Abstract
The DNA glycosylase NEIL3 has been implicated in DNA repair pathways including the base excision repair and the interstrand cross-link repair pathways via its DNA glycosylase and/or AP lyase activity, which are considered canonical roles of NEIL3 in genome integrity. Compared with the other DNA glycosylases NEIL1 and NEIL2, Xenopus laevis NEIL3 C terminus has two highly conserved zinc finger motifs containing GRXF residues (designated as Zf-GRF). It has been demonstrated that the minor AP endonuclease APE2 contains only one Zf-GRF motif mediating interaction with single-strand DNA (ssDNA), whereas the major AP endonuclease APE1 does not. It appears that the two NEIL3 Zf-GRF motifs (designated as Zf-GRF repeat) are dispensable for its DNA glycosylase and AP lyase activity; however, the potential function of the NEIL3 Zf-GRF repeat in genome integrity remains unknown. Here, we demonstrate evidence that the NEIL3 Zf-GRF repeat was associated with a higher affinity for shorter ssDNA than one single Zf-GRF motif. Notably, our protein-protein interaction assays show that the NEIL3 Zf-GRF repeat but not one Zf-GRF motif interacted with APE1 but not APE2. We further reveal that APE1 endonuclease activity on ssDNA but not on dsDNA is compromised by a NEIL3 Zf-GRF repeat, whereas one Zf-GRF motif within NEIL3 is not sufficient to prevent such activity of APE1. In addition, COMET assays show that excess NEIL3 Zf-GRF repeat reduces DNA damage in oxidative stress in Xenopus egg extracts. Together, our results suggest a noncanonical role of NEIL3 in genome integrity via its distinct Zf-GRF repeat in suppressing APE1 endonuclease-mediated ssDNA breakage.
Highlights
DNA damage induced by oxidative stress is inevitable and has been implicated in the pathology of human diseases such as cancer and neurodegenerative disorders [1]
We have provided evidence showing (i) that NEIL3 zinc finger-GRF (Zf-GRF) motifs interact with APE1 and proliferating cell nuclear antigen (PCNA) but not APE2 (Fig. 1); (ii) that NEIL3 Zf-GRF repeat associates with shorter single-strand DNA (ssDNA) and has higher affinity to ssDNA compared with one single Zf-GRF motif (Figs. 2–4); and (iii) that APE1 endonuclease activity on ssDNA but not double-strand DNA (dsDNA) is compromised by NEIL3 Zf-GRF repeat (Fig. 5)
These findings suggest a previously uncharacterized negative regulation of APE1 endonuclease on ssDNA by NEIL3 Zf-GRF repeat
Summary
DNA damage induced by oxidative stress is inevitable and has been implicated in the pathology of human diseases such as cancer and neurodegenerative disorders [1]. NEIL3 interacts with APE1 and PCNA as well as telomere protein TRF2 to repair telomere DNA damage during S phase, it remains elusive how exactly NEIL3 regulates and/or cross-talks with APE1 [30]. We reveal unexpectedly that NEIL3’s Zf-GRF repeat but not ZF1 negatively regulates APE1 AP endonuclease activity on ssDNA but not dsDNA. These findings clearly reveal previously uncharacterized features of the ZfGRF repeat compared with a single Zf-GRF motif. We elucidate the distinct regulation of NEIL3 Zf-GRF motifs in genome integrity via the negative regulation of APE1 endonuclease activity on ssDNA to avoid unnecessary DNA breakages
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