Biology of Extreme Radiation Resistance: The Way of radiodurans

Anita Krisko1 and Miroslav Radman1,2

1Mediterranean Institute for Sciences, 21000 Split, Croatia 2Faculte´ de Me´decine, Universite´ Rene´ Descartes—Paris V, INSERM U1001, 75015 Paris, France Correspondence: [email protected]

The bacterium Deinococcus radiodurans is a champion of extreme radiation resistance that is accounted for by a highly efficient protection against proteome, but not , damage. A well-protected functional proteome ensures cell recovery from extensive radiation damage to other cellular constituents by molecular repair and turnover processes, including an efficient repair of disintegrated DNA. Therefore, cell death correlates with radiation- induced damage, rather than DNA damage, in both robust and standard species. From the reviewed biology of resistance to radiation and other sources of oxidative damage, we conclude that the impact of protein damage on the maintenance of life has been largely underestimated in biology and medicine.

everal recent reviews comprehensively pres- rather than the genome. A cell that instantly Sent the extraordinary bacterium Deinococ- loses its genome can function for some time, cus radiodurans, best known for its biological unlike one that loses its proteome. In other robustness involving an extremely efficient words, the proteome sustains and maintains DNA repair system (Cox and Battista 2005; Bla- life, whereas the genome ensures the perpetua- sius et al. 2008; Daly 2009; Slade and Radman tion of life by renewing the proteome, a process 2011). The aim of this short review of the biol- contingent on a preexisting proteome that re- ogy of D. radiodurans is to single out a general pairs, replicates, and expresses the genome. In concept of the primacy of biological function addition to the functional integrity of the pro- (proteome) over information (genome) in the teome, small metabolites and other cofactors maintenance of life. A cell dies when its vital for catalysis and protein interactions are equally functions performed by the proteome cease, important for proteome functionality. Howev- for example, because of the direct loss of prote- er, chemical damage to cofactors is not a likely ome functionality or via the loss of membrane primary bottleneck in survival because of their integrity, whereas genome integrity is required high molar concentrations, compared with pro- (in addition to an active proteome) for the per- teins. And, finally, it is the proteome that syn- petuation of cells that have survived. But sur- thesizes metabolites and imports vital metal co- vival itself depends primarily on the proteome factors and . Although obvious, the concept

Editors: Errol C. Friedberg, Stephen J. Elledge, Alan R. Lehmann, Tomas Lindahl, and Marco Muzi-Falconi Additional Perspectives on DNA Repair, Mutagenesis, and Other Responses to DNA Damage available at www.cshperspectives.org Copyright # 2013 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a012765 Cite this article as Cold Spring Harb Perspect Biol 2013;5:a012765

1 A. Krisko and M. Radman that the prime target in cell degeneracy and Mattimore and Battista (1996) proposed a death is proteome activity—ensuring all vital more plausible hypothesis that the resistance to functions including genome integrity—is con- radiation is a by-product of a primary selection spicuously absent in biological and medical sci- for resistance to . Moreover, such a ences. This overview of the biology of a prokary- hypothesis is sensible because some other rare otic cell that survives conditions lethal to other unrelated , as well as some , species is to define and elaborate a general con- plants, and small animals, for example, bdelloid cept of sustainability of life that applies to all rotifers and tardigrada, also acquired coincident living cells. resistance to desiccation and radiation. In other words,mechanismsofrecovery fromdesiccation damage ensure recovery from radiation damage. But why did the selection for resistance to desic- ROBUSTNESS AND LIFESTYLE OF cation operate on Dra rather than on all other Deinococcus radiodurans (DRA) bacteria? The answer might liein the lifestyle and Since its anecdotal discovery as a contaminant ecological distribution of Dra. in radiation-sterilized corned beef cans (Ander- Unlike the well-known enterobacteria, Dra son et al. 1956; Duggan et al. 1963), Deinococcus is ubiquitous in nature but its populations are radiodurans (Dra) has fascinated biologists by minor compared with other bacteria occupying its extraordinary resistance to ionizing radia- the same ecological niches. The likely reason is tion. Radiation biology—the study of how ra- that other bacteria overgrow Dra in Nature, diation alters and destroys life—brought about because in the standard laboratory media they the birth of a DNA-centered molecular biology. grow faster than Dra. It looks as if Dra made an Thus, when it was shown that (1) Dra DNA is as “investment” in the efficiency of survival (ro- sensitive to radiation-induced breakage as the bustness), whereas other bacteria made an in- of other bacteria (Burrell et al. 1971; vestment in the efficiency of growth (reproduc- Bonura and Smith 1976; Ge´rard et al. 2001), and tion). The nature of such an “investment” is (2) the analyses of its genome and proteome discussed below. Robustness is the characteris- showed nothing extraordinary (White et al. tic of individual cells and does not imply win- 1999), some fundamental queries surfaced (be- ning the race, at the level of populations, with low). Because the extreme radiation resistance less robust but faster growing competitors, par- did correlate with an extraordinary capacity to ticularly in mild natural habitats. However, repair massive DNA damage inflicted by ioniz- under harsh life conditions—arid environments ing radiation or light, several ques- such as deserts—desiccated Dra populations tions arose: What is the mechanism of such dominate the less-resilient species by their ca- spectacular DNA repair capacity? Can extreme pacity to regrow after rehydration. resistance to radiation be acquired by other or- The lifestyle and metabolism of Dra suggest ganisms, for instance, human? Why and how that this bacterium is a scavenger of food pro- did robust life evolve, in particular, because no duced by (or composed of ) other (dead) organ- radiation sources on Earth are known that could isms: It feeds on amino from degraded produce doses comparable to those of Dra re- and sugars from degraded polymers sistance? The hypothesis of an extraterrestrial by excreting diverse hydrolytic enzymes. Its origin of Dra (Pavlov et al. 2006) should place mosaic genome composition and remarkable its genome outside the terrestrial phylogenetic genomic variability (below) are consistent with tree (which is not true; see below), unless all an evolved preadaptation to diverse environ- DNA-sequenced terrestrial life was seeded by ments scattered among the deinococcal popula- Dra, that is, descended from a deinococcal pan- tions and species, hence, the ubiquitous pres- spermia. In that case, Dra should be at the root ence of similar deinococcal variants in different of the current DNA-based terrestrial phyloge- habitats (White et al. 1999; for review, see Slade netic tree. and Radman 2011).

2 Cite this article as Cold Spring Harb Perspect Biol 2013;5:a012765 Extreme Radiation Resistance of D. radiodurans

THE ECOLOGY AND THE GENOME OF thrive on extreme conditions—indeed, it does D. radiodurans not grow while desiccated or when heavily irra- diated—but it can reproduce under standard The genome of Dra R1 consists of two large growth conditions after recovering from dam- (3.06 Mb) called and two age inflicted by chronic moderate, or acute in- smaller DNAs (223 kb) called (White tense, exposures to cytotoxic conditions. This et al. 1999). Sequence-wise, the Dra genome is a “resurrection” following diverse kinds of dam- mosaic of - and ther- age lethal to most organisms raises the question: mophilus-like genomes with some individual How did the multiresistance evolve? Was it by that could have been acquired even from many parallel strategies, each selected for a spe- other kingdoms of life. Such interkingdom hor- cific resistance, or by a single polyvalent strategy izontal transfer could originate from DNA- generating resistance to many different condi- scavenging events facilitated by the high natural tions lethal to most species? The latter seems to transformability of Dra. Genome sequencing be the case of Dra (see below). of 37 natural isolates (Deinove, pers. comm.) showed a very high diversity that, astonishingly, ROBUST DNA REPAIR IN D. radiodurans appears similar worldwide (see Slade and Rad- man 2011). There is even a factor of two in the The science of molecular biology was dominat- genome size and gene content between the ed by the notion of information, its storage, smallest and largest sequenced deinococcal ge- transmission, and evolution as encrypted in nomes (Jung et al. 2010; Deinove, pers. comm.). the nucleotide sequence of nucleic acids. But The ubiquitous ecological distribution of the biological information is relevant to life deinococcal diversity may account for the re- only to the extent of its translation into useful sults of genome analyses in the following way. biological functions performed, directly or in- The desiccated bacteria are constituents of the directly, by proteins; that is, selection operates dust occasionally blown up by the winds into on phenotypes. Because proteins repair DNA, the atmosphere and stratosphere. where bacte- the destiny of genetic information depends di- ria from different geographic origins mix while rectly on the function of dedicated proteins. being exposed to UVC light 100 to 1000 times Thus, when the extent of DNA breakage by - more intense than on Earth’s surface. Theyeven- izing radiation was studied in Dra, it came as a tually rehydrate when falling back on Earth with surprise that it is the same as in the most sensi- the rain and snow (this is how Francois-Xavier tive bacterial and animal cells; that is, there is Pellay in our laboratory collects robust bacteria) no special protection of DNA to account for and—depending on their genomic constitu- its resistance to radiation (Burrell et al. 1971; tion—develop, or not, in the ecological niches Bonura and Smith 1976; Ge´rard et al. 2001). into which they happen to fall. Indeed, the most What seemed to correlate with radiation re- efficient cellulose degraders are deinococci sistance is the extraordinary capacity to repair found growing in the waist of the wood-sawing hundreds, sometimes thousands, of radiation- industry (Deinove, pers. comm.). induced DNA double-strand breaks (DSBs) per The resistance of D. radiodurans is not ex- cell, whereas standard species’ cells can repair clusive to radiation and desiccation but extends only a dozen (Cox and Battista 2005; Slade and also to many toxic chemicals and conditions Radman 2011). Therefore, at the time, it had (Lown et al. 1978). Therefore, Dra is called a seemed reasonable to anticipate the discovery polyextremophile, a robust “generalist,” to be of a novel repair mechanism or of improved distinguished from specialized “smart” repair proteins that are more efficient with an evolutionary redesign of their proteome in Dra than in most other species. (e.g., proteins purified from are Eventually, the basic mechanism of DSB re- thermostable in vitro) (see Smole et al. 2011). pair in Dra was discovered and called extended Unlike specialized extremophiles, Dra does not synthesis-dependent strand annealing (ESDSA)

Cite this article as Cold Spring Harb Perspect Biol 2013;5:a012765 3 A. Krisko and M. Radman

(Fig. 1) whose elementary act is similar to the A NEW PARADIGM FOR ALL SPECIES: THE synthesis-dependent strand annealing (SDSA) PROTEOME RATHER THAN THE GENOME (Zahradka et al. 2006), well known to operate IS THE PRIME TARGET IN RADIATION- in other species including eukaryotic meiosis, in INDUCED CELL DEATH which DNA is broken by a topoisomerase. The term “extended” refers to the capability of per- When the extensive analysis of the mechanism forming the elementary act of DSB repair many and genes/proteins involved in the repair of hundred times in the same cell. DSBs in Dra showed no novelty (Makarova et al. 2000; Omelchenko et al. 2005; Slade et al. 2009), the puzzle of a “smart” mechanism of repair turned into a puzzle of unexplained effi- ciency of a standard DNA repair system. Neither the specific activity of purified key repair pro- 1 teins nor their cellular amounts could account for the extreme efficiencyof repair (see Slade and Radman 2011). The puzzle of Dra robustness remained until several old studies revived and ESDSA 2 extended by the recent research from Michael Daly’s group (Daly et al. 2007) changed the par- adigm: Radiation-induced oxidative damage to proteins inactivates their function, for example,

3 DNA repair and other vital functions, becoming the principal cause of the sensitivity of the stan- dard organisms. Dra has a way of protecting its proteins from oxidative damage, thereby pre- serving their activity. This paradigm got support from research in Daly’s and our laboratories and 4 withstands rigorous experimental tests. Reactive species (ROS) and, conse- quently, protein carbonylation (PC)—a widely 5 used marker of irreversible oxidative proteome damage—are found at much lower levels in un- irradiated Dra than in other bacteria and so- Figure 1. Extended synthesis-dependent strand an- matic cells of animal species (Table 1) (Krisko nealing (ESDSA) pathway for reassembly of broken and Radman 2010; Krisko et al. 2012). Further- DNA in D. radiodurans. Several genomic copies pres- more, the dose ranges of g rays and UVC light ent in D. radiodurans undergo radiation-induced ran- that saturate PC in Escherichiacoli show no effect dom DNA double-strand breakage, producing nu- on carbonylation of the Dra proteome (Table 1) merous fragments. The end-recessed fragments (step 1) prime synthesis use the homologous regions of (Krisko and Radman 2010). Only, and precisely partially overlapping fragments as a template (step at doses of radiation that become lethal, does 2), presumably through a moving D-loop (bracketed there commence an increase in PC such that intermediate). The strand extension can run to the the killing and PC curves coincide for both end of the template, producing fragments with long, E. coli and Dra, albeit at widely different dose newly synthesized (red) single-stranded overhangs ranges (Table 1) (Krisko and Radman 2010). (step 3) that can a priori engage in several rounds Radiation-induced PC levels saturate at the of extension until they find a complementary partner strand (steps 4 and 5). Hydrogen bonds are indicated same level in E. coli and Dra, meaning that the only for interfragment associations. Long linear frag- intrinsic susceptibility of proteins to carbonyl- ments mature into unit-size circular chromosomes ation is similar in the two species (Fig. 2) and by (crossover). that a proteome protection system consumed by

4 Cite this article as Cold Spring Harb Perspect Biol 2013;5:a012765 Extreme Radiation Resistance of D. radiodurans

Table 1. Dose ranges of g rays and UVC light that saturate PC in E. coli show no effect on carbonylation of the Dra proteome

Dose (Gy) 0 200 400 900 1500 2500 5000 10,000 15,000 20,000 E.coli

D.radiodurans

radiation is present at much higher levels of duced PC is dominant: Apparently, a diffusible activity in Dra than in E. coli cells. When ex- entity from the Dra cell extract protects E. coli tracts of broken E. coli and Dra cells are exposed proteins equally effectively as its own proteins. to radiation, the same difference in PC is found That protective entity is composed of moie- as with intact cells (Krisko and Radman 2010). ties smaller than 3 kDa (Daly et al. 2010; Krisko Mixing of the two extracts before radiation and Radman 2010) and acts by preventing ROS showed that the Dra resistance to radiation-in- production, or neutralizing ROS, induced by

1.0

0.8

0.6 Killing 0.4

0.2

012345678 nmol Carbonyls/mg protein

Dra killing by UV Dra killing by γ A. vaga killing by γ Eco killing by UV Eco killing by γ C. elegans killing by γ

Figure 2. Single correlation between cell killing and protein carbonylation. A similar correlation is observed for the two bacterial (E. coli and D. radiodurans) and the two animal (bdelloid rotifer Adineta vaga and the worm Caenorhabditis elegans) species of widely different radiation resistance. (Data from Krisko and Radman 2010; Krisko et al. 2012.)

Cite this article as Cold Spring Harb Perspect Biol 2013;5:a012765 5 A. Krisko and M. Radman and UVC light as measured ultrafiltrate. In addition to the abundance of by intracellular dihydrorhodamine fluorescence complexes, they have found elevated (Krisko and Radman 2010). concentrations of orthophosphate, nucleotides, and their derivatives, as well as amino acids and peptides. The radioprotective properties MULTIPLE SYSTEMS of the D. radiodurans ultrafiltrate have also IN D. radiodurans been assigned to the abundance of metabolites Based on whole-genome comparisons, there is like TCA cycle products (Daly et al. 2010). in Dra a remarkable abundance of genes encod- Apart from low ROS production in Dra, ing catabolic enzymes including phosphatases, there are several additional metabolic properties nucleases, and proteases, which would be ex- that contribute to its robustness: for instance, pected to give rise to the pool of small antioxi- highlyefficient proteolysis, import of exogenous dant molecules found in the Dra ultrafiltrate amino acids and peptides, and the conversion (Daly et al. 2010). There are several mechanisms of glucose into precursors for dNTPs, as well as for maintenance of low ROS levels in Dra that carbohydrate and polyphosphate storage (see contribute to its resilience. Theycan be belong to Slade and Radman 2011). D. radiodurans is a at least three independent, but complementary proteolytic bacterium and, as such, contains re- groups: (1) increased ROS-scavenging and ROS- dundant mechanisms for protein degradation detoxifying activities (high levels of constitu- and amino acids catabolism. Interestingly, pro- tive catalase and superoxide dismutase activity, teolytic activities are induced following ionizing increased amount of manganese complexes, and radiation (Daly et al. 2010). The degradation of small antioxidant molecules) (Makarova et al. proteins produces amino acids and peptides 2000; Daly et al. 2010); (2) altered metabolic that can be reused during the energetically de- activities that result in a decreased ROS produc- manding recovery period, thus enabling an ef- tion, for example, glyoxylate bypass of the TCA ficientdamage repairandfinallysurvival(Omel- cycle (Liu et al. 2003; Ghosal et al. 2005); and (3) chenko et al. 2005). Furthermore, the pool of reduction of the proteins containing Fe-S clus- amino acids and peptides has been suggested ters, as well as reduction of the number of respi- to have a role as ROS scavengers during irradia- ratory chain enzymes (Ghosal et al. 2005). tion (Daly et al. 2010). The source of the large Divalent iron is a source of production of amino and peptide pool can be accounted the most reactive ROS, the hydroxyl radical. As a foralsobytheirimport:Namely,Drahasasmany consequence of the redundancy of the antioxi- as 90 ABC transporters dedicated to amino acid dant mechanisms in Dra, single pathways can be and peptide uptake (de Groot et al. 2009). inactivated without significantly compromising Glucose metabolism also contributes to the the resilience of the bacterium. For example, remarkable resistance of D. radiodurans.For inactivation of the deinococcal pigment deino- example, mutants with G6PDH deficiency (up- xanthin, one of the most potent regulated in the wild type during the postirra- known to date, has very little effect on the resil- diation recovery) are more sensitive to UV-in- ience of Dra (Lemee et al. 1997; Ji 2010). On the duced , as well as H2O2 and other hand, the production of deinoxanthin mitomycin C (Zhang et al. 2005). Glucose can and the cytosolic antioxidant pyrroloquinoline be metabolized into NADPH, which can be a quinone in E. coli increases its resistance to ox- precursor of dNTPs as well as a cofactor in the idative damage (Misra et al. 2012). Moreover, production of glutathione and thioredoxin. As Dra extract of molecules smaller than 3 kDa mentioned above, ROS production in Dra is provides antioxidant protection to E. coli pro- presumably also reduced owing to fewer pro- teins in vitro (Daly et al. 2010; Krisko and Rad- teins with iron–sulfurclusters. A negative corre- man 2010) and human Jurkat T cells in culture lation between the proportion of iron–sulfur (Daly et al. 2010). The group of Michael Daly cluster proteins and radiation resistance has has characterized the composition of the Dra been observed over several species. For example,

6 Cite this article as Cold Spring Harb Perspect Biol 2013;5:a012765 Extreme Radiation Resistance of D. radiodurans the radiation-sensitive bacterium Shewanella whelming proteome protection against oxida- oneidensis encodes 65% more iron–sulfur clus- tive damage resides in a small-molecular-weight ter proteins compared with Dra (Ghosal et al. fraction of the cytosol. It is this constitutive 2005). protection, present in cells before irradiation, One of the mechanisms maintaining low whose exhaustion determines the sensitivity ROS levels in Dra is the activity of the manga- of the species to radiation and other oxidative nese complexes. A strong correlation has been damage. shown between intracellular Mn/Fe concentra- Indeed, in all bacterial and animal species tion ratios and bacterial resistance to radiation, tested (E. coli, D. radiodurans, C. elegans, and in which the most resistant bacteria contained A. vaga), we have found a characteristic quanti- about 300 times more Mn2þ and about three tative relationship between g- and UVC-light- times less Fe2þ than the most radiation-sensi- induced cell mortality and total protein carbon- tive bacteria (Daly et al. 2007). However, the ylation, regardless of their intrinsic potential nature of Mn-facilitated resistance to ionizing to resist oxidative damage as seen at the level radiation was at first unclear, and the question of PC saturation upon high radiation exposures persisted why many bacteria that encode a nor- (Fig. 2) (Krisko and Radman 2010; Krisko et al. mal complement of repair functions are killed 2012). This observation suggests that the func- by doses of ionizing radiation that cause little or tionality of the proteome of all tested species no DNA damage? is similarly sensitive to incurred PC; however, A potential answer can be found in the re- all species do not accumulate oxidative protein sults showing that the amount of protein dam- damage at the same radiation exposures. The age caused by a given dose of g radiation is very basis of the remarkable robustness of resilient different for resistant and sensitive bacteria species is predominantly the antioxidant pro- (Daly et al. 2007). High levels of protein pro- tection of the cellular proteome. tection during irradiation correlate with high Having established that PC indeed corre- intracellular Mn/Fe concentration ratios and lates with cell mortality over a wide range of high levels of resistance, whereas proteins in ra- doses of two different cytotoxic agents, the key diation-sensitive cells were highly susceptible question remained: Is high PC the cause or the to radiation-induced oxidation. When cell ex- consequence of the process of cell death? Even tracts are devoid of the small-molecular-weight the activity of refrigerated purified proteins (,3 kDa) moieties by ultrafiltration, then D. must be routinely protected by antioxidants radiodurans and E. coli proteomes are equally (e.g., DTT); therefore, it is likely that the decay susceptible to radiation-induced PC, and the of cellular functions culminating in cell death (re)addition of Dra ultrafiltrate bestows the re- is a direct result of the accumulated oxidative silience to radiation-induced PC to either of the damage to the proteome. To measure the prote- two species’ proteomes (Krisko and Radman ome’s biosynthetic efficacy independently of 2010). It is obvious that the biosynthetic “invest- cell death, we have used a “parasite” of E. coli, ment” providing prevention against oxidative the bacteriophage l, as a quantitative probe. proteome damage, required for radiation and Upon entering the host cell, phage l DNA hi- desiccation resistance, is quite significant and jacks biosynthetic machineries of E. coli and evolved under selective pressure for robustness. directs them to its own reproduction. That in- volves host cell replication and transcription and translation machineries, all of which require PROTEIN, NOT DNA, DAMAGE PREDICTS multiple protein interactions and catalytic reac- DEATH BY RADIATION tions expected to be sensitive to PC. Exposure of The above experiments and the observation that E. coli to ionizing radiation or UVC light before purified individual proteins from resistant and infection with undamaged bacteriophage l re- standard species appear to be similarly suscep- sults in a progressive loss of phage production tible to oxidation in vitro argue that the over- correlating with PC levels better than cell death

Cite this article as Cold Spring Harb Perspect Biol 2013;5:a012765 7 A. Krisko and M. Radman

A B 100 7 100 7 nmol Carbonyls/mg protein

–1 –1 nmol Carbonyls/mg protein 10 6 10 6

10–2 10–2 5 5 10–3 10–3 4 4 10–4 10–4 3 3 10–5 10–5

10–6 2 10–6 2 Survival (C.F.U. and infective centers) and infective Survival (C.F.U. 10–7 1 centers) and infective Survival (C.F.U. 10–7 1 0 500 1000 1500 2000 2500 0 100 200 300 400 Dose of γ irradiation (Gy) Dose of UV irradiation (J/m2)

Survival of C.F.U. Survival of I.C. nmol Carbonyls/mg protein

Figure 3. Correlations between radiation-induced protein carbonylation (filled square), cellular capacity to produce phage l (open circle), and cell death (filled circle) in E. coli. (A) g-radiation. (B) UVC radiation. (Data from Krisko and Radman 2010.)

(Fig. 3). In other words, this experiment showed volved in DNA repair, proteostasis, and struc- that an intact phage genome fails to replicate tural maintenance. This conclusion is the con- and express when host cell proteome incurs a sequence of the fact that dedicated proteins critical amount of oxidative damage. repair DNA, and not vice versa. This paradigm The chemistry of DNA damage in cells ex- is fundamental in its obviousness (no living cell posed to ionizing radiation and UV light is dis- can function correctly with an oxidized prote- tinctly different. Exposure to ionizing radiation ome) and, if it is true, must be universal, that is, primarily generates DNA strand breaks and base hold also for human cells. damage, whereas exposure to UV light primar- ily generates photoproducts (Friedberg et al. EVOLVED RADIATION RESISTANCE IN E. coli 2004). The cellular responses to these types of DNA damage are also distinctive. Therefore, the Is proteome protection the molecular basis of observation that saturation levels of PC are the the radiation resistance also in other species? To same in D. radiodurans and E. coli heavily ex- provide an answer to this question, the groups posed to either source of damage shows that of John Battista and Michael Cox have produced oxidative damage is a common correlate of tox- radiation-resistant strains of E. coli by directed icity and that the intrinsic susceptibility to PC evolution in the laboratory (Harris et al. 2009). of the proteomes of these two bacterial species is Genome sequencing of several independently indistinguishable. However, the doses at which obtained radiation-resistant E. coli strains has increases in PC and cell killing commence are revealed more than 60 acquired mutations in always coincident, suggesting that protein dam- nonoverlapping genes that do not reveal genes age—rather than DNA damage—might be the already associated with recognizable radio pro- fundamental chemistry of cell killing (Krisko tection or repair mechanisms. However, we have and Radman 2010). The same conclusion was found that both tested strains of radioresistant reached by Daly and coworkers (Daly et al. 2007; E. coli show the same correlation between radi- Daly 2009). Thus, biological responses to geno- ation-induced loss of biosynthesis, mortality, mic insults depend primarily on the integrity and PC levels as seen in irradiated D. radiodur- of the proteome, which includes proteins in- ans and wild-type E. coli. Because neither gene

8 Cite this article as Cold Spring Harb Perspect Biol 2013;5:a012765 Extreme Radiation Resistance of D. radiodurans acquisition nor any Deinococcus-like features Is it reasonable to make ad hoc connections evolved during selection of resistant E. coli in between the biological effect of oxidative protein the test tube, it is likely that many distinct mo- damage caused by radiation and the same dam- lecular mechanisms exist for increasing the re- age caused by (or just accompanying) aging (see sistance of bacteria to ionizing radiation, UV Richardson 2009)? Is protein damage the cause light, and other toxic agents, but necessarily in- or the consequenceofcellulardegeneracycaused volve acquisition of proteome protection against by radiation or aging? The deleterious cellular oxidative damage. This conclusion is supported effects of the inactivation of the proteasome by the finding that the reproductive death by that degrades oxidized proteins (Servant et al. ionizing radiation correlates with induced PC 2007; Bayot et al. 2010)arguesthat accumulating both in radiation-sensitive (C. elegans) and ra- protein damage is likely the original cause of diation-resistant (A. vaga) invertebrates (Krisko general degradation of cellular functions. et al. 2012). We posit that studying the most robust or- ganisms can bring about the definition of mo- lecular “wear and tear” mechanisms in standard CONCLUSIONS organisms. Such mechanisms are likely to be Wedraw the following general conclusions from relevant to aging and age-related diseases. There the reviewed studies of naturally and experi- is a possibility that such knowledge and the mentally evolved radiation resistance. Protein availability of highly efficient natural antioxi- activities sustain life, whereas the genome per- dants from robust species could have a major petuates life by maintaining the levels of active impact on public health. proteins. Hence, the acute cell killing occurs by The pioneering work of Earl Stadtman and destroying the proteome’s functional integrity, associates in the late 1980s showed that the ex- not necessarily genome integrity, because even ponentially accumulating oxidative damage to an excessively damaged genome can be fully re- the proteome is the best biomarker and the likely paired by an active proteome, even if it takes fundamental cause of aging and age-related dis- days (Slade et al. 2009), ensuring the perpetua- eases because it correlates in the same exponen- tion of cellular life. Obviously, well-protected tial way with the fraction of life span of different proteome activities perform all vital cell func- species (extending from 3 weeks to 100 years) tions including protein turnover, genome main- (Oliveret al. 1987) and with the Gompertz curve tenance, and its expression. D. radiodurans pre- of incidence of disease and death in human and sents a spectacular example. If this paradigm animal populations (Sas et al. 2012). We pro- holds also for mammalian cells, then an effective pose here the concept that aging and age-related therapy by tumor cell killing should tar- diseases are the emerging phenotypes of accu- get the proteome, or both the proteome and the mulating proteome damage whose severity and genome, rather than the genome alone. complexity increase with time. Current research in the cell biology of aging deals usually with the complicated consequences of aging, but noth- PERSPECTIVES: PROTEIN DAMAGE AND ing seemsto contradict the hypothesisthat aging AGING has a simple cause with increasingly complex Progressive cell dysfunction and massive cell consequences. Aging and age-related diseases death are at the basis of aging of multicellular could be progressively complex phenotypes of organisms. A cell dies when it loses its activities accumulating proteome damage. and/or structures necessary for vital cellular functions. Studying Dra has shown that life de- ACKNOWLEDGMENTS pends acutely on proteome quality (function), whereas membrane integrity (structure—cellu- This review is supported by a generous gift larity) has long been the standard diagnostic of from Jean-Noe¨l Thorel, who also supported all cell death. quoted unpublished work at the Mediterranean

Cite this article as Cold Spring Harb Perspect Biol 2013;5:a012765 9 A. Krisko and M. Radman

Institute for Life Sciences. M.R. receives support Ge´rard E, Jolivet E, Prieur D, Forterre P. 2001. DNA from the two affiliated institutions (University protection mechanisms are not involved in the ra- dioresistance of the hyperthermophilic archaea Pyrococ- of Paris Vand Institut National pour la Sante´ et cus abyssi and P. furiosus. Mol Genet Genomics 266: la Recherche Me´dicale). A.K. receives support 72–78. from the Mediterranean Institute for Life Sci- Ghosal D, Omelchenko MV, Gaidamakova EK, Matro- ences. We thank Dr. Jean-Paul Leonetti and Dr. sova VY, Vasilenko A, Venkateswaran A, Zhai M, Kos- tandarithes HM, Brim H, Makarova KS, et al. 2005. Jacques Biton of the Paris-based Deinove com- How radiation kills cells: Survival of Deinococcus radio- pany for allowing us to quote the company’s durans and Shewanella oneidensis under oxidative stress. unpublished work. FEMS Microbiol Rev 29: 361–375. 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