Catalytic and Structural Insights into Neil3-Dependent Unhooking of Endogenous Abasic DNA Crosslink

Abasic (Ap) sites arise frequently in genomic DNA and can form interstrand crosslinks (Ap-ICLs) that block DNA replication and threaten genome stability. The DNA glycosylase NEIL3 is required for replication-coupled repair of Ap-ICLs, yet its catalytic mechanism has remained unclear, as biochemical studies report lyase-dependent strand cleavage whereas cellular systems indicate incision-free unhooking. Here, we show that the catalytic outcome of NEIL3 is determined by the N-terminal processing of its NEI domain. Using biochemically and structurally defined NEI variants, we demonstrate that a native-like processed form (V2M), in which valine 2 is replaced by an initiating methionine, efficiently unhooks Ap-ICLs by releasing the crosslinked strand without generating toxic DNA strand breaks, and without {beta}- or {delta}-elimination. In contrast, an unprocessed form (M1) exhibits elevated Ap-lyase activity and generates strand breaks. Time-resolved Schiff-base trapping in the presence of a reducing agent reveals distinct high-molecular-weight intermediates during Ap-ICL unhooking. A crystal structure of NEIL3 bound to native-like substrate in form of a single-stranded DNA identifies features underlying its preference for fork-like substrates. Together, these findings reconcile previously conflicting models of NEIL3 function and define a mechanistic framework for replication-coupled repair of endogenous crosslinks, Ap-ICL, preserving fork integrity.