Mock Orc2. NPE sperm pre-rc replication

Átírás

1 Time (min): HSS + M Orc2 HSS NPE sperm pre-rc repliction c % Repliction HSS + sp NPE Orc2 Fig. S1: M13 ssdna-induced destruction of does not require ORC. S1: M13 ssdna ws incuted in mock- or Orc2-depleted HSS nd rections were nlyzed s in Figure 2., cross-recting nd serving s loding control. S1: Schemtic of DNA repliction in the solule, nucleus-free system 1. Sperm chromtin is incuted in HSS to form pre-rcs, followed y ddition of highly concentrted nucleoplsmic extrct (NPE) which supports origin firing from pre-rcs ssemled in HSS. S1c: As ORC is not required for ssdna repliction 2, we tested whether ORC ws functionlly depleted from HSS y testing the ility of the mock- nd Orc2-depleted HSS from Fig. S1 to support sperm chromtin repliction. HSS ws incuted with sperm chromtin (3,000/μL finl) for 30, followed y ddition of NPE. The percentge of input DNA replicted fter 60 is plotted. 1. Wlter, J., Sun, L. & Newport, J. Regulted chromosoml DNA repliction in the sence of nucleus. Mol Cell 1, (1998). 2. Crpenter, P. B., Mueller, P. R. & Dunphy, W. G. Role for Xenopus Orc2- relted protein in controlling DNA repliction. Nture 379, (1996). Wlter_Supplementry Figure S Nture Pulishing Group

2 % loded: totl chrom U + uffer + p27 Kip totl sol. totl sol extrct d % loded: 30 totl chrom c + uffer + p27 Kip 115 PCNA U RCC chromtin e PCNA His-Cdc45 Cdc45 rpcna (ng) chromtin His-Cdc45 (ng) molr rtio Rtio of PCNA trimer: Cdc chromtin Wlter_Supplementry Figure S Nture Pulishing Group

3 Figure S2: is uiquitinted predominntly on chromtin. S2, d: Sperm chromtin ws incuted in LSS for 30 with 1 μm p27 Kip, to prevent repliction initition (), or with no inhiitor (d), isolted, nd lotted. 100% loding is the equivlent of 4 μl of extrct (lnes 1-4) or of the chromtin-ound protein from 4 μl of extrct (lne 5). S2: Repliction ws initited in the nucleus free-repliction system 1 y incution of sperm chromtin (3,000/μL finl) in HSS followed y ddition of NPE in the presence of 250 μm methylted uiquitin nd 70 nm r WT in the presence (lnes 3-4) or sence (lnes 1-2) of 1 μm p27 Kip for 20 minutes. Smples were withdrwn efore (lnes 1 nd 3) or immeditely fter (lnes 2 nd 4) centrifugtion to remove the chromtin nd lotted for., cross-recting nd. Arrows indicte uiquitinted forms of. S2c: The chromtin removed from smples 1 nd 3 in () ws lotted s indicted. S2e: Sperm chromtin ws incuted in LSS for the indicted times nd lotted next to the indicted mounts of rpcna or his-tgged rcdc45. The mount of PCNA nd Cdc45 on chromtin ws clculted t ech time point y densitometry, nd the molr rtio of PCNA trimers to Cdc45 is plotted. The experiment ws performed 4 times, with representtive dt set shown. 1. Wlter, J., Sun, L. & Newport, J. Regulted chromosoml DNA repliction in the sence of nucleus. Mol Cell 1, (1998). Wlter_Supplementry Figure S Nture Pulishing Group

4 WB: PI Imm % lod: DDB1 5 DDB XDDB Fig. S3: Chrcteriztion of -XDDB1 serum. S3: 0.25 ng of XDDB ws proed with 1:2,000 dilution of pre-immune (PI) or immune serum (Imm). Imges re of 10-second film exposure. S3: DDB1 depletion from LSS. - or DDB1-depleted LSS ws lotted for DDB1. 100% lod equls 1 μl LSS., cross-recting nd. Wlter_Supplementry Figure S Nture Pulishing Group

5 Time (min): control + MG132 + phidicolin + cffeine + p27 Kip Fig. S4: Shown re the full Western lots from Figure 2A spnning region of ~50-90 kd. The position of in ech pnel is indicted. Wlter_Supplementry Figure S Nture Pulishing Group