Skip to main content
. 2021 Mar 25;9:635383. doi: 10.3389/fcell.2021.635383

FIGURE 2.

FIGURE 2

Ddc2-Rad9 fusions activate Rad53 in meiosis. (A) TCA-precipitated meiotic cell extracts of the indicated strains were analyzed by western blotting using anti-HA, anti-Flag, anti-H3K79-3me (tri-methylation), anti-Dmc1, and anti–α-tubulin antibodies.32P-Rad53 represented 32P-incorporation to Rad53 in the ISA assay, and was quantified as shown Figure 1A (fifth panels). Cell extracts derived from 4 × 106 cells were loaded for the ISA assay. Wild-type (USY543/544), DDC2-RAD9 (USY544/661), DDC2-RAD9 spo11 (USY783/414), and DDC2-RAD9 rad53-KD (USY545/720) were examined. (B) Cell extracts were prepared from the strains expressing C-terminal 3x HA-tagged Ddc2 (USY83/84; DDC2-HA) and N-terminal 3x HA-tagged Rad9 (USY20/35; HA-RAD9) from their native promoters; Ddc2-Rad9 (USY544/671; DDC2-RAD9) and 3x HA-tagged Rad9 (USY544/661; OE-HA-RAD9) from the DMC1 promoter; no HA-tagged wild-type strain (USY543/544); wild type. Rad53 was not tagged with Flag in DDC2-HA and HA-RAD9 strains. Flag-Rad53 was expressed from the native promoter. (C) Rad53 activation was examined in various strains as in (A). Wild-type (USY543/544), DDC2-RAD9 (USY544/671), DDC2-rad9-Y798A (USY544/776), DDC2-RAD9 dot1 (USY768/526), DDC2-RAD9 h3-K79R (USY770/693), and DDC2-rad9-K1088M (USY544/797) were examined. Results of an independent experiment are shown in Supplementary Figure 2C. (D) Rad53 activation was examined in various strains as in (A). Wild-type (USY543/544), DDC2-RAD9 (USY544/671), DDC2-RAD9 mec1 (USY495/785), DDC2-rad9-7A (USY544/667), and DDC2-RAD9 rad53-KD (USY4520) were examined. Results of an independent experiment are shown in Supplementary Figure 2D.