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Background DNA oxidatively damaged by reactive oxygen species is repaired by base excision repair (BER) pathway proteins, with DNA glycosylases removing damaged or mismatched bases in the first step of BER. KsgA is a multifunctional protein that exhibits the activities of two enzymes, DNA glycosylase and rRNA dimethyltransferase. The structure-function relationship of the KsgA protein in cellular DNA repair remains unclear because the domains required for KsgA to recognize DNA have not been identified. Purpose To clarify the mechanisms by which KsgA recognizes damaged DNA and to identify the DNA-binding site, which exists in KsgA. Methods A structural analysis and in vitro DNA-protein binding assay were performed. The C-terminal function of the KsgA protein was investigated in vitro and in vivo. Results The 3D conformations of KsgA, MutM, and Nei were compared at UCSF Chimera. The root mean square deviation of KsgA (214-273) and MutM (148-212) and that of KsgA (214-273) and Nei (145-212) were 1.067 and 1.188 Å, both less than 2 Å, suggesting that the C terminal of KsgA is spatially similar to the H2TH domains of MutM and Nei. The full-length KsgA protein and KsgA lacking 1-8 or 214-273 amino acids were purified and used in gel mobility shift assays. KsgA exhibited DNA-binding activity, which was lost in the C-terminally deleted KsgA protein. Spontaneous mutation frequency was measured using a mutM mutY ksgA-deficient strain, and the results obtained showed that the mutation frequency was not suppressed by KsgA lacking the C-terminal region, whereas it was in KsgA. To assess dimethyltransferase activity, kasugamycin sensitivity was assessed in wild-type and ksgA-deficient strains. Plasmids carrying the full-length ksgA gene and C-terminal deletion gene were introduced into ksgA-deficient strains. KsgA lacking the C terminus restored dimethyltransferase activity in the ksgA-deficient strain as well as KsgA. Conclusion The present results confirmed that one enzyme exhibited two activities and revealed that the C-terminal (214-273) amino acids of KsgA were highly similar to the H2TH structural domain, exhibited DNA-binding activity, and inhibited spontaneous mutations. This site is not essential for dimethyltransferase activity.

Apurinic/apyrimidinic (AP) sites are one of the most frequent DNA lesions. AP sites inhibit transcription and DNA replication, and induce cell death. AP endonucleases are key enzymes in AP site repair. Several types of AP endonucleases have been reported, such as AP endonuclease 2 (APEX2) and ribosomal protein P0 (P0). However, it is not known how the functions and roles differ among AP endonucleases. To clarify the difference of roles among AP endonucleases, we conducted biochemical analysis focused on APEX2 and P0 homologues in Ciona intestinalis. Amino acid sequence analysis suggested that CiAPEX2 and CiP0 are AP endonuclease homologues. Although we could not detect AP endonuclease or 3′-phosphodiesterase activity, these two purified proteins exhibited 3′-5′ exonuclease activity. This 3′-5′ exonuclease activity was sensitive to ethylenediaminetetraacetic acid (EDTA), and the efficiency of this activity was influenced by the 3′-terminus of substrate DNA. Both CiAPEX2 and CiP0 degraded not only a 5′-protruding DNA end, but also nicked DNA, which is generated through AP endonuclease 1 (APEX1) cleavage. These two genes partially complemented the growth rate of AP endonuclease-deficient Escherichia coli treated with hydrogen peroxide. These results indicate that 3′-5′ exonuclease activity is an evolutionarily conserved enzymatic activity of APEX2 and P0 homologues and this enzymatic activity may be important for AP endonucleases. Electronic supplementary material The online version of this article (10.1186/s41021-017-0087-7) contains supplementary material, which is available to authorized users.

Background To observe the efficacy and safety of adding glimepiride to established insulin therapy in poorly controlled type 2 diabetes (T2D) and to assess the relationship of changes in the serum high-molecular weight (HMW) adiponectin levels and glycemic control after glimepiride treatment. Methods Fifty-six subjects with poorly controlled insulin-treated T2D were randomly assigned to either the glimepiride-added group (the group A, n = 29) or the insulin-increasing group (the group B, n = 27) while continuing current insulin-based therapy. Glycosylated hemoglobin (HbA1c) value, daily insulin dose, body weight, waist circumference, plasma lipid concentration, serum HMW adiponectin level and the number of hypoglycemic events were evaluated before and after treatment. Results At the end of study, insulin doses were significantly reduced, and the mean HbA1c, fasting blood glucose (FBG) and 2-hour postprandial blood glucose (P2BG) were improved greater in the group A compared with the group B. The serum HMW adiponectin levels were significantly increased in the group A compared with the group B. Most importantly, we found that changes in HbA1c were inversely correlated with changes in serum HMW adiponectin in the group A (r = −0.452, p = 0.02). Conclusions Adding glimepiride to current insulin treatment led to better improvement in glycemic control with a significant smaller daily insulin dose, and the increases in the serum HMW adiponectin levels may directly contribute to improvement glycemic control.

We investigated the effects of 6- and 10-T static magnetic fields (SMFs) on the expression of protooncogenes using Western blot immunohybridization methods. We used a SMF exposure system, which can expose cells to a spatially inhomogeneous 6 T with a strong magnetic field (MF) gradient (41.7 T/m) and a spatially homogeneous 10 T of the highest magnetic flux density in this experiment. HL-60 cells exposed to either 6- or 10-T SMF for periods of 1 to 48 h did not exhibit remarkable differences in levels of c-Myc and c-Fos protein expression, as compared with sham-exposed cells. In contrast, c-Jun protein expression increased in HL-60 cells after exposure to 6-T SMF for 24, 36, 48, and 72 h. These results suggest that a homogeneous 10-T SMF does not alter the expression of the c-jun, c-fos, and c-myc protooncogenes. However, our observation that exposure to a strong MF gradient induced c-Jun expression suggests that a strong MF gradient may have significant biological effects, particularly regarding processes related to an elevation of c-jun gene expression.