Alleviation of C⋠C Mismatches in DNA by the Escherichia coli Fpg Protein.

DNA polymerase III mis-insertion may, where not corrected by its 3'→ 5' exonuclease or the mismatch repair (MMR) function, result in all possible non-cognate base pairs in DNA generating base substitutions. The most thermodynamically unstable base pair, the cytosine (C)⋅C mismatch, destabilizes adjacent base pairs, is resistant to correction by MMR in Escherichia coli, and its repair mechanism remains elusive. We present here in vitro evidence that C⋅C mismatch can be processed by base excision repair initiated by the E. coli formamidopyrimidine-DNA glycosylase (Fpg) protein. The k cat for C⋅C is, however, 2.5 to 10 times lower than for its primary substrate 8-oxoguanine (oxo8G)⋅C, but approaches those for 5,6-dihydrothymine (dHT)⋅C and thymine glycol (Tg)⋅C. The K M values are all in the same range, which indicates efficient recognition of C⋅C mismatches in DNA. Fpg activity was also exhibited for the thymine (T)⋅T mismatch and for N 4- and/or 5-methylated C opposite C or T, Fpg activity being enabled on a broad spectrum of DNA lesions and mismatches by the flexibility of the active site loop. We hypothesize that Fpg plays a role in resolving C⋅C in particular, but also other pyrimidine⋅pyrimidine mismatches, which increases survival at the cost of some mutagenesis.

The Escherichia coli alkA Gene Is Activated to Alleviate Mutagenesis by an Oxidized Deoxynucleoside.

The cellular methyl donor S-adenosylmethionine (SAM) and other endo/exogenous agents methylate DNA bases non-enzymatically into products interfering with replication and transcription. An important product is 3-methyladenine (m3A), which in Escherichia coli is removed by m3A-DNA glycosylase I (Tag) and II (AlkA). The tag gene is constitutively expressed, while alkA is induced by sub-lethal concentrations of methylating agents. We previously found that AlkA exhibits activity for the reactive oxygen-induced thymine (T) lesion 5-formyluracil (fU) in vitro. Here, we provide evidence for AlkA involvement in the repair of oxidized bases by showing that the adenine (A) ⋅ T → guanine (G) ⋅ cytosine (C) mutation rate increased 10-fold in E. coli wild-type and alkA - cells exposed to 0.1 mM 5-formyl-2'-deoxyuridine (fdU) compared to a wild-type specific reduction of the mutation rate at 0.2 mM fdU, which correlated with alkA gene induction. G⋅C → A⋅T alleviation occurred without alkA induction (at 0.1 mM fdU), correlating with a much higher AlkA efficiency for fU opposite to G than for that to A. The common keto form of fU is the AlkA substrate. Mispairing with G by ionized fU is favored by its exclusion from the AlkA active site.

DHA-induced stress response in human colon cancer cells - Focus on oxidative stress and autophagy.

Polyunsaturated fatty acids (PUFAs) are important constituents of the diet and health benefits of omega-3/n-3 PUFAs, especially eicosapentaenoic acid (EPA, 20:5 n-3) and docosahexaenoic acid (DHA, 22:6 n-3) have been well documented in relation to several diseases. Increasing evidence suggests that n-3 PUFAs may have anticancer activity and improve the effect of conventional cancer therapy. The mechanisms behind these effects are still unclear and need to be elucidated. We have examined the DHA-induced stress response in two human colon cancer cell lines, SW620 and Caco-2. SW620 cells are growth-inhibited at early time points by DHA, while the growth of Caco-2 cells almost remains unaffected by the same treatment. Gene expression analysis of SW620 cells treated with DHA revealed changes at early time points; transcripts involved in oxidative stress and autophagy were among the first to be differentially expressed. We find that oxidative stress is induced in both cell lines, although at different time points and to different extent. DHA induced nuclear translocation of the oxidative stress sensor NFE2L2 in both cell lines, indicating an induction of an anti-oxidative response. However, vitamin E did not counteract ROS-production or the translocation of NFE2L2 to the nucleus. Neither vitamin E nor the antioxidants butylated hydoxyanisole (BHA) and butylated hydoxytoluene (BHT) did affect the growth inhibition in SW620 cells after DHA-treatment. Also, siRNA-mediated down-regulation of NFE2L2 did not sensitize SW620 and Caco-2 cells to DHA. These results indicate that oxidative stress response is not the cause of DHA-induced cytotoxicity in SW620 cells. Using biochemical and imaging based functional assays, we found a low basal level of autophagy and no increase in autophagic flux after adding DHA to the SW620 cells. However, Caco-2 cells displayed a higher level of autophagy, both in the absence and presence of DHA. Inhibition of autophagy by siRNA mediated knock down of ATG5 and ATG7 sensitized both SW620 and Caco-2 cells to DHA. Stimulation of autophagy by rapamycin in SW620 and Caco-2 cells resulted in decreased DHA-sensitivity and inhibition of autophagy in Caco-2 cells by chloroquine resulted in increased DHA-sensitivity. These results suggest that autophagy is important for the DHA sensitivity of colon cancer cells and imply possible therapeutic effects of this fatty acid against cancer cells with low autophagy.