An autoinhibitory role for the GRF zinc finger domain of DNA glycosylase NEIL3

Abstract

The NEIL3 DNA glycosylase maintains genome integrity during replication by excising oxidized bases from single-stranded DNA (ssDNA) and unhooking interstrand cross-links (ICLs) at fork structures. In addition to its N-terminal catalytic glycosylase domain, NEIL3 contains two tandem C-terminal GRF-type zinc fingers that are absent in the other NEIL paralogs. ssDNA binding by the GRF-ZF motifs helps recruit NEIL3 to replication forks converged at an ICL, but the nature of DNA binding and the effect of the GRF-ZF domain on catalysis of base excision and ICL unhooking is unknown. Here, we show that the tandem GRF-ZFs of NEIL3 provide affinity and specificity for DNA that is greater than each individual motif alone. The crystal structure of the GRF domain shows the tandem ZF motifs adopt a flexible head-to-tail configuration well-suited for binding to multiple ssDNA conformations. Functionally, we establish that the NEIL3 GRF domain inhibits glycosylase activity against monoadducts and ICLs. This autoinhibitory activity contrasts GRF-ZF domains of other DNA processing enzymes, which typically use ssDNA binding to enhance catalytic activity, and suggests that the C-terminal region of NEIL3 is involved in both DNA damage recruitment and enzymatic regulation.

Microbial Type IA Topoisomerase C-Terminal Domain Sequence Motifs, Distribution and Combination

“…tritici topoisomerase III have been predicted as described in Methods. The modeling of individual zf-GRF domains seemingly followed the three available zf-GRF structures, Xenopus laevie Apex2 C-terminal zf-GRF [ 36 ], human N 6 -methyladenosine N-terminal zf-GRF [ 37 ], and human NEIL3 C-terminal tandem zf-GRF domains [ 38 ]. The two zf-GRF domains (GRF1 and GRF2) are connected by a 40 residue long linker ( Figure 6 a).…”

“…Although the structure and function of each of two individual zf-GRF domains can be predicted to a certain extent, their possible association is unknown, especially in the presence of a 40 residue long linker between them. The two human NEIL3 C-terminal zf-GRF domains are packed against each other with a short 3-residue linker [ 38 ]. The association of the two zf-GRF domains was believed to enhance DNA binding and the binding specificity [ 38 ].…”

“…The two human NEIL3 C-terminal zf-GRF domains are packed against each other with a short 3-residue linker [ 38 ]. The association of the two zf-GRF domains was believed to enhance DNA binding and the binding specificity [ 38 ]. In the prototypical Topo_C_ZnRpt-containing EcTOP1 structure, there are two interacting pairs (D5-D6 and D8-D9) [ 17 ].…”

Noncatalytic Domains in DNA Glycosylases

“…RanBP zinc fingers are mostly regarded as protein-protein-interacting units, as in the Ran-binding proteins in which they are involved in binding Ran/GDP [87]. The GRF zinc-fingercontaining part of human NEIL3 has been crystallized, revealing a β-ribbon structure well suited for binding single-stranded DNA [88]. Interestingly, in the mouse protein, the GRF zinc fingers of NEIL3 efficiently bind single-stranded and forked DNA but inhibit the glycosylase activity, perhaps competing with the catalytic domain for substrate binding [88].…”

“…The GRF zinc-fingercontaining part of human NEIL3 has been crystallized, revealing a β-ribbon structure well suited for binding single-stranded DNA [88]. Interestingly, in the mouse protein, the GRF zinc fingers of NEIL3 efficiently bind single-stranded and forked DNA but inhibit the glycosylase activity, perhaps competing with the catalytic domain for substrate binding [88]. Forked DNA is a preferred substrate for NEIL3, possibly reflecting its role in the repair of stalled replication intermediates [89], and zinc-finger-mediated protein-protein and protein-DNA interactions within the replication fork might be critical for the correct positioning of NEIL3 to repair the lesions encountered during the replication.…”