Expression and characterization of recombinant human NEIL3 from Escherichia coli
The DNA glycosylase NEIL3 is one of a family of proteins that release oxidized bases from DNA, thereby initiating base excision repair. NEIL3 gene expression is normally tightly regulated and expressed only in certain rapidly dividing cells, however, human NEIL3 (hNEIL3) is also highly expressed in cells from metastatic tumours. In this project, full length hNEIL3 and truncations of the cDNA have been cloned into the pETDuet-2 expression vector for subsequent expression and purification. Each of the hNEIL3 truncations codes for the N-terminal Fpg/Nei and H2tH domains essential for DNA glycosylase activity, but lack at least one of four C-terminal motifs unique to NEIL3. Using pETDuet-2, active truncated and full length hNEIL3 have been overexpressed in Escherichia coli and purified. Enzyme assays were performed using oligonucleotide substrates containing one of three oxidised bases, thymine glycol (TG), 5-hydroxyuracil (5-OHU) or 8-oxoguanine (8-oxoG). Results indicate that the recombinant hNEIL3 proteins are active on single-stranded DNA (ssDNA) substrates containing 5-OHU and TG but show only weak activity on 8-oxoG. In contrast, in double-stranded DNA (dsDNA), activity on 5-OHU:G and TG:A was only weakly observed with nominal activity on 8-oxoG:C. The oxidised bases were then placed at three different sites in a model replication fork, at position -4 in ssDNA, at the fork junction (the last nucleotide in the dsDNA) and position +4 in dsDNA. For TG, hNEIL3 incised the fork substrate through β-elimination at all positions and exhibited greater activity at the +4 position than in the equivalent dsDNA substrate. For 5-OHU, a similar level of β-elimination activity was observed at the -4 and fork junction positions, but activity at the +4 dsDNA position was predominantly by β,δ-elimination when the full length hNEIL3 protein was used. Again, only weak activity was observed on 8-oxoG fork substrates and a similar incision pattern was observed to that for 5-OHU. Therefore, the results indicate that hNEIL3 acts as a bifunctional DNA glycosylase, either through β- or β,δ-elimination depending on the substrate lesion. For the first time it is shown that the C-terminal domains can influence the bifunctional DNA glycosylase activity of hNEIL3. These studies will help to define the biochemical function of this unique protein in both normal and malignant human cells.
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