2′-Deoxyinosine

29522920

Deamination of adenine can occur spontaneously under physiological conditions to generate the highly mutagenic lesion, deoxyinosine (hypoxanthine deoxyribonucleotide, dI). In DNA, dI preferably pairs with cytosine rather than thymine and results in A:T to G:C transition mutations after DNA replication. The deamination of adenine is enhanced by ROS from exposure of DNA to ionizing radiation, UV light, nitrous acid, or heat. In Escherichia coli, dI repair is initiated by endonuclease V (endo V; nfi gene product) nicking but a complete repair mechanism has yet to be elucidated. Using in vitro minimum component reconstitution assays, we previously showed that endo V, DNA polymerase I (pol I), and E. coli DNA ligase were sufficient to repair this dI lesions efficiently and that the 3′-5′ exonuclease of pol I is essential. Here we employed a phagemid-based T-I substrate mimicking adenine deamination product to demonstrate pol I proofreading exonuclease is required by the endo V repair pathway both in vitro and in vivo. In vivo we found that the repair level of an nfi mutant (11%) was almost 8-fold lower than the wild type (87%). while the polA-D424A strain, a pol I mutant defective in 3′-5′ exonuclease, showed a high repair level similar to wild type (both more than 80%). Using additional C-C mismatch as strand discrimination marker we found that the high level of dI removal in polA-D424A was due to strand loss (more than 60%) associated with incomplete repair. Thus, pol I proofreading exonuclease is the major function responsible for dI lesion removal after endoV nicking both in vitro and in vivo. Finally, using MALDI-TOF to analyze single-nucleotide extension product we show that the pol I proofreading exonuclease excises only 2-nt 5′ upstream of endo V incision site further honing the role of pol I in the endoV dI dependent repair pathway.

Copyright ? 2018 Elsevier B.V. All rights reserved.

DNA polymerase I 3′-5′; DNA repair; Deoxyinosine; Endonuclease V repair; In vivo repair assay; MALDI-TOF mass spectrometry; Reconstitution repair in vitro; Repair mechanism; exonuclease

DNA polymerase I proofreading exonuclease activity is required for endonuclease V repair pathway both in vitro and in vivo.

Su KY1, Lin LI1, Goodman SD2, Yen RS3, Wu CY3, Chang WC3, Yang YC1, Cheng WC4, Fang WH5.

2018 Apr

27194215

Aptamers can be chemically modified to enhance nuclease resistance and increase target affinity. In this study, we performed chemical modification of 2′-deoxyinosine in AS1411, an anti-proliferative G-rich oligodeoxynucleotide aptamer, which binds selectively to the nucleolin protein. Its function was augmented when 2′-deoxyinosine was incorporated at positions 12, 13, 15, and 24 of AS1411, respectively. In addition, double incorporation of 2′-deoxyinosine at positions 12 and 24 (FAN-1224dI), 13 and 24 (FAN-1324dI), and 15 and 24 (FAN-1524dI) promoted G-quartet formation, as well as inhibition of DNA replication and tumor cell growth, and induced S-phase cell cycle arrest. In further animal experiments, FAN-1224dI, FAN-1324dI and FAN-1524dI resulted in enhanced treatment effects than AS1411 alone. These results suggested that the position and number of modification substituents in AS1411 are critical parameters to improve the diagnostic and therapeutic function of the aptamer. Structural investigations of the FAN-1524dI/nucleolin complex structure, using molecular dynamics simulation, revealed the critical interactions involving nucleolin and 2′-dI incorporated AS1411 compared with AS1411 alone. These findings augment understanding of the role of 2′-deoxyinosine moieties in interactive binding processes.

Bioactivity of 2'-deoxyinosine-incorporated aptamer AS1411.

Fan X1, Sun L2, Wu Y1, Zhang L1, Yang Z1.

2016 May 19