IntroductionAdenosine triphosphate (ATP)-dependent chromatin remodeling (nucleosome remodeling) complexes regulate and control nucleosome positioning for transcription, DNA replication, and DNA repair. These complexes are categorized into the switch/sucrose non-fermentable (SWI/SNF), imitation switch (ISWI), INO80, and chromatin helicase DNA-binding (CHD) families according to the specific ATPase present1. Recent studies have reported various cancer cells to exhibit extremely high frequencies of mutations in the genes involved in chromatin remodeling2,3. The tumor-suppressive activity of remodeling factors can be attributed to their role in transcriptional regulation1. Furthermore, we and other groups have reported that the SWI/SNF- and ISWI-family chromatin-remodeling complexes are essential for non-homologous end joining (NHEJ) and homologous recombination (HR) in the occurrence of DNA double-strand break (DSB) repair, and this involvement in DSB repair has also been suggested to contribute to tumor suppression2,4,5,6,7,8.Five different ISWI-family complexes harboring SNF2H as the catalytic subunit have been identified in human cells: ATP-dependent chromatin assembly and remodeling factor (ACF), nucleolar remodeling complex (NoRC), RSF, WSTF-ISWI chromatin-remodeling complex (WICH), and chromatin assembly complex (CHRAC)9. The ISWI-family complexes remodel chromatin in order to control various cellular processes including transcription, DNA replication, and DNA repair1. It is unclear how ISWI moves nucleosomes during cellular processes, including for DNA repair, although two different chromatin-remodeling activities have been detected for this complex in vitro; namely, nucleosome assembly and nucleosome sliding1,9,10,11. However, the remodeling activities required for each cellular function, including DSB repair, are poorly understood.One protein of the CHRAC family, CHRAC17 (an H2B-type histone-fold protein) is also included the DNA polymerase ε complex (Pol ε) family, with the alternative name of POLE3. In vitro, CHRAC17 has been shown to form a complex with CHRAC15—an H2A-type histone-fold protein—to form an H2A/H2B-type histone-fold protein complex, which promotes the nucleosome-binding activity and remodeling activities of ACF1-SNF2H10,11. In Polε, CHRAC17 forms a histone chaperone complex with POLE4, an H2A-type histone-fold protein, which then combines with POLE1 and POLE2 to form the Pol ε complex12,13. It remains unclear whether CHRAC17 coordinates the functions of CHRAC and Pol ε in the same cellular processes, including DSB repair. Furthermore, the role of Pol ε in DSB repair remains elusive in humans and is poorly understood even in yeast12. This study elucidates the mechanism of HR—which plays an important role in genome stability—by evaluating the role of CHRAC and the Pol ε complex in DSB repair.ResultsCHRAC accumulates at DNA DSB sites via CHRAC17In the context of cellular functions, including DSB repair, the functional relationship between the CHRAC15-17 and SNF2H-ACF1 catalytic subunit complex is poorly understood. It can be expected that CHRAC17 would accumulate at DSB sites because ACF1 binds the CHRAC15-17 complex via direct interaction with CHRAC1710,11. We previously demonstrated that SNF2H-ACF1 catalytic subunits accumulate at DSB sites via ACF15. Similar to one of the catalytic subunits, GFP-ACF1 (Fig. 1a, lower) GFP-CHRAC17 (Fig. 1a, upper) accumulates at DSB sites immediately after induction of DSBs by laser micro-irradiation. On the other hand, GFP-CHRAC15 was weakly recruited to laser micro-irradiated DSB sites (Supplementary Fig. S1a).Fig. 1: Chromatin assembly complex is recruited to DNA double-strand break sites via interactions between CHRAC17 and γH2AX under the control of ATM signaling.Full size imagea Green fluorescent protein (GFP)-chromatin assembly complex protein 17 (CHRAC17) accumulated at double-strand break (DSB) sites immediately after induction of DSB by laser micro-irradiation in H1299 cells. After laser micro-irradiation, cells were incubated for 10 min and immunostained with anti-γH2AX. b Results of chromatin immunoprecipitation (ChIP) analysis with immunoglobulin G (control) or antibodies against γH2AX, or CHRAC17 performed 0 or 12 h after transfection of the I-SceI expression plasmid into dA3-1 H1299 cells. The relative enrichment of proteins at each time point is presented, showing CHRAC17 recruitment at DSB sites. c Accumulation of GFP-CHRAC17 at DSB sites was not affected by knockdown of ATP-dependent chromatin assembly and remodeling factor 1 (ACF1). H1299 cells expressing GFP-CHRAC17 were laser micro-irradiated and subjected to real-time recording of protein accumulation at laser-irradiated sites. (left) Quantification of real-time recording of signal intensity on laser-micro-irradiated sites. (right) Intensity of GFP-CHRAC17 at 200 s after laser micro-irradiation. Data are presented as mean ± standard deviation of two independent biological replicates. d Knockdown of CHRAC17 reduced the accumulation of GFP-ACF1 at DSB sites. H1299 cells expressing GFP-ACF1 were subjected to laser micro-irradiation and real-time recording of protein accumulation at laser-micro-irradiated sites. (left) Quantification of real-time recording of signal intensity on laser-micro-irradiated sites by FLUOVIEW software (Olympus). (right) Intensity of GFP-ACF1 at 200 s after laser micro-irradiation. Data are presented as mean ± standard deviation of two independent biological replicates. e Knockdown efficiency of CHRAC17 is shown on a western blot. f Schematic diagrams of CHRAC17-deletion mutants. g The recruitment of CHRAC17 deletion mutants at DSB sites. H1299 cells expressing CHRAC17 deletion mutants were subjected to laser micro-irradiation and real-time recording of protein accumulation at laser-micro-irradiated sites. (Upper) Representative images. (bottom) Quantification of real-time recording of signal intensity on laser-micro-irradiated sites by FLUOVIEW software (Olympus). Intensity of GFP-CHRAC17 at 200 s after laser micro-irradiation. Data are presented as mean ± standard deviation of two independent biological replicates. h ATM signaling promotes CHRAC17 recruitment to DSB sites. H1299 cells expressing GFP-CHRAC17 were subjected to laser micro-irradiation and real-time recording of protein accumulation at laser-micro-irradiated sites. Quantification of real-time recording of signal intensity on laser-micro-irradiated sites by FLUOVIEW software (Olympus). Intensity of GFP-CHRAC17 at 60 s after laser micro-irradiation. All statistical analyses were performed using GraphPad Prism, with significance defined as P