Figure 5. CARM1 slows replication forks through its interaction with PARP1.

(A) A schematic representation of the CARM1 variants tested in this study. (N-term) N-terminal region; (C-term) C-terminal region; (WT) CARM1^WT; (R168A) CARM1^R168A. (B) CARM1-SFB or vector-SFB was transfected into HEK293T cells. Two days after transfection, cells were treated with 10 μM PARPi (Olaparib) for 2.5 h. SFB-tagged CARM1 variants were immunoprecipitated using streptavidin magnetic beads (M280). Levels of the indicated protein were analyzed by Western blot. (C) HEK293T cells were transfected with plasmids expressing SFB-tagged CARM1^WT (WT) or CARM1^B/C for 48 h. Cells were treated with 3 mM HU for 120 min and then released in EdU for 30 min. Proteins at restarting forks were captured by iPOND, and levels of indicated proteins were analyzed by Western blot. (D) MCF10A cells that inducibly express CARM1^WT-HA, CARM1^B-HA, or CARM1^C-HA were transfected with CARM1 siRNA for 48 h. One day after transfection, cells were treated with 1000 ng/mL, 25 ng/mL or 40 ng/mL doxycycline for 24 h to induce expression of CARM1^WT-HA, CARM1^B-HA, or CARM1^C-HA. Levels of the indicated proteins were analyzed by Western blot. (E) Cells described in (D) were analyzed by DNA fiber assay as indicated. The length of replication tracts (n=200) was measured (left panel). Black bars indicate the mean tract length. Mean tract length was determined from three independent experiments (n=3, right panel). Statistical significance was determined by Mann-Whitney test: (****) P<0.0001; (ns) not significant.