactivation, oxygen and reducing power. This lifestyle exposes plants to particularly high levels of plant. genotoxic stress that threatens genomic integrity, leading to mutation, developmental arrest and cell death. Plants, which with algae are the only photosynthetic eukaryotes, have evolved very effective pathways for DNA damage signalling and repair, and this review summarises our current understanding of these processes in the responses of plants to genotoxic stress. We also identify how the use of new and emerging technologies can complement established physiological and ecological studies to progress the application of this knowledge in biotechnology. New Phytologist (2005) 168: 511–528 © The Authors (2005). Journal compilation © New Phytologist (2005) doi: 10.1111/j.1469-8137.2005.01548.x

linkages and the purine and pyrimidine bases that contribute I. Introduction informational content to the genome – can suffer damage. The genome of a living organism is subjected to a wide variety The biological consequences arising from any damage to the of genotoxic stresses from both endogenous (intracellular) and genome depend upon the chemical nature of the alteration environmental origin during its lifetime. All of the primary to the structure of the DNA and can vary from the innocuous components of DNA – the sugar residues, phosphodiester to the highly mutagenic. Ultimately, the toxicity or mutagenicity

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of the incurred damage to the genome will depend upon a result of the cumulative effects of temperature, moisture the efficiency of the cellular mechanisms that have evolved to and oxygen levels pertaining over the time period of storage. sense, recognise and eliminate the damage and the accuracy of Accumulation of chromosomal damage and/or an inability to the pathways available to the cell to repair the lesion. repair such damage during the imbibition period appear to be The importance to plants of such appropriate and effective significant factors contributing to loss of seed viability during responses to potential mutagenic events is emphasised upon storage (Cheah & Osborne, 1978). The relatively easy detec- examination of a plant’s lifestyle. Plants, unlike most higher tion of chromosome breakage or translocations has enabled eukaryotes, lack a reserve germ line, with gametes arising from cytogenetic studies of DNA damage. These gross chromosomal undifferentiated meristematic cells on completion of the alterations are potentially lethal events but probably occur sporophytic part of the plant life cycle. These progenitor cells at low frequency. have passed through numerous rounds of DNA replication Several attempts have been made to quantify other types of and cell division before gamete formation. This provides the damage to the DNA of the eukaryotic genome arising from opportunity for mutations to arise in the genome through endogenous sources, but these studies have been focused mainly errors in the process of DNA replication during the cell cycle and on animal cells. Cellular metabolites can react with genomic DNA long-term exposure of their genome to damaging endogenous including the intracellular methyl group donor S-adenosyl and environmental mutagens. The sessile, photosynthetic methionine, which acts on DNA like a weak alkylating agent. nature of a plant’s lifestyle also means that plants have had to Chromosomal proteins offer no significant protection to the evolve different strategies to animals in order to minimise the DNA in chromatin against cytotoxic alkylation of purines by effects of harmful genotoxic environmental agents, such as solar S-adenosyl methionine, although these modifications occur at ultraviolet (UV) irradiation, to which they are continually a rate which is estimated to be ∼20 times lower than the rate exposed. The observation that mutation rates seen in long-lived of depurination (Lindahl & Barnes, 2000). The level of damage plant species, such as trees, are not unexpectedly high indicates to mammalian DNA arising from these endogenous sources that the activities responsible for maintaining the integrity highlights the extent to which the cell continually needs to employ of the genome in plant somatic cells must be very efficient its armoury of DNA repair pathways to maintain the integrity (Hays, 2002). However, there is evidence that in exceptional of the genome. Water represents one of the main sources of circumstances – such as in mangrove trees (Rhizophora mangle), damage to the genome and DNA is inherently unstable in the where numerous cell divisions over many years separate one aqueous environment. The spontaneous nonenzymatic cleavage generation from the next – that such mutations can accumulate of glycosyl bonds in DNA and the hydrolytic deamination of to significant levels in plant meristems (Klekowski & Godfrey, cytosine bases accounts for a considerable amount of damage 1989; Britt, 1998). to the mammalian genome on a daily basis (Table 1). Several One of the earliest suggestions that loss of plant cell viability thousand oxidative adducts (chemically altered bases) may also could arise from spontaneous mutations in the genome accumulate in the DNA of animal cells (Table 1) as a result of of cells arising from endogenous (intracellular) sources came oxidation of bases in DNA by biological oxidants such as from studies on seeds of Crepis tectorum L. (Navashin, 1933). reactive oxygen species (ROS) (Beckman & Ames, 1997), Seeds stored for 6 yr at ambient temperature germinated poorly and the efficient removal of these oxidative products requires and produced seedlings that were weak or that had developmental effective excision repair pathways to operate in the cell. abnormalities, including albinism. The mutant phenotypes The few studies which have attempted to quantify the scale arising from seed ageing resembled those produced by high-dose of DNA damage in plant cells have demonstrated the extent to X-ray treatment, including translocations involving two or which the plant genome can be damaged by endogenous and more chromosomes (Navashin, 1933). The mutation rate was exogenous genotoxins. Environmental stresses including drought, accelerated by increasing the temperature at which the dehy- high and low temperatures and increased UV-B owing to strato- drated seeds were stored (Navashin & Shkvarnikov, 1933). spheric ozone depletion result in significant reductions in both Subsequent studies some 35 years later (Abdallah & Roberts, crop quality and productivity largely mediated via the production 1969) demonstrated that chromosome damage appearing during ROS. In plant cells, ROS are the primary cause of single-strand the ageing of seeds, including those of major crop species, was breaks (SSBs) in the DNA, either directly, through destruction

Table 1 Estimates of endogenous DNA Events per damage in animal cells arising from Process day per cell Reference spontaneous hydrolysis, ROS and metabolite induced damage to the genome Depurination 2–10 000 Lindahl & Nyberg (1972) Cytosine deamination 100–500 Lindahl & Nyberg (1974) Alkylation (S-adenosylmethionine) ≈600 Lindahl & Barnes (2000) Oxidative adducts ≈150 000 Beckman & Ames (1997)

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Table 2 Estimates of accumulation and repair of apurinic/apyrimidinic exposure to high levels of ionising radiation in its lifetime. (AP) sites in DNA during early germination of maize seeds (from However, the explosion which occurred in the Number 4 Dandoy et al., 1987) reactor at the Chernobyl nuclear power plant in the Ukraine

in April 1986 and the extensive radioactive fallout which fol- AP sites per 106 AP sites per nucleotides in DNA Status genome per cell lowed resulted in the contamination of large tracts of inhab- ited agricultural land in Belarus, the Ukraine and Russia, and 38 Quiescent unimbibed seed 9.5 × 104 scattered areas beyond these limits. This disaster has provided 150 20-h imbibed seed 3.75 × 105 a fresh impetus and relevance to studies on the effects of ionising radiation in ecosystems. This contaminated region has produced a unique radioecological situation over a large tract of deoxyribose units, or by covalent modification of bases. of land (over 600 km2) where both habitation and agriculture Repair of potentially mutagenic modified bases is mediated by have been prohibited. Here, nuclear pollution of the environ- excision repair pathways that are themselves an additional ment can be studied and the effects correlated with the genetic secondary source of SSBs. ROS continually arise within hazards to animal and plant life. plant cells as a result of normal oxidative cellular processes and The initial fallout exposed plants to radiation doses of over present a continuous danger to the integrity and viability of 60 Gy, and pine trees near to the power plant were killed by the cell even in the absence of external stresses. this dose. In general, woody species appeared to be more The seed may provide an ideal ‘model’ system through which susceptible to the killing or damaging effects of radiation than to investigate the effects of a variety of endogenous DNA herbaceous species did, but the molecular basis for this differ- damaging agents and environmental stresses on genome ence is unknown. In spite of these initial high levels of radia- integrity. From the early stages of development on the mother tion, plants have continued to grow since the accident, even plant, the seed experiences a range of developmentally pro- in the most contaminated areas. Several investigations into grammed and unprogrammed oxidative, water and tempera- the effects of radiation on plant genome stability and on the ture stresses which are potentially damaging to the genome. In ability of plants in this area to survive and adapt to the dam- the case of orthodox seed, maturation drying reduces mois- aging effects of radiation have been performed (summarised ture content to 5–15% and production of a new seedling is in Kovalchuk et al., 2004). Wheat and rye grown in the exclu- subsequently initiated by rehydration and germination sion zone exhibited high levels of chromosomal aberrations, (Bewley & Black, 1994). In maize seeds, the maturation drying/ indicating significant damage to the genome of these plants rehydration cycle results in the appearance of thousands from the ionising radiation (Kovalchuk et al., 2000). Seeds of of SSBs in the genome of each cell (Dandoy et al., 1987; Arabidopsis thaliana that survived high levels of exposure to Table 2), which can result in cell cycle arrest (Reichheld et al., ionising radiation produced plants that were more resistant to 1999). Significantly, this study (Dandoy et al., 1987) meas- radiomimetic agents or free radical producing agents than ured only SSBs arising indirectly from base damage by base plants grown from seed from Arabidopsis exposed to lower excision repair (BER) pathways. Consequently, this figure radiation levels (Kovalchuk et al., 2004). In addition, Arabi- is an underestimate of the number of SSBs appearing in the dopsis seeds collected from plants grown in the contaminated genome because it takes no account of those arising directly in area 5–6 yr after the explosion produced plants that were the DNA from sugar damage by ROS during imbibition. The more resistant to mutagens than plants from the same sites increase in apurinic/apyrimidinic sites (AP sites) seen in maize collected 3–4 yr after the explosion, reflecting ongoing adap- radical tip cells during the first 20 h of imbibition (Table 2) tation within these populations to survival in the face of con- reflects repair of damaged bases either already present in tinued chronic exposure to the effects of ionising radiation. the DNA of radical tip cells in the mature quiescent embryo or A series of elegant experiments utilised a recombination which appear in the genome of these cells during early imbi- substrate comprised of a disrupted β-glucuronidase (uidA) bition. These AP sites need to be repaired before the onset of reporter gene integrated into the Arabidopsis genome to study S-phase of the cell cycle, which in these maize embryos does genome stability (Kovalchuk et al., 1998). These transgenic not occur during the 36-h imbibition period studied. plants have proved to be useful and sensitive bioindicators of The DNA of living organisms has been subjected to the the effects of ionising radiation arising from nuclear fallout damaging effects of ionising radiation throughout evolution. and have provided a unique insight into the remarkable Although ionising radiation energy can directly cause both adaptability of plants to respond to and survive severe acute SSBs and DSBs (double-strand breaks, the most lethal form and chronic periods of genotoxic insult. Arabidopsis plants of damage) in DNA, a major source of spontaneous damage growing in the highly contaminated exclusion zone showed arises from radiolysis of water to produce ROS, including the adverse effects on seed germination rates and an increasing highly damaging hydroxyl radical (Friedberg et al., 1995). mutation frequency, which was also observed in the progeny Fortunately, a living organism will rarely, if ever, encounter plants from these seeds, reflecting the highly toxic effects of either short bursts (acute) or lengthy (chronic) periods of the levels of ionising radiation (up to 6000 Ci km−2) in the

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polluted soil (Kovalchuk et al., 1998). Chronic exposure to A third UV component of the electromagnetic spectrum, UV- ionising radiation increased both the incidence of strand C (200–280 nm) fails to penetrate the biosphere because it is breaks in the genome of exposed Arabidopsis plants and the strongly absorbed by oxygen and ozone in the atmosphere. The frequency of somatic intrachromosomal homologous recom- genotoxic effects from solar radiation arise from a combination bination (HR) events in the genome of transgenic plants, as of UV-A/visible-light-induced photosensitisation reactions and detected through reconstitution of β-glucuronidase activity UV-B-induced dimer production between adjacent pyrimidine on histochemical staining of plants (Kovalchuk et al., 1998). residues in a DNA strand. Photosensitisation reactions cause Somewhat surprisingly, HR rates declined in Arabidopsis plants oxidative damage to bases in DNA and, to a lesser extent, exposed to the highest levels of ionising radiation (above 1000 abasic sites and DNA strand breaks. UV-B photons induce Ci km−2), but this may not reflect a simple decline in overall the formation of two major types of pyrimidine dimer, the recombination rates within these plants. This observation cyclobutane pyrimidine dimer (CPD) and the pyrimidine– could be accounted for by an increase in the level of the pyrimidone (6–4) photoproduct (6–4PP) (Fig. 1a). The 6– alternative, less precise, illegitimate recombination pathway 4PP may occasionally convert to the Dewar isomer, which that may result in an inactive β-glucuronidase gene even after is formed by photoisomerisation of the 6–4PP by light of a recombination event. Further investigations showed a very wavelengths longer than 290 nm. Quantitative estimations of low frequency of extrachromosomal HR events in plants from the relative occurrence of the different forms of pyrimidine Chernobyl seed collections compared with controls (Kovalchuk dimers in plant tissues after exposure to UV-B indicate that et al., 2004). Collectively, these studies suggest that the survival CPDs constitute the majority of these dimers being formed at adaptation response to genome damage arising from chronic 9 times higher yield than 6–4PPs (Dany et al., 2001). ionising radiation effects in plants is initially to up-regulate Exposure of plants to elevated levels of UV-B radiation is high precision repair via intrachromosomal HR whilst down- detrimental to plant growth, development, morphology and regulating extrachromosomal recombination events, thereby metabolism (Dany et al., 2001). Removal of these pyrimidine preserving genome integrity by restricting potentially harmful dimers is necessary for plant survival (Landry et al., 1997) genome rearrangements. because their presence in the genome is both mutagenic and cytotoxic owing to their ability to block the progress of DNA and RNA polymerases along the DNA during replication and II. Photoreactivation transcription. However, there is some evidence that plants Solar UV radiation reaching the Earth’s surface comprises mainly may tolerate a low level of CPDs in their genome (Britt et al., UV-A (315–400 nm) and UV-B (280–315 nm) wavelengths. 1993; Britt & Fiscus, 2003; Schmitz-Hoerner & Weissenbock,

Fig. 1 UV-B induced DNA damage and repair. (a) Structures of the two major UV- induced DNA photoproducts typified by the formation of cyclobutane pyrimidine dimers and pyrimidine–pyrimidone (6 – 4) photoproducts between adjacent thymine bases in a DNA strand. (b) Simplified overview of the reaction catalysed by the CPD photolyase enzyme. A blue light photon is absorbed by the MTHF chromophore. Energy from the excited MTFH* is then used to excite the fully reduced flavin FADH–, which in turn participates in a cyclic electron transfer step, resulting in the splitting of the pyrimidine dimer into two pyrimidine monomers and conversion of the flavin coenzyme back into the active two electron fully reduced state.

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2003). This probably reflects the activity of specialised second chromophore is either 5,10 methenyltetrahydrofolate translesion polymerases which function in the nonmutagenic (MTHF) or 8-hydroxy-7,8-didemethyl-5-deazariboflavin bypass of UV-B-induced photoproducts persisting in the (8-HDF), which, whilst not essential for activity, are far more genome (Hays, 2002; Kozmin et al., 2003). These lesions are efficient than FADH as a photoantenna through which to removed from DNA in the plant cell by either relatively inef- absorb blue (350–450 nm) photons (Sancar, 2003). Plants ficient excision repair pathways (the so-called ‘dark repair’ appear to contain the reduced pterin MTHF in their CPD pathways which are independent of light and which are photolyase as the second chromophore (Waterworth et al., described later) or via efficient enzymatic photoreactivation 2002). In a light-independent reaction, the photolyase binds processes which depend upon light of wavelength 350– to a pyrimidine dimer lesion which has ‘flipped out’ of the 450 nm (Taylor et al., 1996). DNA structure (Berg & Sancar, 1998; Carell et al., 2001) and The photoreactivation reaction is catalysed by a DNA forms a stable enzyme–substrate complex (Fig. 1b). The folate photolyase enzyme that binds to UV-damaged DNA and uses chromophore of photolyase absorbs a blue light photon a blue light (350–450 nm) photon to power cyclic electron and transfers the excitation energy of the photon to FADH–, transfer to split the pyrimidine dimer ring structure and promoting a one-electron transfer from FADH– to the enzyme- restore the bases to their normal undamaged form (Sancar, bound pyrimidine dimer complex (Fig. 1b). The covalent 2003). In natural ecosystems, light in the visible region of linkage between the adjacent pyrimidines of the CPD is split the spectrum always accompanies solar UV-B irradiation; and the two pyrimidine bases return to their original states in thus the photolyase catalysed reaction constitutes an exquisite the DNA, with the simultaneous transfer of an electron back light-dependent, error-free mechanism for repair of UV-B- to FADH and regeneration of the active two-electron-reduced induced DNA damage without the need for any base or form FADH– (Sancar, 2003). Thus the overall reaction does nucleotide excision step. Distinct photolyase enzymes repair not involve either the gain or loss of an electron. A similar either the CPD or the 6–4PP lesion, with the enzyme respon- mechanism is suggested to operate for 6–4 photolyases, but sible for recognising the 6–4PP also able to recognise and with the inclusion of a step involving thermal conversion of repair the Dewar isomer but at reduced efficiency compared the 6–4PP to an unstable oxetane intermediate before the with the 6–4PP (Carell et al., 2001). Almost all organisms, photochemical reversion steps (Sancar, 1996). with the possible exception of placental mammals, produce a Levels of UV-B-induced DNA damage and stage of plant CPD photolyase, but the presence of a 6–4 photolyase has development are both factors which influence the pathways been detected only in a select number of species, including employed by plant cells to remove UV-B-induced photoprod- insects and plants (Table 3; Todo et al., 1996; Nakajima et al., ucts from their genome. Low levels of pyrimidine dimers 1998). (< 30 dimers per 106 bases) are removed from alfalfa seedlings The reaction mechanisms by which CPD photolyases and via error-free photoreactivation mechanisms, with little con- 6–4 photolyases repair the respective pyrimidine dimers are tribution from excision repair pathways (Quaite et al., 1994). similar. All photolyases contain two cofactors, one of which is At higher DNA damage levels, additional repair capacity always the two-electron-reduced form of FAD (FADH–), the appears to be needed, and both photoreactivation and universal photocatalyst in all photolyases (Sancar, 2003). The excision repair pathways are involved in removal of pyrimidine

Table 3 Arabidopsis NER and photolyase genes and the corresponding ethyl methane sulfonate (EMS) mutants

Human Yeast Arabidopsis AGI code Mutant Human Yeast Arabidopsis AGI code Mutant

XPA Rad14 ? XPG Rad2 AtXPG/RAD2 At3g28030 uvh3/uvr1 XPC Rad4 At5g16630 ERCC1 Rad10 AtERCC1 At3g05210 uvh7 hHRD23 Rad23 At1g79650 CSA Rad28 At2g18760 At1g16190 At3g02540 At5g38470 XPB Rad25 AtXPB1 At5g41370 CSB Rad26 At1g27840 AtXPB2 At5g41360 At1g19750 XPD Rad3 AtXPD At1g03190 uvh6 hCEN2 AtCEN2 At4g37010 XPF Rad1 AtRAD1 At5g41150 uvh1 Photolyases PHR1 CPD photolyase At1g12370 uvr2 6–4 photolyase At3g15620 uvr3 (PHR)(UVR3)

Characterised genes are displayed in bold type. Genes identified by database analysis are taken from Kunz et al. (2005).

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dimers from the genome. There are also contrasting gene America, is the only region where a well-developed terrestrial expression patterns for photoreactivation and excision repair ecosystem is affected by ozone depletion, and this area has genes in different tissues during plant development. Although been the subject of intensive research into the effects of photoreactivation mechanisms are widely distributed throughout elevated solar UV-B irradiation on plants in the environment both meristematic and nonproliferating cells in above ground (Ballaré et al., 2001). Using Gunnera magellanica as an indig- organs in plants, the expression of excision repair pathway enous ‘model’ herbaceous plant, these studies demonstrated transcripts is much higher in proliferating than nonprolifera- that steady-state levels of CPDs in leaf tissue were linearly tive tissue (Kimura et al., 2004). In Arabidopsis, the highest correlated with UV dose at ground level. Increased solar UV-B levels of CPD and 6–4PP photolyase proteins are found in levels in early spring had a subtle but significant inhibitory floral tissues, where they may serve to minimise formation of effect on leaf growth and morphology in G. magellanica mutagenic lesions in germline DNA because long-term expo- and in other herbaceous species studied but had no effect on sure to high UV-B levels may have deleterious effects on the growth of woody perennials. This effect of UV-B contrasts genomes of the cells in plant reproductive tissues (Ries et al., with that of γ-irradiation, for which woody species appear 2000). Repair of UV-B-induced damage to the genome is more susceptible than herbaceous species (Kovalchuk et al., expected to be more critical in young growing seedlings where 2004). These observations indicate distinct species differences DNA replication is associated with elevated levels of cell divi- in response to enhanced UV-B irradiation levels, and in the sion. However, young Arabidopsis seedlings contain very low case of G. magellanica may reflect the low CPD repair capacity levels of CDP photolyase protein compared with mature leaf of this species compared to other species studied (Ballaré tissue indicating an important role for DNA repair in differ- et al., 2001). These and other studies (Stapleton & Walbot, entiated tissues (Waterworth et al., 2002). In contrast, expres- 1994; Ries et al., 2000; Britt & Fiscus, 2003) have also sion of excision repair genes in wheat and Arabidopsis is higher demonstrated that the production of UV-absorbing molecules in proliferating cells compared with developmentally mature such as flavanols and sinapate esters by plant cells in response cells (Taylor et al., 1996; Waterworth et al., 2002; Kimura to elevated UV-B and white light levels is only partially effec- et al., 2004). tive in protecting the plant genome against the damaging CPD photolyase gene expression in Arabidopsis tissues is effects of enhanced solar UV-B irradiation. inducible by white light (Ahmad et al., 1997), whereas 6–4PP Elevated UV-B levels may also be accompanied by global photolyase gene expression is constitutive (Waterworth et al., warming in the early part of the 21st century (Moody et al., 2002). However, white light intensity does affect the rate of 1997). Photolyase-mediated repair of UV-B-induced DNA removal of both CPD and 6–4PP photoproducts from wheat damage has been reported to be sensitive to temperature leaf DNA. Dimer removal rates decrease with decreasing light increases above ambient (Pang & Hays, 1991; Takeuchi et al., fluences (Taylor et al., 1996), with 6–4PP removal affected to 1996), which could have important implications for crop a lesser extent than CPD removal, indicating either a decrease yield and biomass production via detrimental effects on in CPD gene transcription or a more rapid turnover of CPD morphology and growth. However, this temperature sensitiv- photolyase protein than 6–4PP photolyase under low light ity of the CPD photolyase, at least for Arabidopsis, may vary conditions or the more effective removal of 6–4PPs by light- with ecotype because it appears more significant in Columbia independent excision repair pathways. Significantly, the rate than in Landsberg erecta (Pang & Hays, 1991; Waterworth of CPD photolyase protein turnover is slow in Arabidopsis et al., 2002). The residual CPD photolyase activity present in (Waterworth et al., 2002), which implies that there will Arabidopsis grown at temperatures up to 37°C would still be always be a low level of CPD photolyase protein present in sufficient to cope with the anticipated increased levels of UV- tissues from field-grown plants or plants experiencing normal B-induced DNA damage sustained under these growth con- light/dark cycles. Therefore these plants will be capable of ditions (Waterworth et al., 2002). However, the same may photoreactivation upon first exposure to sunlight, of which not be true for all species, especially genetically inbred crop UV-B is an inevitable component. The white light induction species. The long-term effects of increased UV-B irradiation of plant CPD photolyase combined with the CPD protein levels accompanying depletion of the stratospheric ozone turnover rate in cells may explain why a delay and initial slow layer on genome stability within plant populations is difficult rate of repair of CPDs has been seen in plants exposed to UV- to predict with any certainty. However, in addition to photo- B and white light after prolonged periods in the dark (Chen reactivation and excision repair pathways, evidence is now et al., 1994). emerging that HR mechanisms may also contribute to the Thinning of the stratospheric ozone layer in the latter part repair of UV-B-induced DNA damage in Arabidopsis in of the 20th century has resulted in elevated solar UV-B irra- conditions of long-term exposure of plants to UV-B (Ries diation levels reaching the Earth’s surface (Madronich et al., et al., 2000). Further research is required to determine whether 1998) and stimulated significant research into the biological other species have retained the protective mechanisms against consequences of these increased UV-B irradiation levels on the adverse effects of UV-B irradiation and global warming on ecosystems. Tierra del Fuego, at the southern tip of South genome integrity that have been found in Arabidopsis.

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Fig. 2 Pathway for BER in human cells. (a) Short-patch repair. Base damage is removed by a DNA glycosylase or a bifunctional DNA glycosylase-endonuclease and the AP site is cleaved by AP endonuclease. POLβ removes the deoxyribose sugar if necessary and fills in the gap. In mammals the single-stranded gap is sealed by the XRCC1-LIG3 complex. This pathway may be active in plant cells, although there is no LIG3 homologue. (b) Long-patch repair. After base removal and APE nicking, the replicative DNA polymerase δ/ε complex fills the gap and displaces several nucleotides adjacent to the AP site. Endonuclease activity removes the overhang and the nick is re-joined by LIG1. Adapted from Sancar et al. (2004).

damaged base by a DNA glycosylase enzyme that is specific for III. Excision repair pathways: BER, NER, mismatch the particular base adduct (Caldecott, 2001; Fromme et al., repair 2004). Several DNA glycosylases have been characterised in Many forms of DNA damage affect only one of the two strands plants, including a 3-methyladenine-specific DNA glycosylase of the duplex. Highly accurate repair of these DNA lesions is (Santerre & Britt, 1994), which showed highest expression facilitated using the sequence information provided by the com- in tissues undergoing rapid cell division and growth (Shi et al., plementary strand. This process is initiated by the recognition 1997). Some glycosylases have a secondary activity, cutting and excision of the damaged region and completed by repair the DNA backbone on the 3′ side of the abasic (AP) site synthesis and ligation. There are distinct excision repair pathways (Fig. 2). For example, repair of the guanine oxidation product (termed dark repair pathways in plants) that are highly conserved 7,8-dihydro-8-oxoguanine (8-oxoG) in Arabidopsis is catalysed through evolution. by the bifunctional 8-oxoG DNA glycosylase/AP lyase (Dany & Tissier, 2001; Garcia-Ortiz et al., 2001). 8-oxoG is read as thymidine, giving rise to G–T mutations. This damaged base 1. Base excision repair can also be repaired by AtMMH, the Arabidopsis homologue Metabolic by-products and environmental genotoxins react of Escherichia coli MutM, indicating redundancy in plant with cellular DNA to produce SSBs and a variety of different BER pathways (Ohtsubo et al., 1998; Murphy & Gao, 2001). base adducts. Experimentally, base modification is widely DNA glycosylase activity results in release of the damaged exploited in plant mutagenesis, which uses ethyl methane base adduct, leaving an AP site which is a substrate for the SSB sulfonate (EMS) to produce alkylation products, including repair pathway (Fig. 2). In mammalian cells, the AP site can be O6-ethylguanine. This modified base is recognised as adenine further processed by formation and repair of a single nucleotide by the DNA replication machinery, causing G–A mutations gap (short-patch repair). In the alternative long-patch repair, (Vidal et al., 1995; Greene et al., 2003). Whereas some base AP endonuclease (APE) cuts the sugar phosphate backbone 5′ adducts are miscoding, others, including 3-methyladenine, to the AP site (Babiychuk et al., 1994; Sancar et al., 2004). block replication and transcription and are highly cytotoxic. Repair is completed by DNA polymerase complex Polδ/ε, A wide range of damaged bases can be eliminated from the PCNA and FEN1, also active in replication, which displaces genome by BER. This pathway is initiated by removal of the and resynthesises 2–10 nucleotides 3′ to the abasic site.

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In cases where the damaged base is removed by a bifunc- both excision and recombinational repair pathways (Amor tional DNA glycosylase/AP lyase the subsequent 5′ incision et al., 1998; Babiychuk et al., 1998; Babiychuk et al., 2001). by APE results in the release of the deoxyribose moiety. In mammalian cells, the resulting gap can be filled by short-patch 2. Nucleotide excision repair repair mediated by the POLβ-XRCC1-LIG3α pathway. POLβ has intrinsic deoxyribose (dRP) lyase activity, and this enzyme In contrast to BER, NER can act on a wide range of substrates can also remove the sugar left by a monofunctional DNA as this pathway detects modifications indirectly by conforma- glycosylase, and the DNA polymerase activity fills the single tional changes to the DNA duplex rather than relying on the nucleotide gap (Caldecott, 2001). The short-patch repair appears recognition of specific DNA damage products. NER targets the to be less well conserved, and there are no clear plant homologues damaged strand and removes a 24–32 base oligonucleotide of DNA polymerase β (POLβ) or of DNA ligase III. However, containing the damaged product (Fig. 3). DNA synthesis and rice DNA polymerase λ, which shares a similar N-terminal ligation completes the repair process (Sancar et al., 2004). domain with human Polλ, has in vitro dRP-lyase activity, indicat- This highly conserved DNA repair pathway is also found in ing a potential POLβ-like role in BER (Uchiyama et al., 2004). plants and was initially characterised by EMS mutagenesis. Interestingly, an Arabidopsis XRCC1-like protein lacks the These experiments defined a number of uvr and uvh comple- domains responsible in mammalian XRCC1 for interaction mentation groups that showed hypersensitivity to UV-C, some with POLβ and LIG3α but retains a conserved BRCT domain of which were also hypersensitive to γ-irradiation (Jenkins which may mediate interaction with poly(ADP-ribose)polymerase et al., 1995; Jiang et al., 1997). In addition to the NER genes (PARP) (Doucet-Chabeaud et al., 2001; Taylor et al., 2002; identified in these genetic screens, many putative homologues Uchiyama et al., 2004). There are at least two PARP activities of the evolutionarily conserved NER components can be in plants and, whilst the functions of these proteins remain found in plant genome databases (Table 3; The Arabidopsis uncertain, they may have a role in DNA damage sensing in Genome Initiative, 2000; Kunz et al., 2005).

Fig. 3 Pathway for NER in human cells. In global genomic repair, DNA damage is recognised through binding by the XPC/ RAD23 complex which recruits the TFIIH transcription factor. Transcription-coupled repair (not shown) occurs where RNA polymerase stalls at the site of DNA damage and is independent of XPC. Helicase activity of the TFIIH components XPD and XPB unwinds the damaged region and the endonucleases XPG and XPF/ERCC1 allow the release of the single-stranded oligonucleotide. Repair synthesis is catalysed by the POLδ/ε complex and completed by LIG1-mediated ligation. Adapted from Sancar et al. (2004).

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NER is catalysed by six multiprotein complexes, and the the Saccharomyces cerevisiae nomenclature as AtRAD1, and the initial DNA damage detection step can occur by two different full-length gene was shown to be partially functional in yeast processes dependent on the transcriptional activity of the rad1– mutants (Gallego et al., 2000). Arabidopsis uvh1–1 mutants damaged region. Global or general genomic repair (Fig. 3) is are deficient in AtRAD1 and display a small reduction in CPD initiated by damage recognition mediated by the xeroderma repair and hypersensitivity to DNA damage (Fidantsef et al., pigmentosum group C (XPC)/hHR23B complex (Thoma 2000; Gallego et al., 2000; Liu et al., 2000). A Lily (Lilium & Vasquez, 2003). In contrast, transcription-coupled repair longiflorium) ERCC1 homologue was able to complement the (TCR) is independent of XPC and is initiated when RNA MMC hypersensitivity of ERCC1-deficient Chinese hamster polymerase II stalls at the site of DNA damage (Mu & Sancar, ovary cells (Xu et al., 1998). Mouse ERCC1 mutants are embry- 1997). TCR in mammals requires the Cockayne syndrome onic lethal, whereas an Arabidopsis uvr7–1 mutant containing (CS) CSB and CSA proteins, which may help remove the a premature stop codon in the AtERCC1 gene is viable, although stalled RNA polymerase complex and facilitate DNA repair hypersensitive to UV-C and γ-irradiation (Hefner et al., 2003). (Svejstrup, 2002; de Waard et al., 2004). RAD1/ERCC1 has additional roles in the removal of DNA In general genomic NER in humans, the XPC/hHR23 com- ‘flap’ structures formed during recombination. Differences in plex is stabilised by hCEN2, and Arabidopsis plants deficient recombination frequencies between inverted chromosomal in AtCEN2 show decreased repair of UV-C-induced DNA repeats in atercc1 and wild-type plants showed that AtERCC1 damage in vitro (Molinier et al., 2004). XPC/hHR23B also has a role in the major HR pathways responsible for gene binding recruits TFIIH, which functions in the cell as an RNA conversion in plants (Dubest et al., 2004). The gap generated polymerase II transcription factor in addition to its role in by the endonuclease activities of XPG and the AtRAD1/ repair. A rice hHR23B homologue was first identified through ERCC1 complexes is filled by the replicative DNA polymerase its interaction with the transcription factor VP1 (Schultz & complex of Polδ/ε, PCNA, RFC and RPA and the phos- Quatrano, 1997). The TFIIH transcription factor complex phodiester backbone rejoined by DNA ligase – probably AtLIG1 contains the helicases XPB and XPD which unwind the (Taylor et al., 1998). DNA, allowing binding of XPG with the concomitant release of XPC (Fig. 3). Unlike most organisms, Arabidopsis has two 3. Mismatch repair isoforms of XPB with similar expression patterns, and both genes are able to complement repair defects in the corre- During genome replication, the replicative DNA polymerases sponding Saccharomyces cerevisiae mutant (Costa et al., 2001; accurately select their deoxynucleotide substrates and use their Morgante et al., 2005). Despite this apparent redundancy, atxpb1 associated proof-reading functions to remove misincorporated mutant plants show hypersensitivity to alkylating agents and nucleotides. Together, these checking mechanisms realise a fidelity to ROS, and display delayed development, especially during level of one incorrect base incorporated per 106−107 base pairs germination and early seedling growth (Costa et al., 2001). In in replicating DNA. However, this level of accuracy is still contrast with Arabidopsis XPB, human XPB and yeast RAD25 insufficient for effective genome integrity maintenance, and a are single-copy genes and null mutations are lethal, indicating mismatch repair (MMR) mechanism which is highly conserved the essential role of the TFIIH transcription factor in develop- between species subsequently removes the vast majority (99.9%) ment. The comparatively mild phenotype of atxpb1 mutants of the errors remaining after polymerase proofreading to reduce may suggest that AtXPB2 partially compensates for the lack the error rate to one misincorporated base per 109−1010 nucleotides of AtXPB1 activity or that the truncated AtXPB1 transcript in the nascent DNA chain (Leonard et al., 2003). MMR may expressed in the mutant lines may encode a protein with residual also have an important role in recognising mismatches at sites activity (Costa et al., 2001). The 5′–3′ TFIIH helicase com- of recombination between DNA sequences, thereby reducing ponent, XPD, is encoded by a single-copy gene in Arabidopsis the rate of occurrence of recombination events which might lead and null mutants are lethal (Liu et al., 2003). However, a point to inappropriate chromosome rearrangements or interspecies mutation in AtXPD in the uvh6–1 Arabidopsis line resulted in hybridisation (Wu et al., 2003). reduced growth, yellowing leaves and a slight decrease in the rate The eukaryotic homologues of the evolutionarily conserved of repair of UV-induced 6–4 photoproducts (Liu et al., 2003). prokaryotic MutS proteins involved in MMR are the MSH After unwinding of the DNA by the helicase action of XPD protein subunits. Their role in MMR is to recognise the rare and XPB, the RPA / XPA complex is recruited (Tapias et al., 2004). sporadic mismatches in replicating DNA and to prevent the This is followed by endonuclease activity of XPG, which nicks establishment of mutations in the genome by discriminating the damaged DNA strand 3′ to the lesion (Fig. 3), and an between the existing correct nucleotide in the template strand Arabidopsis xpg mutant, uvh3–1, which shows hypersensitivity and the incorrect nucleotide inserted into the replicating DNA to UV-C consistent with this role of AtXPG in NER (Liu et al., strand (de Wind & Hays, 2001). In plants, heterodimeric proteins 2001). The 5′ incision is catalysed by the XPF-ERCC1 nuclease involving MSH2, MSH3, MSH6 and MSH7 are responsible complex, allowing the release of the damaged DNA stretch as for executing this initial stage of MMR. MSH2, 3 and 6 a 24–32 nucleotide oligomer. Arabidopsis XPF is named using homologues are found in all eukaryotes but MSH7 is unique

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Fig. 4 Simplified overview of DNA mismatch repair in Arabidopsis. Initial recognition of the mismatch lesion by the appropriate ATMSH heterodimer (s) is followed by a step in which an enzyme complex, possibly involving AtMLH1. PMS2, discriminates between the nascent and template DNA strands, promoting unwinding of the DNA helix, excision of the nascent strand to a point beyond the mismatch lesion and culminating in resynthesis and ligation steps to fill in the gap in the nascent DNA strand.

to plants. Arabidopsis also contains a homologue of the yeast this strand. Finally, a high-fidelity DNA polymerase fills in the MSH1 protein which is absent from mammalian cells. AtMSH1 stretch of gapped DNA and a DNA ligase, probably DNA appears to be targeted to mitochondria but its role in MMR ligase 1 in plants (Taylor et al., 1998), rejoins the DNA ends or mitochondrial genome stability has yet to be established (Fig. 4). Arabidopsis contains orthologues of MLH1, PMS2 (Hays, 2002). In Arabidopsis, the hetreodimeric proteins and also a further MLH orthologue, MLH3 (Hays, 2002), and AtMSH2.MSH6 (termed AtMutSα) and AtMSH2.MSH7 these proteins may play a role in the coupling of mismatch (AtMutSγ) recognise mismatched bases (Fig. 4) but differ in recognition to nascent strand identification and excision steps specificity of which mismatches are recognised (Wu et al., 2003). in MMR in the plant cell (Fig. 4). AtMSH2.MSH3 (AtMutSβ) recognises regions of DNA with extrahelical loops but not mispairs, similar to the human IV. DNA double-strand break repair orthologue (Culligan & Hays, 2000). Use of Arabidopsis Atmsh2 lines in which AtMSH2 is inactivated has demonstrated a critical There are two fundamental mechanisms for recombination requirement for efficient MMR in germline cells but a less which are required for the repair of DNA DSBs. HR uses critical role for MMR in differentiated somatic cells (Hoffman an identical or very similar DNA sequence as a template for et al., 2004). Arabidopsis has two other proteins resembling the repair of a DSB. Illegitimate recombination, also termed MSH homologues MSH4 and MSH5 (Hays, 2002). The nonhomologous end joining (NHEJ) recombines DNA largely Arabidopsis MSH4 homologue (AtMSH4) has been shown to independent of the sequence. These pathways are responsible function at an early stage in meiotic recombination (Higgins for balancing genome stability against the generation of genetic et al., 2004), but a role for either AtMSH4 or AtMSH5 in diversity. MMR has yet to be established. In the final step of MMR in animal cells, an MLH1.PMS2 1. Homologous recombination heterodimer plays a key role in discriminating between the template and nascent DNA strands, and then specifically HR is important in DNA repair in somatic plant cells and promotes the excision of a stretch of nascent strand DNA also occurs during meiosis. There are three models for HR, containing the misincorporated nucleotide, with the excision which are largely based on work done in yeast (reviewed in process terminating just beyond the mismatched base on Aylon & Kupiec, 2004; Krogh & Symington, 2004).

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Fig. 5 Pathways for double-strand break (DSB) repair in plants. (a) Nonhomologous end joining is catalysed by a complex of KU70 and KU80 which binds the exposed DNA ends. The DSB may then be processed by the MRE11/RAD50/NBS1 (MRN) complex before ligation by the LIG4/ XRCC4. Other pathways of illegitimate recombination are likely to be present in plant cells. Homologous recombination (HR) is initiated by extensive resection to form long 3′ tails which are coated with RPA. Formation of a RAD51 filament promotes homology search and strand invasion. In meiotic cells, HR occurs largely by the DSBR model (b), resulting in Holliday junction formation and crossing over. In somatic plant cells, HR predominantly occurs by synthesis-dependent single-strand annealing (c), in which DNA synthesis using a homologous template results in sequence overlap across the region of the break allowing re-annealing and break repair. Adapted from Krogh & Symington (2004).

DNA double-strand break repair (DSBR) model Meiotic aligned homologous chromosomes rather than sister chromatids. recombination is best described by the double-strand break Initially, the ends of the duplex at the DSB are extensively repair (DSBR) model (Fig. 5b). DSBR is initiated by a DSB and resected to produce long 3′ tails that invade the homologous mediates Holliday junction formation, crossing over between chromosome. DNA synthesis is primed from the invading

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strand using the homologous chromosome as a template. indicating that, at least in cells in G1 phase of the cell cycle, Ligation of the invading strand with the other side of the DSB HR plays a very minor role in DSB repair in plants (Puchta, results in the formation of a joint molecule containing two 1999). However, in situations where homologous sequences Holliday junctions which can be resolved with or without crossing are readily available, for example if a break occurs between over (Fig. 5b). Recombination between sister chromatids is tandem repeats, a third of DSBs are repaired by SSA and inhibited in meiotic cells, promoting the genetic mixing between around 7% are repaired by SDSA (Siebert & Puchta, maternal and paternal homologous chromosomes. Meiotic 2002; Orel et al., 2003). But, despite the close proximity of recombination in plants has been the subject of recent reviews homologous sequences, the majority of repair was catalysed (Bhatt et al., 2001; Caryl et al., 2003; Jones et al., 2003; by the NHEJ pathway. The frequency of recombination Schuermann et al., 2005). between sister chromatids is harder to measure and it is still unclear how significant this pathway is in DSB repair in plants Synthesis-dependent strand annealing (SDSA) A second model (Schuermann et al., 2005). DNA damage, including that induced for HR, termed synthesis dependent strand annealing (SDSA) by UV-B irradiation and bleomycin treatment, significantly plays an important role in DSB repair in somatic cells (Aylon increases recombination frequencies. Increased recombination & Kupiec, 2004; Puchta, 2005). This pathway can use a sister may result from a combination of factors, including activation chromatid, homologous chromosome or ectopic region of of the HR pathway by signalling pathways on detection of homology in the genome as a recombination substrate. As DNA damage, the formation of HR-repair substrates and with the DSBR model, SDSA is initiated by a 3′ resection an indirect effect of chromatin remodelling around the sites of forming a long single-stranded DNA tail that invades a DNA damage, making the recombination substrate more homologous duplex and primes DNA synthesis. In contrast accessible to the HR machinery (Ries et al., 2000; Molinier et al., to the DSBR model, the newly synthesised DNA then re- 2004). Similar explanations may account for the observed anneals with the other side of the DSB, repairing the break increases in somatic HR found in DNA repair mutants including and avoiding the formation of the joint molecule (Fig. 5c). uvr2–1 (CPD-photolyase), atrad50 and atcen2. SDSA reduces the likelihood of Holliday junction formation and crossing over, which would be highly mutagenic if this Molecular mechanism of HR Meiotic and somatic recom- occurred between ectopic regions of homology (Paques & bination pathways share similarities at the molecular level Haber, 1999). However, Holliday junction formation can occur (Caryl et al., 2003; Schuermann et al., 2005). In yeast and during SDSA and crossing over would not be mutagenic if mammals, HR is catalysed by RPA and the RAD52 epistasis restricted to sister chromatids. An attractive mechanism proposes group (RAD51, RAD52, RAD54 and the MRN complex of that crossing over only occurs between regions of extensive RAD50, MRE11 and yeast XRS2/human NBS1). RAD51 is homology, thus reducing the chances of chromosomal similar to the bacterial recombinase RecA and binds the 3′ translocations arising from ectopic recombination (Inbar ssDNA tails, catalysing strand invasion. Arabidopsis HR null & Kupiec, 1999; Inbar et al., 2000). Both SDSA and mutants, including AtRAD51, AtRAD50 and AtMRE11, are DSBR-mediated HR may operate in somatic and meiotic completely sterile owing to severe meiotic defects; meiocytes of cells, although the relative frequencies may differ between these mutant lines show extensive chromosome fragmentation, the different cell types. resulting in nonviable gametes (Li et al., 2004; Puizina et al., 2004; Bleuyard et al., 2005). These mutants also fail to align Single-strand annealing (SSA) A third mechanism of HR may homologous chromosomes (synapsis) during the early stages occur between tandemly repeated sequences, where homologous of meiosis. regions exposed during resection anneal by single-strand Arabidopsis and mammalian HR also involves the RAD51- annealing (SSA) and the intervening sequence is deleted. This like proteins RAD51B, RAD51C, RAD51D, XRCC2 and pathway may be of particular importance on regions of the XRCC3 and the regulatory proteins BRCA1 and BRCA2 genome containing repeated gene arrays. (McIlwraith et al., 2000; Bleuyard et al., 2005). Interestingly, Arabidopsis knockouts in the RAD51 paralogues display HR pathways in plants There is evidence for all three HR hypersensitivity to the DNA cross-linking reagent mitomycin pathways operating in plants and growing evidence that much C (MMC) but little or no hypersensitivity to γ-radiation of the molecular machinery is common between the different (Bleuyard et al., 2005). Hypersensitivity to interstrand DNA HR mechanisms (Puchta, 2005; Schuermann et al., 2005). cross-links (ICLs) suggests a role for HR in the repair of this Recombination frequencies vary greatly, depending on the form of DNA damage, as was found in yeast (Grossmann et al., origin of the donor and recipient sequences. In plant somatic 2001). The mild sensitivity to γ-radiation may suggest that cells, HR-mediated DSB repair using allelic sequences occurs HR has a minor role in DSB repair in plants. The DSB hyper- very rarely, accounting for around 1 in 10 000 repair events sensitivity found in Arabidopsis rad50 and mre11 knockouts (Gisler et al., 2002). Similar frequencies were observed for may reflect the roles of these genes in NHEJ (Gallego et al., the repair of a DSB using ectopic homologous sequences, 2001; Bundock & Hooykaas, 2002).

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2. Nonhomologous end joining V. Molecular responses to genotoxic stress: DNA damage sensing and signalling NHEJ is the major pathway for DSB repair in most higher eukaryote cells. The initial recognition of the DSB may be DNA damage sensing is an essential requirement to minimise mediated by a complex of KU70 and KU80. Mutation of the deleterious effects of genotoxins on cellular growth. Sensing Arabidopsis KU genes results in plants that are hypersensitive and signalling mechanisms promote genomic integrity by to γ-irradiation, bleomycin (which induces strand breaks delaying passage through the cell cycle to allow time for repair in DNA) and methyl methane sulphonate (MMS, a DNA and activation of repair pathways. In the absence of this alkylating agent), consistent with the role of these genes in signalling, DNA replication and mitosis (or meiosis) occurs DSB repair (Riha et al., 2002; West et al., 2002). KU proteins unchecked and exacerbates the DNA damage. For example, have additional roles in eukaryotes in maintaining telomeres, DNA replication can convert SSBs into DSBs, and nuclear providing an interesting link between DNA-damage-related division in the presence of a DSB leads to loss of chromosome DSBs and naturally occurring chromosome ends (Riha et al., fragments and aneuploid daughter cells (Sancar et al., 2004). 2002; Gallego et al., 2003a). The DNA ends may then be Gamma irradiation of Arabidopsis plants results in an processed (possibly by the MRN complex) to make them increase in the numbers of cells in G2, suggesting the presence suitable substrates for DNA ligase. Processing can involve the of a DNA damage responsive G2/M checkpoint in plants alignment of DNA ends at microhomologies of one or more (Preuss & Britt, 2003; Culligan et al., 2004). This G2 arrest bases and trimming of overhanging DNA ‘flaps’. Ligation is appears to be a controlled event rather than a direct effect of then catalysed by a complex of DNA ligase IV and XRCC4 DNA damage because mutants can be isolated that overcome (West et al., 2000). Although Arabidopsis NHEJ mutants this checkpoint, allowing entry into mitosis and radioresistant show clear hypersensitivity to DSB-inducing agents, plants are growth (Preuss & Britt, 2003). In yeast and mammals, the still able to grow in the presence of relatively high concentrations phosphoinositiol-3 kinase-like kinases (PI3KK), which have of bleomycin, suggesting that alternative repair pathways protein rather than lipid substrates, play important roles in are active in Arabidopsis. This supposition is supported by signalling DNA damage within the cell (Durocher & Jackson, several lines of evidence, including KU70-independent NHEJ 2001). These include ATM (ataxia telangiectasia-mutated, between chromosomes in atku70 and attert double mutants Mec1 in S. cerevisiae), ATR (ATM-Rad3-related) and the and the rapid repair of DSBs in Arabidopsis NHEJ mutants mammalian DNA-dependent protein kinase catalytic subunit assayed by single-cell electrophoresis (Angelis, West & (DNA-PKcs). Plant homologues of ATM and ATR play Bray, unpublished results; Heacock et al., 2004). Further important roles in the response to DNA damage and programmed analysis of chromosome fusions in atku70.attert double DSBs induced during meiosis. and atku70.attert.mre11 triple mutants indicated that KU- Whereas ATM responds primarily to damage causing independent end joining showed greater use of microhom- DSBs, ATR mediates responses to a wider range of genotox- ologies, whereas pathways independent of both MRE11 and ins, especially those that interfere with DNA replication. KU were associated with the insertion of a filler sequence Consistent with this, Arabidopsis atr mutants show only mild (Heacock et al., 2004). Deletions and insertions of filler hypersensitivity to γ-irradiation, but severe growth defects in sequences are often observed during plant illegitimate the presence of hydroxyurea, which inhibits DNA replication recombination, and the insertions may be copied from ectopic by reducing the availability of dNTPs through inactivation of sites in the genome or from extrachromosomal DNA during ribonucleotide reductase (Culligan et al., 2004). This con- plant transgene integration into the host genome (Kirik et al., trasts with mammals, in which mutations in ATR are lethal 2000). The insertion of filler DNA suggests a mechanism (Cortez et al., 2001). In addition, aphidicolin, an inhibitor of for illegitimate end joining involving a synthesis-dependent DNA polymerases δ and ε, used at concentrations that dis- annealing-like reaction, although given the differences in rupt rather than inhibit DNA replication, causes G2 arrest requirements for homology this pathway presumably differs in wild-type but not in atatr mutant plants (Culligan et al., mechanistically from SDSA-mediated HR (Puchta, 2005). 2004). Some overlap in function exists between Arabidopsis NHEJ in atku70.attert.mre11 triple mutants indicates the ATM and ATR because double mutants are completely presence of novel recombination pathway(s) which may sterile, whereas atr mutants are normal and atm mutants be responsible for the rapid DSB repair observed in NHEJ have reduced fertility (Culligan et al., 2004). mutant backgrounds (Heacock et al., 2004). Interestingly, The sensing mechanisms that activate ATR integrate a Agrobacterium-mediated transformation frequencies of NHEJ wide range of insults to genomic DNA into a single damage mutants show little or no difference to wild-type plants, response. The DNA damage signal resulting in ATR activa- indicating that KU-dependent NHEJ is not essential for tion in mammals is ssDNA, which is present at collapsed transgene integration, in marked contrast to the situation in replication forks, in recombination intermediates and is formed yeast (van Attikum et al., 2001; Friesner & Britt, 2003; during excision repair pathways. Recognition of ssDNA is Gallego et al., 2003b; van Attikum et al., 2003). mediated by RPA, ATR and ATRIP (Zou & Elledge, 2003).

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In contrast to ATR, activation of ATM occurs largely in An intriguing recent discovery relevant to DNA repair has response to DSBs, and in mammals requires the MRN com- arisen from investigations on Arabidopsis plants homozygous plex (Falck et al., 2005; Lee & Paull, 2005). Arabidopsis plants for recessive mutant alleles of the organ fusion gene HOT- deficient in ATM are hypersensitive to γ-radiation and display HEAD (HTH). This study has presented evidence for the extensive chromosome fragmentation during meiosis, leading existence of a pathway in Arabidopsis for the non-Mendelian to reduced fertility (Garcia et al., 2003). Arabidopsis atm inheritance of sequence information maintained outside the mutants fail to induce transcription of DNA repair genes plant genome (Lolle et al., 2005). The pathway utilises a type following γ-irradiation, consistent with the role of ATM in DNA of ‘memory’ mediated possibly via an ancestral stable RNA- damage signalling in plants. This ATM-mediated signalling is sequence cache similar to that which permits RNA silencing rapid in Arabidopsis, resulting in transcriptional induction of effects. Most crucial is the observation that the extragenomic AtRAD51 within 30 min. Mammalian ATM phosphorylates information in this RNA cache can persist over several plant a number of proteins involved in DNA repair and cell cycle generations. Thus the RNA components of this cache repre- control, including p53, NBS1, the cell cycle checkpoint pro- sent a library of useful allelic sequences containing genetic teins CHK1 and CHK2 and the histone H2A variant termed information that has proved to be functionally important for H2AX (Shiloh, 2003). Phosphorylation of Arabidopsis H2AX survival of the plant. Specifically this ‘genetic memory’ within occurs within 20 min of irradiation and is largely dependent the RNA cache can be used by the plant cell to restore delete- on ATM during M-phase and ATR during S-phase (Friesner rious mutations arising in key genes back to their ancestral et al., 2005). Phosphorylated histone H2A has proven to be a functional wild-type sequence, even in the absence of a wild- powerful marker for DSBs in situ in yeast and mammalian type allele of the gene in the nuclear genome. This landmark systems, and the application of this approach to plants will discovery thus represents an as yet ill-defined, but exquisitely enhance our understanding of repair processes. unique, DNA repair mechanism distinct from all pathways The combined and partially overlapping activities of ATM known to date and could also be present in other organisms and ATR in plants results in the phosphorylation of target in addition to plants. The ramifications of this exciting discov- proteins and activation of checkpoints and DNA repair proc- ery for DNA repair processes are numerous and significant, esses. Downstream targets of DNA damage signalling include and elucidation of the detailed mechanism of this pathway the transcriptional activation of genes involved in DNA repair and how and when it is used by plants is eagerly awaited. and the cell cycle (Chen et al., 2003; Garcia et al., 2003). The proteomics era has brought an increasing awareness Putative substrates of Arabidopsis PI3KKs have been charac- that most proteins exert their function by way of protein– terised recently and include the checkpoint complex protein interactions and that enzymes are often held in tightly AtRAD9, AtRAD1-like and AtHUS1 (the 9–1−1 complex) controlled regions of the cell by such interactions. The control and the DSB-inducible AtRAD17 checkpoint protein of subcellular localisation of proteins and their interaction (Heitzeberg et al., 2004). RAD17 forms an RFC-like com- with specific protein partners in vivo are important parame- plex which loads the 9–1−1 complex onto chromatin. This ters that provide clues to their function and regulation. It is complex forms a ‘sliding clamp’ analogous to PCNA and may now becoming clear that eukaryotes employ extensive and function to recruit further repair factors and initiate signalling dynamic multiprotein complexes to perform DNA replication, pathways (Bermudez et al., 2003). repair and recombination. Additionally, there is substan- tial evidence that the distribution of DNA recognition and protein–protein interaction domains amongst the putative VI. Conclusions and future prospects components of DNA replication and repair complexes in The ability of plants to perceive and respond to environmental plants is clearly distinct from that in mammals. Integration of stresses which compromise genomic integrity is crucial, not approaches based on functional genomics and proteomics can only to the survival of the individual plant but also to future complement established whole-plant physiological and eco- generations. The identification and characterisation of the logical studies to accelerate characterisation of components of molecular components that detect specific DNA damage intracellular signal transduction pathways that mediate the products and the elucidation of how these signals trigger plant’s response to DNA damage. A fundamental knowledge responses that lead to cell cycle arrest and promote DNA repair of plant DNA damage sensing, signalling and repair pathways will reveal the molecular mechanisms by which intracellular is essential for a more directed approach to the improvement receptors give rise to appropriate DNA damage responses. of crop species to tolerate environmental genotoxins. Further- Many similarities have emerged in the comparative study more, an understanding of these pathways will help elucidate of plant and animal responses to genome damage. However, the mechanism by which transgenes insert into the host plant several key components integral to the animal signalling pathway genome. Such knowledge will impact on biotechnology appear to be absent from plants, indicating that fundamental because it may lead to the development of more controlled differences exist between distantly related phyla in the recogni- methodologies, leading to site-specific T-DNA insertion tion and signalling of DNA damage. during plant transformations. Arabidopsis plants mutated in a

New Phytologist (2005) 168: 511–528 www.newphytologist.org © The Authors (2005). Journal compilation © New Phytologist (2005) Tansley review Review 525 range of key DNA repair genes are now becoming available damage, but not for the integration of Agrobacterium T-DNA. Nucleic and their use combined with major advances in protein ana- Acids Research 31: 4247–4255. lytical technologies will hasten progress towards these aims. Aylon Y, Kupiec M. 2004. New insights into the mechanism of homologous recombination in yeast. Mutation Research 566: 231–248. The complex organisation and compartmentalisation of Babiychuk E, Cottrill PB, Storozhenko S, Fuangthong M, Chen Y, plant cells makes it possible that the molecular behaviour of O’Farrell MK, Van Montagu M, Inze D, Kushnir S. 1998. Higher plants proteins in vitro may be different to that in planta. Therefore possess two structurally different poly (ADP-ribose) polymerases. 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