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

R Scott Williams,Alyssa A Rodriguez,Jessica L Wojtaszek,Briana H Greer,Tuhin Haldar,Kent S Gates,R.Scott Williams,Brandt F Eichman

doi:10.1074/jbc.ra120.015541

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 that 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.

Highlights

The NEIL family of DNA glycosylases, which include NEIL1, NEIL2, and NEIL3, are important for repair of oxidative DNA damage in vertebrates [1]
interstrand cross-link (ICL) repair is initiated by unhooking the tethered strands, either by dual incisions on one strand by endonucleases associated with Fanconi anemia and nucleotide excision repair, or alternatively by cleavage of the cross-linked nucleotide by a specialized DNA glycosylase [5, 31]
Neither purified recombinant GST (Fig. 1D) nor a NEIL3 GST–RanBP/NPL4-type ZF (NZF) domain fusion (Fig. 1E) bound DNA with appreciable affinity. These results indicate that the NEIL3 GRF12 domain binds single-stranded DNA (ssDNA) with no apparent specificity for a fork structure and that the tandem GRF motifs bind with higher affinity and specificity than either GRF motif alone

Summary

Results

The individual NEIL3 GRF motifs have been shown to bind ssDNA [28], but the DNA binding specificity of the tandem. Because the mNEIL3–GD has base excision activity on its own [8, 17], we tested the mechanism by which the GRF–ZF inhibits catalytic activity by adding the purified GRF12 domain in trans to mNEIL3–GD enzymatic reactions containing ssDNA and fork substrates harboring a single DHT residue (Fig. 5A). Compared with reactions that contained no GRF12 protein, the presence of an equimolar amount of GRF12 caused a severe reduction in DHT excision kinetics (Fig. 5B) To test whether this reduction of enzymatic activity was caused by competition of GD and GRF binding to the DNA substrate, we performed the same reaction with the GRF12 K522E/K568E double mutant (GRF12mut). Similar to the result with ssDNA, the addition of GRF12, but not GRF12mut, caused the same inhibition of glycosylase activity against both DHT fork substrates, indicating that the GRF domain has no effect on the preference for leading-strand damage at forks (Fig. 5C)

Discussion

C AP lag ICL

Experimental procedures