Model of abasic site DNA cross-link repair; from the architecture of NEIL3 DNA binding domains to the X-structure model

Abstract

Covalent DNA interstrand crosslinks are toxic DNA damage lesions that block the replication machinery that can cause a genomic instability. Ubiquitous abasic DNA sites are particularly susceptible to spontaneous cross-linking with a base from the opposite DNA strand. Detection of a crosslink induces the DNA helicase ubiquitination that recruits NEIL3, a DNA glycosylase responsible for the lesion removal. NEIL3 utilizes several zinc finger domains indispensable for its catalytic NEI domain repairing activity. They recruit NEIL3 to the repair site and bind the single-stranded DNA. However, the molecular mechanism underlying their roles in the repair process is unknown. Here, we report the structure of the tandem zinc-finger GRF domain of NEIL3 and reveal the molecular details of its interaction with DNA. Our biochemical data indicate the preferential binding of the GRF domain to the replication fork. In addition, we obtained a structure for the catalytic NEI domain in complex with the DNA reaction intermediate that allowed us to construct and validate a model for the interplay between the NEI and GRF domains in the recognition of an interstrand cross-link. Our results suggest a mechanism for recognition of the DNA replication X-structure by NEIL3, a key step in the interstrand cross-link repair.

Structural and biochemical insights into NEIL2’s preference for abasic sites

“…The MAP processing resulting from endogenous E. coli expression is sufficient to cleave Met1 in most DNA glycosylases, but NEIL3 necessitates MAP supplementation in order to obtain an active enzyme ( 43 , 44 ). This finding is congruent with the very long time course required for the chemical crosslinking reaction to reach completion when the N-terminal methionine is still present ( 42 ).…”

“…Zoom panels show NEIL1 orthologs have two void-filling loops, including one with a residue (R118) to stabilize the intrahelical orphaned base whereas NEIL2 has the shorter Asn182 containing loop. ( C ) Comparison to NEIL3 (orange) in complex with AP-site trapped ssDNA hairpin (PDBID 7Z5A ( 42 )). Asp133 is equivalent to Asn182 in NEIL2 and was proposed to contribute to ssDNA requirement of NEIL3 via charge repulsion.…”

“…A comparison to the structure of NEIL3 covalently trapped to an AP-site shows that the truncated second void-filling loop harbors an aspartate (Asp133) in the position analogous to Asn182 in NEIL2 (Figure 6C ) ( 42 ). The Asp133 position in human NEIL3 is one of two acidic residue substitutions that were proposed earlier to contribute to its strict ssDNA preference through electrostatic repulsion with the DNA phosphate backbone ( 40 ).…”

“…The Asp133 position in human NEIL3 is one of two acidic residue substitutions that were proposed earlier to contribute to its strict ssDNA preference through electrostatic repulsion with the DNA phosphate backbone ( 40 ). While the NEIL3 complex structure provides global insight into the general orientation of the DNA interacting with the enzyme, it is important to note that the nature of the cross-linked complex is likely artifactual, as the chemical trapping reaction to the AP-site involved the N-terminal Met1 ( 42 ). Fpg/Nei glycosylases require post-translational processing by methionine aminopeptidase (MAP) to remove the N-terminal methionine, making the N-terminus, and active site nucleophile, residue number 2 (Pro2 in NEIL1 and NEIL2; Val2 in NEIL3).…”

Biological Functions of the DNA Glycosylase NEIL3 and Its Role in Disease Progression Including Cancer

“…In 2009, Krokeide et al first expressed and purified the human NEIL3 protein [ 4 ]. In contrast to other members of the Fpg/Nei family of proteins, they found that the N-terminal end of the human NEIL3 protein has an integral Fpg/Nei-like core protein structural domain (GD), consisting mainly of the N-terminal domain, a helix-2-turn-helix (H2TH) motif and a zinc finger DNA-binding motif [ 5 , 27 , 28 ]. Additionally, in the N-terminal structural domain of NEIL3, valine replaces the central catalytic role of proline; it also has been shown that activation of the glycosylase activity of NEIL3 requires the removal of the N-terminal methionine [ 5 ].…”