Toxicity via dim er bypass inhibition of replication and transcription

Figure 1. UV-induced DNA damage presents a challenge both to the viability of the plant (through inhibition of transcription) and to its genetic stability.

epidermal pigments (and cuticular waxes) protect the cells of the leaf mesophyll varies widely between species. Virtually no biologically effective UV-B penetrates through the epidermis of the needles of a wide variety of pines [7]. In contrast, the epidermis of many herbaceous species is not as effective an absorber of UV, with as much as 40% of the incident UV penetrating to the cells of the mesophyll [8]. Expression of UV-absorbing pigments is induced by environmental UV-B. Plants also alter their development in many ways in response to UV (reviewed in [9]); some of these responses may enhance UV resistance, while others may be symptoms of stress. Some flavonoid pigments (for instance anthocyanins), may play multiple roles in stress reduction, acting not only as UV absorbing agents [10] and antioxidants [11], but also as absorbers of visible light. This shading effect may act to protect tissues from the damaging effects of photoinhibition during the assembly of the photosynthetic apparatus in emerging leaves and during its disassembly in senescing leaves [12].

In most cases, reproductive (meristematic) tissues are tucked away and shielded from UV at least until flowering, at which point the pollen grains, but not the ova, are exposed as they emerge from the anther and make their way to the stigma. Thus it is not clear whether UV exposure during the growth of the plant contributes to the mutational load of the 'germline'. Recent studies, assaying recombination between tandem repeats, suggest that physiologically relevant exposures to UV-B can induce 'germline' mutations of this type in plants, and the full extent of solar UV-B's contribution to "spontaneous" mutation rates has yet to be established [13-15]. However, the causal links between the UV treatment and the recombination events observed remain unclear; the same types of mutations can be induced by other stresses, such as viral infection [16]. If UV-B does contribute significantly to the mutational load in the 'germline', the effect would likely be seen on a population and/or generational scale. For example, mismatch-repair defective Arabidopsis lines accumulate point mutations and small insertion/deletions, but are phenotypically normal within the first few generations [17]. In later generations, however, the overall fitness of the population begins to decline; a higher incidence of phenotypic abnormalities and extinctions are observed, suggesting the population is less fit. Nonetheless, the possible effects of solar UV on the long-term genetic stability of plants, and any roles for DNA repair or damage tolerance pathways in reducing this effect, remains an open question.

3. Repair of UV-induced DNA damage I: Photoreactivation

There is no natural environment in which plants are exposed to ultraviolet radiation without also being exposed to visible light. It has long been observed that most organisms exhibit enhanced resistance to UV if subsequently exposed to visible light. This phenomenon, termed "photoreactivation" is due to the presence of a light-dependent pathway for the reversal of pyrimidine dimers. While most DNA damage products are repaired via a variety of "remove and replace" excision repair mechanisms, pyrimidine dimers are one of the few lesions that are both common and consequential enough to merit a specialized pathway for their direct reversal, through the action of photolyases.

Photolyases specifically bind to either CPDs or 6-4 dimers, but not both, as the structures of the lesions are very different. Arabidopsis expresses both 6-4 and CPD-specific photolyases, and one would assume that most plants possess both classes of photolyase. All photolyases are monomelic and carry 2 chromophores. A flavin cofactor (FADH-) acts as a transient electron donor to reverse the crosslink between the bases. A second chromophore acts as an antenna pigment to excite the electron donor. This antenna pigment is methenyltetrahydrofolate or 8-deazaflavin, depending on the species and enzyme, and it largely determines the action spectrum of the enzyme. Photolyases bind their cognate lesions even in the absence of light, but once a photon (in the UV-A to blue range) is absorbed, the lesion is reversed and the enzyme dissociates from the DNA [18].

Some plant photolyases are regulated by visible light, and by UV-B. Arabidopsis CPD photolyase activity is only detected when plants have been exposed to visible light prior to, as well as during, the period of repair [19]. This is because the transcription of the gene is regulated by white light (and by UV-B) [20-22]. However, the gene is not simply induced by light; high-level expression of both the protein and the mRNA seem to require day/night cycling. Continuous exposure to white light results in a gradual decrease in protein level over a period of several hours [21]. Similar effects, suggestive of diurnal regulation, have been observed in cucumber [23]. This induction of photolyase activity at dawn, and subsequent decrease in expression later in the day, makes sense in terms of optimizing expression in relation to UV flux. On the other hand, the 6-4 photolyase is constitutively expressed; it isn't clear why both proteins would not be regulated in the same way. The mechanism regulating expression of photolyase is still poorly understood, but a recessive UV-resistant mutant of Arabidopsis, uvil, has been identified that does not require prior exposure to light to express the CPD photolyase [22]. This mutant appears to be upregulated for a variety of UV repair pathways, as it also exhibits an enhanced rate of dark repair of 6-4 products (see nucleotide excision repair, below). In Chlamydomonas, a second gene (PHR1) in addition to the CPD photolyase gene itself (PHR2) is required for full activity of the CPD photolyase protein [24]. Although the PHR1 gene might represent a post-translational regulatory step, at present it appears to be a protein cofactor required for maximal CPD binding activity of the Chlamydomonas photolyase.

Work in the Arabidopsis seedling, in rice, and in alfalfa indicates that photoreactivation greatly enhances the rate of removal of dimers. Although in the absence of photoreactivating (blue/near UV) light dimers are slowly eliminated from bulk DNA (and 6-4 products are generally observed to be repaired more quickly than CPDs), the half life of both CPDs and 6-4

products is greatly reduced by blue light [19,25-28]. Plants grown in the presence of photoreactivating radiation will eliminate the majority of both 6-4 products and CPDs within hours, or in some cases minutes, of their induction.

The plant genomes are divided into three membrane-bound compartments, the nucleus, the plastid, and mitochondrion. The PHR2 CPD photolyase of Chlamydomonas includes a plastid targeting sequence, and is required for the repair of both the plastid and nuclear genomes. In contrast, the Arabidopsis and cucumber CPD photolyases lack predicted organellar targeting sequences. Whether the organellar genomes of higher plants are subject to photoreactivation is a matter of debate; although sequence-specific analysis of CPD photoreactivation in Arabidopsis seedlings indicated no significant photoreactivation of the organellar genomes [19], analysis of bulk DNA repair in expanding leaves, as well as the continued replication of organellar DNAs in this organ, suggests that organellar DNAs might be subject to photoreactivation [25].

4. Repair of UV-induced DNA damage II: Nucleotide excision repair

Most types of DNA damage cannot be directly reversed, but are instead excised, and the resulting gap filled using the undamaged strand as a template, resulting in error-free repair. A variety of lesion-specific glycosylases have evolved that recognize commonly occurring oxidized or alkylated bases and so initiate "base excision repair" [29]. An additional mechanism, termed nucleotide excision repair (NER), recognizes, with varying efficiencies, a wide variety of damage products. In organisms such as Humans that lack photolyase activity, NER is a critical pathway for repair of UV-induced damage. As mentioned above, in Xeroderma pigmentosum patients defective in NER, exposure to sunlight results in hospitalization, and skin cancers appear at an early age.

Plants, in contrast, possess very efficient photolyases, capable of reversing dimers within minutes to hours of their induction. However, plants also possess a nucleotide excision repair (NER) pathway that is capable of excising dimers, particularly 6-4 photoproducts. Genetic and genomic analysis indicates that the plant NER pathway is homologous to that of mammals and fungi and unrelated to the bacterial system [30-35]. However, continued classical genetic analysis has unearthed at least one additional factor required for NER in Chlamydomonas; a homolog of this gene, rexl is also found in higher plants, fungi, and mammals [36]. The NER pathway, illustrated in Figure 2, involves damage recognition, unwinding of the damaged DNA, binding of two distinct endonucleases to the 5' and 3' ends of the damaged strand, incision, removal of the damaged oligonucleotide, resynthesis using the undamaged strand as a template, and finally ligation of the resulting contiguous 5' and 3' ends. The coordinate actions of perhaps a dozen proteins are required to recognize damage and complete the excision step. In mammals, fungi, and bacteria, NER is at once promoted and complicated by the presence of a stalled RNA polymerase at the lesion. RNA polymerase clearly plays a role in damage recognition (damage on the transcribed strand of a transcribed region is repaired more efficiently than damage on the untranscribed strand or a nontranscribed region) [37,38], but additional protein factors are required to either push the polymerase away from the lesion or, alternatively, target it for proteolysis (to make way for repair or lesion bypass proteins) [39,40]. Transcription-coupled repair has not yet been demonstrated to take place in plants, but given its presence in bacteria, fungi, and mammals, it is quite likely that it occurs in plants too.

Figure 2. Nucleotide excision repair. Damage recognition and excision by NER requires the coordinated actions of approximately a dozen proteins. Damage products are bound by XPA, XPC, RPA, and TFIIH. The XPB and XPD helicases of TFIIH then act to create a "repair bubble". Guided by RPA, the 5' (ERCC1/XPF) and 3' XPG endonucleases nick the damaged strand. The damaged oligonucleotide and repair factors are removed. Finally (not shown) DNA polymerases delta/alpha accurately copy the undamaged strand and DNA ligase seals the remaining nick. In transcription-coupled repair, primary recognition is via RNA polymerase II, and does not require XPC.

Figure 2. Nucleotide excision repair. Damage recognition and excision by NER requires the coordinated actions of approximately a dozen proteins. Damage products are bound by XPA, XPC, RPA, and TFIIH. The XPB and XPD helicases of TFIIH then act to create a "repair bubble". Guided by RPA, the 5' (ERCC1/XPF) and 3' XPG endonucleases nick the damaged strand. The damaged oligonucleotide and repair factors are removed. Finally (not shown) DNA polymerases delta/alpha accurately copy the undamaged strand and DNA ligase seals the remaining nick. In transcription-coupled repair, primary recognition is via RNA polymerase II, and does not require XPC.

The significance of NER in the repair of UV-induced damage is unclear. It is possible that NER, even in the presence of photoreactivation, plays a role in the removal of transcription-blocking dimers or of other UV-induced lesions, as at least some NER-defective mutants do exhibit a UV-sensitive phenotype even in the presence of photoreactivating light [41]. Whether NER plays any role in limiting UV-induced mutagenesis in plants remains to be determined.

5. "Damage tolerance": the repair of daughter strand gaps

Replicative polymerases are fastidious enzymes, refusing to polymerize past a noninformational lesion like a pyrimidine dimer. Stalled replication forks in turn induce cell cycle checkpoints, arresting progression through the cell cycle, and so blocking both cell division and, perhaps, the endoreduplicative cycle. In mammals prolonged arrest can result in the induction of cell death, and sunburn is a manifestation of the collective suicide of skin cells undergoing this response to UV irradiation [42]. In haploid cells (such as yeast) permanent arrest is the genetic equivalent of death. For this reason irradiated yeast and bacteria will first respond to damage by inducing repair, and if, for some reason, repair is not completed, they will proceed into a secondary "damage tolerance" phase in which stalled DNA polymerases will be rolled back or removed from sites of damage, and a second set of more specialized and less precise polymerases upregulated to perform "dimer bypass"; the polymerization of DNA past a damaged site [43]. While all bypass polymerases, operating on damaged substrates, are less accurate than normal replicative polymerases operating on undamaged substrates, some, such as the Y-family polymerase pol eta (Rad30 in yeast, XPV in mammals) still correctly interpret many commonly encountered lesions, and thus are loosely termed "error-free" bypass polymerases [44]. Pol eta enables cells to complete DNA synthesis in spite of the persistence of DNA damage, in a relatively risk-free manner. This permits haploid cells to survive at the expense of a small risk of mutation. Loss of pol eta, in yeast, results in enhanced UV-induced mutagenesis at the UV-induced mutagenesis hotspots TC and CC, indicating that this protein acts to protect yeast from the mutagenic effects of UV [44]. XPV, the human pol eta, plays a very important role in resistance to UV-induced mutagenesis in humans [45], generally bypassing pyrimidine dimers correctly. Loss of XPV results in hypersensitivity to both the induction of sunburn (as cells actively induce death in response to persisting stalled replicative polymerases) and the induction of skin cancer (as far more error-prone polymerases step into the breach generated by the loss of pol eta), and through the induction of recombinational repair [46]).

In contrast, the Y-family polymerase encoded by the umuC and umuD genes of E. coli is actually required for UV-induced mutagenesis; defects in this gene result in the near-complete loss of UV-induced mutagenesis, but this comes at the expense of a slight increase in sensitivity to the lethal effects of UV [47]. Presumably UmuC/D is employed by E. coli as part of a last ditch effort to restore DNA replication at those sites (such as dimers opposite daughter strand gaps) that cannot be repaired in an error-free fashion.

The B-family error-prone polymerase zeta (whose subunits are Rev3 and Rev7 in yeast) extends mismatched primers (including those generated by other bypass polymerases) and is required for >98% of UV-induced mutagenesis in yeast [48]. Arabidopsis mutants defective in atrev3 exhibit hypersensitivity to both the lethal effects of UV and its effects on DNA replication. UV-hypersensitivity was observed in the dark and under photoreactivating conditions, as well as hypersensitivity to the crosslinking agent mitomycin C, suggesting a role in the tolerance and/or repair of closely opposed or crosslinked lesions. These types of lesions cannot be directly repaired by NER, as there is no undamaged strand available to act as a template for repair replication [26].

6. Cell cycle responses to UV-induced DNA damage

UV doesn't induce mutations directly. It is the process of DNA replication, and the error-prone nature of replication past dimers, that generates mutations. In addition, replication blocks generated by dimers can induce the formation of daughter-strand gaps, and a second round of replication at these gaps can produce double-strand breaks, leading to translocations, and/or deletions. Therefore, an efficient cellular response to DNA damage requires more than just the induction of DNA repair. Cells arrest cell cycle progression in response to damage, single stranded gaps, and replication blocks, delaying entry into S-phase or mitosis. These genome-surveillance mechanisms, known as cell-cycle "checkpoints", permit the cell to repair its DNA

before progression through the cell cycle makes a bad situation worse. Although the term checkpoint suggests a particular point of inhibition, such as the transition from G1 to S-phase, a damage-induced "checkpoint" may also initiate at any point during the cell cycle at which DNA replication and/or integrity is monitored. These responses have been well characterized in yeast and mammalian systems, and are proving to be conserved in plants.

Multiple cell-cycle checkpoints can be activated throughout the process of DNA replication, including the initiation of DNA replication (G1/S), within S-phase, and at the end of DNA replication at the G2/M boundary. In many cases, these responses are activated via the same core molecular mechanisms, although the effect on cell cycle arrest and the downstream molecular players involved can be quite different, depending on the phase on the cell cycle.

Ideally, before initiating DNA replication, a cell would repair UV-induced damage generated during G1, thereby restoring the template for DNA replication. Persistence of UV-induced photoproducts could lead to the stalling of the replication fork, followed by replication restart downstream of the lesion, resulting in a daughter-strand gap. A damage product opposite a gap is no longer a substrate for excision repair, and a repair option has been lost. However, although yeast and mammalian cells do exhibit cell cycle responses to UV-irradiation in G1, there is no direct evidence that this is in response to DNA damage [49], and it is often unclear whether this arrest (measured as the delay of onset of S phase) is actually occurring at the G1/S boundary (i.e., in response to stalled replication forks rather than to UV-induced damage or repair intermediates) rather than being a true, intra-Gl, response to the presence of dimers or their repair intermediates.

As cells initiate DNA replication, the replication machinery itself acts as a scanning mechanism for replication blocking agents. Stalling of a replication fork, the resulting persistence of single stranded DNA, generates a signal that globally inhibits replication origin firing, inducing both inhibition of further replication and the repair of disrupted replication forks through recombinational mechanisms [50]. This constitutes an "intra-S checkpoint". The persistence of ssDNA also blocks progression into M phase (S/M arrest), as can damage induced during G2 (G2/M arrest). Repair of these gaps prior to M phase is advantageous as there is the option for homologous recombinational repair with the fully aligned sister chromatid. Failure to repair gaps at this stage in the cell cycle will inevitably lead to either the formation of double-strand breaks or the filling of the gap by a possibly error-prone bypass polymerase. All of these responses share a requirement for the PI 3-kinase-like protein kinases ATM and ATR (and, in vertebrates, DNA PKcs) [51]. Because the functions of ATM and DNA-PKcs are to some extent DSB-specific, we will focus our discussion here on damage/replication block recognition and signal transduction by ATR [52].

Studies of the gene products involved in DNA damage-induced checkpoints in yeast or mammalian systems show that they are highly conserved in structure and function. In response to UV-induced DNA damage, the ATR-Chk1-Cdc25 pathway is primarily active, with ATR playing a central, regulatory role (Figure 3). ATR (also called MEC1 in S. cerevisiae and Rad3 in S. pombe) is a protein kinase that bridges sensing of DNA damage to downstream direct effectors of cell cycle control [50,53,54]. DNA damage is generally sensed through specific recognition of RPA-bound single-stranded DNA (ssDNA), which accumulates if replication forks are blocked, or during DNA-repair processing of damaged DNA. At least two molecular pathways are involved; one includes a PCNA-like complex of proteins called the 9-1-1 complex (for Rad9, Husl, and Radl), the Rad17-RFC complex, RPA (replication protein A which binds ssDNA) and ATRIP (ATR Interacting Protein), while the other consists of a more direct interaction of RPA, ATR and ATRIP with ssDNA or damaged DNA [50]). These protein interactions promote the recruitment of checkpoint signaling machinery, including ATR, into an active state. Once active, ATR phosphorylates the protein kinase Chk1 leading to a cascade of downstream events that specifically regulate the cell cycle. For example, G2-phase phosphorylation of Chk1 leads to the inhibitory phosphorylation of cdc25. This form of cdc25 is unable to activate the mitotic promoting cyclinB/cdc2 kinase, thus blocking progression into mitosis [50,55].

In plants, the study of cell-cycle checkpoints in response to DNA damage has lagged behind that of yeast and animals, but recent progress has established the existence of such checkpoints and some of the players involved in the signal transduction pathway have been identified. Growth responses suggestive of damage-dependent cell-cycle arrest were first observed in plants as the arrest of growth following gamma-irradiation of plant seeds [56,57]. These "gamma plantlets", grown from irradiated seeds, were healthy and responded normally to environmental stimuli, but didn't produce postembryonic leaves. This suggested a regulatory effect specifically affecting dividing cells, rather than a direct toxic effect. Recent studies of Arabidopsis mutants defective in DNA ligase IV, which is involved in repairing DNA double

Figure 3. General overview of UV-induced cell-cycle check-point activation mechanisms. The protein kinase ATR plays a central role by bridging the sensing of ssDNA (left pathway) or stalled replication forks (right pathway) to downstream effectors of cell cycle progression. Not all of the molecular players are shown for simplicity. Phosphorylation by ATR-ATRIP of Chk1 leads to activation of direct regulators of cell cycle progression at the G1/S transition, S-phase progression, or the G2/M transition (adapted from [50]).

strand breaks, display a more pronounced gamma-plantlet effect versus wild-type plants [35, 58]. These mutants form gamma plantlets at lower doses of gamma irradiation than do wildtype plants, suggesting that the gamma arrest response is induced by persisting DNA damage (in this case double strand breaks) rather than through direct detection of radiation, or by damage to some other cellular component.

The isolation of an Arabidopsis mutant (termed "sog" for "suppressor of gamma plantlet") specifically defective in cell-cycle response to IR-induced DNA damage [59] indicates that this DNA damage-induced inhibition of cell-cycle progression in plants is indeed a programmed response to damage, rather than a direct effect of a compromised genome. As would be expected, although sog mutants are resistant to the inhibitory growth-effects of persisting IR-induced DNA damage, they are hypersensitive to its mutagenic effects.

Genes involved in damage-induced cell-cycle checkpoint pathways have also been isolated through reverse-genetic approaches. Arabidopsis encodes an ortholog of ATR, and recent studies have shown that this gene, like its yeast and animal counterparts, is required for G2 arrest in response to replication inhibitors [60]. Plants defective in ATR were hypersensitive to UV-B light, as measured by root growth. Furthermore, while wild-type root meristem cells displayed arrest in G2 in response to aphidicolin [60] and UV-B light (Culligan and Britt, unpublished), the atr mutant plants were defective in this arrest. This indicates that ATR's function as a detector of replication blocks has been conserved in plants. However, while ATR is an essential gene in mammals, it is not essential for plants. atr null homozygotes exhibit normal growth rate and full fertility [60].

Other mutants in orthologs of the ATR-dependent pathway have been described in Arabidopsis, such as Rad9 (of the 9-1-1 complex), Rad17 (involved in recognition of DNA damage [61]), ATM (Ataxia Telangiectasia Mutated, a paralog and functional partner of ATR) [62] and BRCA1 (breast cancer susceptibility [63]). Specific DNA damage-induced effects on the cell cycle have not yet been described in these mutants, but their DNA damaging agent hypersensitivity suggests that their role in these processes may be conserved.

Finally, plants, and other organisms, exhibit responses to UV that are unrelated to the induction of DNA damage. These may be triggered by UV receptors (analogous to other wavelength dependent developmental responses in plants) or they may be induced by damage to other cellular components.

7. Do plants exhibit an apoptotic response to DNA damage?

A further response to UV-light DNA damage in animal cells is p53-dependent programmed cell death. Plants, like fungi, lack a p53 homolog, and probably also lack the sensitive apoptotic response to UV. Given that fact that plants cannot be killed by cancer (as, even in long-lived plants, cancers cannot metastasize), there is probably no adaptive role for such a phenomenon in plants. However, very high, non-physiological doses of UV-C have been shown to induce an apoptotic-like response in Arabidopsis leaves, including characteristic DNA-laddering and Caspase-like activities [64,65]. In Arabidopsis plants defective in ATR, the replication-blocking agent aphidicolin induces an apoptotic-like response, in which the nuclei of meristematic cells first become condensed and then are lost entirely [60]. Both of these lines of evidence, particularly the laddering observed by Danon et al., suggest that a programmed cell death response to DNA damage may exist in plants. However, induction of this type of cell death in plants may require very high levels DNA damage, as DNA-repair defective mutants in Arabidopsis, even when challenged with exogenous DNA-damaging agents, do not show obvious signs of apoptosis [35,66]. The response observed in the two studies cited above may represent an ordered disassembly of a hopelessly compromised cell, rather than the selective elimination of perfectly viable, but possibly precancerous, cells observed in mammals.

8. Functional significance of DNA repair in terms of plant growth

Much progress has been made in the last 15 years in understanding plant DNA repair and damage tolerance mechanisms. Given the existence of plants defective in specific repair or tolerance pathways, it should be possible to determine the relevance of these pathways to resistance to solar UV. Humans defective in nucleotide excision repair cannot survive continued exposure to sunlight. Can plants with similar defects survive under natural conditions? Are both NER and photoreactivation important contributors to survival? What about dimer bypass? Does UV-B-induced mutagenesis in the 'germline' contribute the plant population's long-term fitness? Some of these questions have been addressed in the laboratory; all of the abovementioned mechanisms contribute to UV resistance, but their relevance to growth in the field remains unclear. Plants are exquisitely sensitive to the spectral balance of light, particularly as it affects UV resistance [9,67], and it is very difficult (and expensive) to reproduce natural light balances in the growth chamber. In addition, many researchers use unnaturally intense and short-term exposures to UV to address issues of sensitivity. Given current interest in the effects of ozone depletion, it is important to understand how plants tolerate their constant exposure to UV and how adaptable they might be to elevations in ambient UV-B. If the results of these experiments are to be relevant to plant growth under natural conditions, they must be performed either in the field (with UV-absorbing filters, or carefully monitored supplemental lighting, or through changes in UV ratios generated by changes in elevation) or under very carefully controlled and monitored artificial lighting. Experiments testing the effects of natural UV on plant growth have been performed out of doors (under UV-B transparent vs. UV-B opaque filters) and results often, but by no means always, suggest that ambient UV-B does have a small but measurable effect on, for example, crop yield [3,68-71].

Results of the single "ambient UV" field study on repair defective Arabidopsis plants suggested that the effects of deficiencies in photoreactivation, nucleotide excision repair, or both processes were surprisingly slight [71]. In dramatic contrast to NER-defective mammals, repair defective Arabidopsis lines exhibited only a slightly enhanced sensitivity to solar UV-B. The effects of the tt5 mutation, which eliminates production of flavonoid pigments, were slightly more impressive. Plants defective in both sunscreen biosynthesis and DNA repair were strikingly sensitive to solar UV-B, with obvious effects within a day of exposure to unfiltered sunlight [71]. Thus the difference between Humans and Arabidopsis in terms of the importance of DNA repair in resistance to the effects of ambient UV-B might be ascribed to the synthesis, in Arabidopsis, of very effective natural sunscreens. However, it might also be due to the fact that plants do not actively induce cell death in response to biologically relevant levels of DNA damage.


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