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New Technology May Reveal Mechanisms of Radiation Resistance in Deinococcus Radiodurans

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New Technology May Reveal Mechanisms of Radiation Resistance in Deinococcus Radiodurans Commentary New technology may reveal mechanisms of radiation resistance in Deinococcus radiodurans Jan Mra´ zek* Department of Mathematics, Stanford University, Stanford, CA 94305-2125 dvances in DNA sequencing technol- genic, red-pigmented bacterium. Although late numerous double-strand DNA breaks Aogy in the past decade have resulted technically Gram-positive, D. radiodurans during irradiation and invokes its extra- in rapid production of DNA data includ- features plasma and outer membranes and a ordinary DNA repair facility to reconstruct ing more than 70 complete prokaryotic multilayer cell envelope reminiscent of its genome from the remaining fragments genomes and several eukaryotic genomes Gram-negative bacteria (5). Its radioresis- (11, 12). Similar repair mechanisms are also currently available. This accumulation of tance makes it one of the leading candidates apparently involved in radioresistance of sequence data has provided opportunities for bioremediation of radioactive waste sites Pyrococcus furiosus (13) and possibly other for development of proteomics. Pro- that contain hazardous mixtures of radio- hyperthermophilic archaea. Many bacteria teomic analyses focus on composition of nuclides, heavy metals, and other toxic including D. radiodurans possess multiple the protein mixture extracted from a cell chemicals. Engi- copies of their chro- culture and its changes in response to neered strains of D. mosomes and can radiodurans are ca- recover from a lim- environmental disturbances or during the D. radiodurans’ radioresistance cell cycle. A standard scenario is as fol- pable of transform- ited number of dou- lows: First, extract the complex protein ing these mixtures to makes it one of the leading ble-strand DNA mixture from cell cultures growing under less hazardous ones candidates for bioremediation breaks by reassem- controlled conditions. Then run a two- by degrading some bling an intact chro- of radioactive waste sites. dimensional PAGE to separate different of the toxic compo- mosome from over- proteins into a large number of often nents (6, 7). Under- lapping fragments partly overlapping spots. Finally, identify standing molecular via homologous re- the spots and assign them to known pro- mechanisms that allow cells to survive dam- combination. However, multiple copies of teins or genes (e.g., ref. 1). This last step aging effects of UV radiation is also of chromosomes do not themselves confer ra- COMMENTARY is facilitated by use of MS (e.g., ref. 2). interest in medical research, especially in diation resistance. D. radiodurans can repair Routine use of MS technology in pro- conjunction with ongoing environmental more than 100 double-strand DNA breaks teomics was made possible by complete changes that allow more UV radiation to without lethality whereas most other bacte- genome sequencing. A complete genomic reach the Earth’s surface (8). ria with multiple copies of chromosomes are sequence can be used to identify all puta- Although the complete genomic DNA unable to repair more than a few double- tive proteins encoded in the genome. This sequence of D. radiodurans is now known strand breaks (14). It is possible that D. limits the number of possible peptide frag- (9), the molecular mechanisms of its radia- radiodurans uses this type of repair in a ments expected to be found in the proteins tion resistance are not well understood. Ra- different way than other organisms. Homol- extracted from the cells (3) and, given diation causes complex damage to various ogous recombination is promoted by the sufficient accuracy of the MS measure- cell components, including nucleic acids, RecA protein, which plays an important role ments, the mass of a fragment can often be proteins, and membranes. The damage can in the radiation resistance of D. radiodurans. unambiguously associated with a specific be caused directly by the radiation or indi- When D. radiodurans RecA was replaced protein. In this issue of PNAS, Lipton et al. rectly by free radicals generated by it. At the with a RecA gene from Shigella flexneri, the (4) use an improved MS technology based DNA level, UV radiation damage is limited resulting D. radiodurans mutant lost its ra- on peptide accurate mass tags for pro- mainly to DNA bases whereas ionizing ra- diation resistance (11). The D. radiodurans teomic analyses of the radiation-resistant diation causes a wider variety of damage, RecA was originally not detected in undam- bacterium Deinococcus radiodurans. The including single- and double-strand breaks. aged cells, leading to speculation that it is method uses Fourier transform ion cyclo- The single-strand breaks, where one DNA actually toxic to the normal cells and is tron resonance (FTICR) experimentation strand is damaged but the other remains expressed only after DNA damage. How- instead of standard tandem MS, which intact, can be repaired by base excision ever, precise accurate mass tag measure- provides greater sensitivity and accuracy, repair where the damaged part of the DNA ments detected RecA under normal growth yielding a more comprehensive coverage is removed and synthesized de novo by using conditions and confirmed its significant in- of the proteome. Thus, the accurate the undamaged strand as a template. In crease after irradiation (4). FTICR measurements applied to D. ra- double-strand breaks, both DNA strands In our laboratory, we identified pre- diodurans identified accurate mass tags are broken at the same site, precluding dicted highly expressed (PHX) genes of D. for 61% of all putative D. radiodurans excision repair. Different strategies can be radiodurans and compared them to other proteins—the most complete proteome used in protecting DNA from the effects of prokaryotes (15). With the exception of coverage to date. radiation. Rubrobacter radiotolerans, an- RecA, the DNA repair proteins of D. D. radiodurans is exceptional in its other radiation-resistant bacterium, appar- capacity to withstand high doses of ionizing ently protects its DNA so the strand breaks and UV radiation that are lethal to virtually do not occur (10). By contrast, D. radio- See companion article on page 11049. all other living organisms. It is a nonpatho- durans has been demonstrated to accumu- *E-mail: [email protected]. www.pnas.org͞cgi͞doi͞10.1073͞pnas.182429699 PNAS ͉ August 20, 2002 ͉ vol. 99 ͉ no. 17 ͉ 10943–10944 Downloaded by guest on September 29, 2021 radiodurans are generally of low predicted converts glucose to precursors of deoxy- investigated, possibly indicating a highly spe- expression levels and their assortment is ribonucleotide triphosphates, which are cialized function for these proteins. not larger than that of Escherichia coli. used in DNA synthesis and repair. It has Although repair processes appear to The D. radiodurans RecA has a very high been proposed that an active pentose play an important role in D. radiodurans’ predicted expression level compared with phosphate pathway is important in D. survival of radiation and desiccation dam- RecA proteins in other prokaryotes, con- radiodurans radiation resistance (18). The age, so far there is no evidence of repair sistent with its possible enhanced role in protein Rsr has been shown to contribute mechanisms unique to D. radiodurans but D. radiodurans. On the other hand, in to D. radiodurans resistance to UV radi- not present in less resistant organisms. In comparison with other prokaryotes, D. ation (although not ionizing radiation). fact, D. radiodurans DNA repair pathways radiodurans stands out in its number of Rsr is a homolog of ribonucleoprotein Ro appear to be less complex and diverse than PHX chaperone, degradation, protease, previously found only in eukaryotes where those of E. coli or Bacillus subtilis. For and detoxification genes. Chaperones as- it binds misfolded 5S rRNA molecules. example, the RecBCD recombinase, sist protein folding and repair misfolded Chen et al. (19) suggested that it could which is a major component of recombi- proteins whereas degradation enzymes contribute to degradation and removal of nation systems in most free-living organ- and proteases digest and recycle dysfunc- damaged RNA molecules. Lipton et al. (4) isms, is not found in the D. radiodurans tional proteins and nucleic acids. Detoxi- detected Rsr in cell cultures growing un- genome (5). However, the function of a fication proteins remove toxic free radi- der most conditions, but not after an large number of D. radiodurans proteins cals from the cell. We (15) speculated that extended period of starvation (4 weeks). has yet to be determined, and some of chaperone, degradation, and detoxifica- D. radiodurans contains a high number of these may turn out to be the missing pieces tion proteins may play an important role intergenic DNA repeats that could con- of the puzzle. Notably, D. radiodurans contains an unusually high number of in response to extensive damage caused by tribute to efficient recombination and en- uncharacterized hypothetical proteins radiation. Degradation and export of hance DNA repair after irradiation (9). whose codon usage is consistent with high damaged DNA after irradiation is an im- Besides its capacity to survive high levels expression levels (15). As an example of portant part of the repair process (12) and of radiation, D. radiodurans can also with- how their data could be used in charac- the same probably applies to damaged stand periods of desiccation. It has been terizing hypothetical proteins, Lipton et al. proteins. suggested that radiation resistance evolved
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