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2012 Osaka

To measure the DNA damage tolerance conferred by each gene, we measured the survival rate in the presence of some DNA damaging agents (such as Mitomycin C and Hydrogen peroxide). Transformants E. coli were exposed to the DNA damaging agents and then incubated for 2 hours. Then, the cells were spreaded on agar plates at different dilutions. The plates were wrapped with aluminum foil and incubated in the dark. Colony-forming units were scored after 16h incubation at 37°C.

Mitomycin C tolerance

PprI did not increase the tolerance alone, while PprI regulates the multiple DNA repair and protection pathways in response to radiation. As E. coli lacks downstream proteins of D. radiodurans, it is expected that PprI would not be able to confer tolerance on its own. PprA, which repairs blunt-ended breaks in DNA lesions, also did not confer tolerance. PprA may not protect against DNA damages induced by MitomycinC. On the other hand, PprM and RecA much increased the tolerance. PprM is known to be a modulator of the PprI-dependent pathway. However, our data indicated that it is protective for E. coli by itself.

We observed increases in viability when certain tolerance genes were combined. For example, PprI by itself did not increase tolerance but boosted the ability of RecA to confer tolerance. This agrees with the role of PprI as an inducer of RecA in radiodurans. The combination of PprM and RecA also showed increase in tolerance. Since PprM is thought to not regulate RecA, this results indicate that PprM may induce or modulate other, unknown proteins and some of these proteins may have homologs in E. coli that benefit from the presence of PprM.

Hydrogen peroxide tolerance

Each PprA and PprM alone decreased tolerance. It may be due to the cost of their expression in the stressful cellular environment at least. On the other hand, both PprI and RecA significantly increased tolerance, which agrees with the role of PprI as an enhancer of enzyme activities of catalases.

We observed increases in viability when certain tolerance genes were combined. PprA or PprM alone did not increase tolerance, the combination of PprI and other radiotolerance genes somehow improved the tolerance. This results confirms that PprI enhance the enzyme activities of catalases.