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. 2021 Jul 9;49(14):8145–8160. doi: 10.1093/nar/gkab585

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© The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 4. — Tpk1 phosphorylates yeast Nej1 at S298. (A, B) Extracts from the Nej1-HA wild-type (WT) and mutant strains harbouring with (+) or without (−) Tpk1 in the presence (+) or absence (−) of HO-induced DSBs were immunoblotted (IB’ed) with anti-HA antibody to detect DNA-damage induced mobility shift of phosphorylated Nej1 (*; A) or with anti-phosphoserine (B) antibody to identify phosphorylated serine. Yeast (Sc) anti-GAPDH (A) or anti-HA (B) were used as a control (Ctrl). (C) Rad53 and γ-H2A foci (n = 200 cells per sample) in BLM-induced WT and tpk1 mutants, with or without mutagenized Nej1, immunostained with anti-Rad53 or anti-γ-H2A antibody. Nuclei stained with DAPI are indicated with dotted lines. Scale bar, 5 μm. (D) Phosphorylated Rad53 (*) and H2A in the indicated WT and tpk1 mutants, with or without Nej1 variant, after 1 hr of recovery from BLM induction was analyzed by probing with anti-Rad53 and anti- γ-H2A antibody, along with yeast (Sc) anti-GAPDH as a control. (E) HO-induced chromosomal (RCF, relative colony formation) DSBs on endogenously expressed Nej1 WT or variants in the presence or absence of tpk1. (F) Accretion of resected ssDNA in the strains with no DNA damage or recovered after HO induction (i.e. 0.3 kb from break site) at varying times points. (G, H) Micrographs (G) and quantification (H; n = 100 cells per sample) of Nej1 and Lif1 nuclear targeting in the BLM-induced Nej1 WT or variants, carrying with or without tpk1, after probing with anti-HA or anti-Lif1 antibody. DNA stained with DAPI. Scale bar, 5 μm. Data for E and F are mean ± SD (n = 3 biological replicates; *P ≤ 0.05 by Student's t test). Molecular masses (kDa) of marker proteins are indicated in panels A, B and E.