The primary objectives of this laboratory are to define the subset of proteins required for the ionizing radiation resistance of Deinococcus radiodurans R1, and characterize the activities of those proteins. D. radiodurans, a non-sporeforming bacterium, has extraordinary tolerance for ionizing radiation. D. radiodurans , a non-sporeforming bacterium, has extraordinary tolerance for ionizing radiation. Exponential phase cultures of D. radiodurans R1 survive 500,000 Rad (5000 Gray) of γ radiation without loss of viability or evidence of mutation. In terms of DNA damage, 5000 Gy γ radiation introduces approximately 200 DNA double strand breaks, over 3000 single strand breaks, and greater than 1000 sites of base damage per genome. This organism does not passively protect its genome from the incident radiation. Instead all available evidence argues that D. radiodurans efficiently and accurately repairs DNA damage.
The D. radiodurans genome has been sequenced in its entirety, and encodes essentially the entire ensemble of DNA repair proteins found in E. coli. With the exception of alkylation transfer and photoreactivation, all of the major prokaryotic DNA repair pathways are represented. This observation is significant, not because it says anything about why D. radiodurans is radioresistant, but because it confirms something long suspected; D. radiodurans possesses unique mechanisms for dealing with ionizing radiation-induced DNA damage. Clearly, the collection of repair proteins identified in D. radiodurans, in and of itself, is not sufficient to confer radioresistance. If it were, E. coli would be as radioresistant. D. radiodurans must encode novel DNA repair proteins or, alternatively, it must use the DNA repair proteins it encodes much more efficiently than more radiosensitive prokaryotes. Either possibility suggests that there are unprecedented mechanisms facilitating this species recovery following exposure to ionizing radiation.
Of the 3187 open reading frames identified in D. radiodurans R1, only 1493 could be assigned a function based on similarity to other gene products found in the protein databases. Of the 1694 proteins of unknown function, 1002 are, at present, unique to D. radiodurans, showing no database match. The secret to understanding the radioresistance of D. radiodurans is presumably found among these proteins of unknown function. The challenge lies in unequivocally determining what proteins are responsible for radioresistance.
Currently, a combination of two approaches (random mutagenesis and DNA microarray-based analysis) is being employed to allow identification of gene products potentially involved in ionizing radiation resistance. Once identified, these gene products are characterized to establish how these proteins contribute to ionizing radiation resistance. In the long term, it is expected that this work will: a) enhance our understanding of D. radiodurans, and b) define novel protein families involved in DNA damage tolerance.
The most recent work from this laboratory is an outgrowth of a DOE-funded project in which a D. radiodurans-specific DNA microarray was used to identify gene products potentially involved in ionizing radiation resistance. D. radiodurans R1’s transcriptional response to a sub-lethal dose of ionizing radiation (IR) was characterized, and 72 genes identified that are up-regulated three fold or higher during the first hour post-irradiation. This expression profile was compared with R1 cultures recovering from desiccation and with an isogenic irrE defective strain exposed to IR. Two key results have come from these comparisons. First, this organism’s remarkable ability to repair IR-induced DNA damage does not appear to be related to massive alterations in gene expression, or large magnitude changes in transcript abundance. Second, we establish that the irrE gene product is a transcriptional activator that regulates at least 23 downstream targets, 16 of which are part of this species’ common response to IR and desiccation. This work focused our attention on a limited number of genes that should confer radiotolerance on D. radiodurans, including a number of genes that encode proteins of unknown function. The five hypothetical genes that were induced to highest level in response to ionizing radiation and desiccation were deleted, and the radioresistance of the resulting strains compared to the R1 parent. All five have been shown to contribute to the ionizing radiation resistance of this species, unequivocally establishing that D. radiodurans’ radioresistance is, at least in part, due to the action of novel proteins.