Part:BBa_K602007:Experience

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Applications of BBa_K602007

2011 Osaka

DNA Damage Tolerance Assay

Several D. radiodurans proteins related to DNA damage repair (PprI, PprM, PprA, RecA) were assayed for their ability to confer damage tolerance to host E. coli cells. Transformed and pre-cultured cells were plated and exposed to varying doses of UV radiation. Plates were then wrapped with aluminum foil to prevent further exposure and incubated for 16h. From colony counts, the survival percentages of irradiated samples relative to controls were calculated and used as an indicator of tolerance.

Results & Discussion

It seemed that neither PprI nor PprA were capable of conferring any DNA damage tolerance in isolation. It is possible that expression of only PprI, a regulatory protein, would not do much to improve cell survival in the absence of it's downstream effectors while PprA, a DNA-binding protein mainly involved in repairing blunt-ended breaks in double-stranded DNA, may not protect against the thymine dimerization induced by UV. In fact, expression of either protein actually decreased tolerance, perhaps due to the increased burden on the cells. PprM did not have a clear positive or negative effect: although data seems to indicate increased survival at high (20 J/m^2) UV energy dosage, it was not significant enough to preclude experimental error. On a positive note, both RecA and the combined part (comprising all four proteins) significantly increased UV tolerance.

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