Abstract
Endonuclease VIII-like (NEIL) 1 and 3 proteins eliminate oxidative DNA base damage and psoralen DNA interstrand crosslinks through initiation of base excision repair. Current evidence points to a DNA replication associated repair function of NEIL1 and NEIL3, correlating with induced expression of the proteins in S/G2 phases of the cell cycle. However previous attempts to express and purify recombinant human NEIL3 in an active form have been challenging. In this study, both human NEIL1 and NEIL3 have been expressed and purified from E. coli, and the DNA glycosylase activity of these two proteins confirmed using single- and double-stranded DNA oligonucleotide substrates containing the oxidative bases, 5-hydroxyuracil, 8-oxoguanine and thymine glycol. To determine the biochemical role that NEIL1 and NEIL3 play during DNA replication, model replication fork substrates were designed containing the oxidized bases at one of three specific sites relative to the fork. Results indicate that whilst specificity for 5- hydroxyuracil and thymine glycol was observed, NEIL1 acts preferentially on double-stranded DNA, including the damage upstream to the replication fork, whereas NEIL3 preferentially excises oxidized bases from single stranded DNA and within open fork structures. Thus, NEIL1 and NEIL3 act in concert to remove oxidized bases from the replication fork.
Highlights
It is estimated that in each cell approximately 10,000 DNA bases are chemically modified every day [1]
We observed only a single band when hNEIL3FL protein was present in the reaction mix, and that the band obtained was of the expected size for human NEIL3 (hNEIL3) complexed to DNA (Figure 2C)
Given previous evidence that the expression of human NEIL1 (hNEIL1) and hNEIL3 is overlapping during S-phase of the cell cycle, this suggested a role for these DNA glycosylases in initiating Base excision repair (BER) during DNA replication
Summary
It is estimated that in each cell approximately 10,000 DNA bases are chemically modified every day [1]. Cells have evolved complex DNA repair mechanisms to respond to specific DNA damage, to prevent mutagenesis and carcinogenesis [2,3,4]. DNA repair mechanism responsible for the removal of chemically altered bases, and the repair of abasic sites and single-strand breaks [5,6]. Removal of damaged bases proceeds via one of eleven lesion-specific DNA glycosylases that cleave the N-glycosylic bond between the deoxyribose sugar and modified base [7]. A fundamental understanding of the biochemical mechanisms of DNA glycosylases in base damage removal within specific DNA substrates generated during normal physiology is essential in elucidating the cellular roles that they play in vivo
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