guanosine-triphosphate and 2--deoxy-5--adenosine-monophosphate

7,8-Dihydro-8-oxo-deoxyguanosine (8oxodG) is a highly premutagenic DNA lesion due to its ability to mispair with adenine. Schizosaccharomyces pombe lacks homologs for relevant enzymes that repair 8oxodG, which suggests that this lesion could be persistent and must be tolerated. Here we show that SpPol4, the unique PolX in fission yeast, incorporates ATP opposite 8oxodG almost exclusively when all nucleotides (ribos and deoxys) are provided at physiological concentrations. Remarkably, this SpPol4-specific reaction could also occur during the NHEJ of DSBs. In cell extracts, misincorporation of ATP opposite 8oxodG was shown to be SpPol4-specific, although RNase H2 efficiently recognized the 8oxodG:AMP mispair to remove AMP and trigger error-free incorporation of dCTP. These data are the first evidence that ribonucleotides can be used safely for 8oxodG tolerance, suggesting that insertion of the highly abundant ATP substrate could be beneficial to promote efficient and error-free repair of 8oxodG-associated DSBs. Moreover, we demonstrate that purified SpPol4 uses 8oxo-dGTP and 8oxo-GTP as substrates for DNA polymerization, although with poor efficiency compared to the incorporation of undamaged nucleotides opposite either 8oxodG or undamaged templates. This suggests that SpPol4 is specialized in tolerating 8oxodG as a DNA template, without contributing significantly to the accumulation of this lesion in the DNA.

Topics: 8-Hydroxy-2'-Deoxyguanosine; Adenosine Triphosphate; Base Pair Mismatch; Deoxyadenine Nucleotides; Deoxyguanine Nucleotides; Deoxyguanosine; DNA End-Joining Repair; DNA Repair; DNA-Directed DNA Polymerase; Guanosine Triphosphate; Ribonuclease H; Schizosaccharomyces; Schizosaccharomyces pombe Proteins

Deoxynucleoside-5'-monophosphates (5'-dNMPs) are the basic components of DNA and are widely used in medicine and as chemical and biochemical reagents. A large amount of effort has been expended to obtain 5'-dNMPs of high quality and at a low cost. However, these procedures are inefficient and inconvenient. In this study, deoxyadenosine-5'-monophosphate (5'-dAMP), 2,6-diaminopurine deoxynucleoside-5'-monophosphate (5'-dDAMP), and deoxycytidine-5'-monophosphate (5'-dCMP) were biosynthesized using recombinant N-deoxyribosyltransferase II (NDT-II), deoxycytidine kinase, and acetate kinase in a one-pot reaction system. The ndt-II gene from Lactobacillus delbrueckii, dck from Bacillus subtilus, and ack from Escherichia coli K12 were overexpressed in E. coli BL21 (DE3). Thymidine was used as the deoxyribose donor; GTP was used as the phosphate donor, and acetyl phosphate was used to regenerate GTP. Under optimized conditions, each 10 mM adenine, 10 mM 2,6-diaminopurine, or 10 mM cytosine were converted into 9.01 mM 5'-dAMP, 8.68 mM 5'-dDAMP, or 6.23 mM 5'-dCMP, respectively. The high yield indicated that this process of biosynthesis of 5'-dAMP, 5'-dDAMP, or 5'-dCMP was efficient and economical, and this one-pot system may also potentially be used for the preparation of other types of 5'-dNMPs.

Topics: Bacillus; Coenzymes; Deoxyadenine Nucleotides; Deoxycytidine Monophosphate; Escherichia coli K12; Gene Expression; Guanosine Triphosphate; Lactobacillus delbrueckii; Metabolic Engineering; Phosphotransferases (Alcohol Group Acceptor); Recombinant Proteins