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 .
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.