Luis J. García-Rodríguez; Giacomo De Piccoli; Vanessa Marchesi; Richard C. Jones; Ricky D. Edmondson; Karim Labib

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Oxford Journals

Institution: University of Warwick
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Oxford Journals
Science & Mathematics
Nucleic Acids Research

A conserved Polϵ binding module in Ctf18-RFC is required for S-phase checkpoint activation downstream of Mec1

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Figure 5.

Disruption of the Pol ϵ binding module of Ctf18-RFC blocks activation of the S-phase checkpoint, downstream of Mec1. (A) Control cells (W303–1a), ctf18–2A (YLG249) and ctf18Δ cells (YVM164) were arrested in G1-phase at 24°C and then released into fresh medium containing 0.2M hydroxyurea for the indicated times. Rad53 hyperphosphorylation was monitored by immunoblotting (upper panels), and the data from three such experiments were then quantified (lower panels; ‘% Rad53 phosphorylation’ was calculated from the ratio of hyperphosphorylated to hypophosphorylated Rad53). (B) Control cells (YLG426), ctf18–2A (YLG423), ctf18Δ (YLG421) and mrc1Δ (YGDP993) were arrested in G1-phase at 24°C, before release for 90 min into S-phase in the presence of 0.2M hydroxyurea. Mcm4–5FLAG was immunoprecipitated from cell extracts and the indicated proteins monitored by immunoblotting.