Developmental Regulation of the Tetrahymena thermophila Origin Recognition Complex
Figure 2
Abrogated intra-S phase checkpoint response in ORC1 knockdown cells.
(A) Elutriated G1 phase wild type (CU428) and ORC1 knockdown (ORC1-KD) cells were treated with 20 mM HU and samples were collected at the indicated intervals for flow cytometry analysis. (B) G1 synchronized cells were released into fresh medium containing 20 mM HU or 0.06% MMS +/− 1 mM caffeine (1 mM) for 4 h. Whole cell lysates were subjected to western blot analysis of Rad51p. (C) G1 synchronized cells were incubated in medium containing 20 mM HU. Whole cell lysates were prepared at timed intervals and subjected to western blot analysis with anti-Rad51 antibody. (D) G1 synchronized cells were incubated in the presence of HU (1–20 mM) for 4 h and subjected to western blot analysis. (E) Alkaline gel electrophoresis of nascent DNA strands accumulated under HU treatment. G1 synchronized cells were cultured in 20 mM HU and genomic DNA was isolated at indicated time points. RIs were released under alkaline condition and resolved in a 1% alkaline agarose gel. RIs from the rDNA 5′ NTS origin region were visualized by Southern blot analysis.