The C-terminus of Nej1 is critical for nuclear localization and non-homologous end-joining

•Nej1-CTR in essential for NHEJ-mediated DNA repair.•Residues K331–K334 serve as a nuclear localization signal in Nej1.•F335 is critical for interactions with Lif1 and NHEJ.

Nej1 is an essential factor in the non-homologous end-joining (NHEJ) pathway and interacts with the DNA ligase complex, Lif1-Dnl4, through interactions with Lif1. We have mapped K331-V338 in the C-terminal region of Nej1 to be critical for its functionality during repair. Truncation and alanine scanning mutagenesis have been used to identify a motif in Nej1, KKRK (331–334), which is important for both nuclear targeting and NHEJ repair after localization. We have identified F335-V338 to be important for proper interaction with Lif1, however this region is not required for Nej1 recruitment to HO endonuclease-induced DNA double-strand breaks in vivo. Phenylalanine at position 335 is particularly important for the role of Nej1 in repair and the loss of association between Nej1 and Lif1 correlates with a decrease in cell survival upon either transient or continuous HO expression in nej1 mutants.