Structural architecture of NEIL3 glycosylase in abasic site DNA repair

Hušková Andrea, Bouřa Evžen, Šilhán Jan

Institute of Organic Chemistry and Biochemistry of the Czech Academy of Science, Flemingovo náměstí 542/2, Praha 6, 16610

DNA interstrand cross-links (ICLs) are very toxic DNA damage lesions covalently connecting the opposite strands. They form impenetrable barriers for the replication machinery during cell division. Abasic site ICLs (Ap-ICLs) are formed spontaneously when an abasic (Ap) site forms a covalent bond with a base located in the opposite DNA strand. This recruits NEIL3 glycosylase, an enzyme responsible for the removal of the Ap-ICLs, however, the molecular mechanism of this repair remains elusive [1,2].

NEIL3 contains three sets of zinc finger domains. First, located on the N-terminus is a catalytic Nei domain removing the Ap-ICL. Second, the ubiquitin-binding (NZF) domain contains a ubiquitin-binding zinc finger. Last, two adjacent Gly-Arg-Phe (GRF) domains are known for their ability to bind single-stranded DNA, but their role in the ICL repair is unclear [3].

We have investigated the recognition of a stalled DNA replication fork by NEIL3. We have solved the NMR structure of GRFs and shown how they recognise single-stranded DNA. We have further revealed how GRFs bind DNA forks with a slight preference for 5’-DNA overhangs. Along with GRFs inter-domain rigidity, our data outline how GRF recognises DNA forks suggesting their role in Ap-ICL repair.

Next, we have solved a crystal structure of mouse NEIL3 Nei domain in complex with the DNA reaction intermediate. With Nei‘s preference for a 3’-DNA overhang, we have outlined the interplay between Nei and GRFs in Ap-ICL repair. Our results suggest how NEIL3 recognises the structure of two collided replication forks, a DNA replication X-structure.


1. Price, N.E., Johnson, K.M., Wang, J., Fekry, M.I., Wang, Y., and Gates, K.S. (2014). Interstrand DNA–DNA Cross-Link Formation Between Adenine Residues and Abasic Sites in Duplex DNA. J. Am. Chem. Soc. 136, 3483–3490.

2. Zhang J, Walter JC. Mechanism and regulation of incisions during DNA interstrand cross-link repair. DNA Repair (Amst). 2014 Jul;19:135-42

3. Semlow, D.R., Zhang, J., Budzowska, M., Drohat, A.C., and Walter, J.C. (2016). Replication-Dependent Unhooking of DNA Interstrand Cross-Links by the NEIL3 Glycosylase. Cell 167, 498-511.e14