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. 2016 Jan 25;7:10393. doi: 10.1038/ncomms10393

Figure 7. Sgo2 regulates replication timing at the subtelomeres by limiting Sld3 loading.

Figure 7

(a) Replication kinetics of the early and late origins in wt and sgo2Δ cells. The locations of the origins on chromosome 2 are shown schematically (top). ars2004 is an early origin and AT2088 is a late origin. tel-21.2 is located at 21.2 kb from tel2R. The non-origin region (non-ori) is located at 30 kb from ars2004. Temperature-sensitive cdc25-22 mutant cells expressing thymidine kinase and the nucleotide transporter were arrested at the G2/M boundary at the restrictive temperature (36 °C) and then released from arrest by a temperature shift to 25 °C in the presence of BrdU (200 μM). At the indicated time points, the amount of HL DNA and LL DNA, which were separated by caesium chloride (CsCl) density gradient centrifugation, was determined by qPCR (middle and bottom). (b) Replication of the subtelomeric late origins is affected in sgo2Δ cells. The strains used in a were released from G2/M arrest and their DNA was labelled with BrdU for 90 min in the presence of hydroxyurea (HU; 10 mM). The BrdU-labelled DNA was purified by immunoprecipitation and quantified by quantitative PCR. Each value was normalized to that of ars2004. Error bars indicate the s.d. (n=3). Asterisks indicate a significant change as compared with the wt strain (P<0.05, two-tailed t-test). (c) Replication of sgo2Δ, bub1-KD (kinase-dead) and h2a-S121 mutants in the presence of HU. The experiments were performed as in b. Error bars indicate the s.d. (left, n=4; right, n=3). Asterisks indicate a significant change as compared with the wt strain (P<0.05, two-tailed t-test). (d) Recruitment of Mcm6 and Sld3 to the replication origins. cdc25-22 nda4-108 mutant cells were released from G2/M arrest and then incubated for 180 min at 20 °C. ChIP analyses using anti-Mcm6 and anti-Flag (for Sld3-Flag) antibodies were performed. Each value was normalized to that of non-ori. Error bars indicate the s.d. (n=3). Asterisks indicate a significant change as compared with the wt strain (P<0.05, two-tailed t-test).