Xenopus origin recognition complex (ORC) initiates DNA replication preferentially at sequences targeted by Schizosaccharomyces pombe ORC

Abstract

Budding yeast (Saccharomyces cerevisiae) origin recognition complex (ORC) requires ATP to bind specific DNA sequences, whereas fission yeast (Schizosaccharomyces pombe) ORC binds to specific, asymmetric A:T-rich sites within replication origins, independently of ATP, and frog (Xenopus laevis) ORC seems to bind DNA non-specifically. Here we show that despite these differences, ORCs are functionally conserved. Firstly, SpOrc1, SpOrc4 and SpOrc5, like those from other eukaryotes, bound ATP and exhibited ATPase activity, suggesting that ATP is required for pre-replication complex (pre-RC) assembly rather than origin specificity. Secondly, SpOrc4, which is solely responsible for binding SpORC to DNA, inhibited up to 70% of XlORC-dependent DNA replication in Xenopus egg extract by preventing XlORC from binding to chromatin and assembling pre-RCs. Chromatin-bound SpOrc4 was located at AT-rich sequences. XlORC in egg extract bound preferentially to asymmetric A:T-sequences in either bare DNA or in sperm chromatin, and it recruited XlCdc6 and XlMcm proteins to these sequences. These results reveal that XlORC initiates DNA replication preferentially at the same or similar sites to those targeted in S.pombe.

Fig. 1. Schizosaccharomyces pombe Orc1, Orc4 and Orc5-bound ATP. Purified ORC-5 complex containing Orc1, 2, 3, 5 and 6 (A) and purified SpOrc4 (B) was subjected to a UV-crosslinking (+UV) in the presence of [α-³²P]ATP. Only Orc1, Orc4 and Orc5 were radiolabeled. ‘Proteins’ were stained with silver. In gels stained with Coomassie Blue (data not shown), the ratio of subunits in the ORC-5 complex was equimolar.

Fig. 2. Schizosaccharomyces pombe Orc proteins exhibited ATPase activity. ORC-5 complex was fractionated by sedimentation through a neutral glycerol gradient. (A) Aliquots from individual fractions were assayed for protein composition by SDS gel electrophoresis followed by silver staining. Molecular weight standards were fractionated in parallel (BSA, 65 kDa; catalase, 250 kDa; thyroglobulin, 700 kDa). (B) Aliquots were assayed for ATPase activity by their ability to release ³²P_i from [γ-³²P]ATP (1 h time point). ‘Minus ORC-5’ was a control without the ORC-5 complex. (C) Ratio of ATPase activity to Orc1 plus Orc5 protein was calculated from densitometry data (SEM was ±10%). (D) SpOrc4 (50 ng) or ORC-5 complex (40 ng) was mixed with 1 µl [γ-³²P]ATP either in the presence or absence of 500 ng S.pombe ARS3002 900 bp DNA fragment (Kong and DePamphilis, 2001) or 500 ng poly(dG-dC)·(dC-dG) and assayed for ATPase activity.

Fig. 3. SpOrc4 inhibited XlORC-dependent DNA replication. (A) The amount of XlOrc2 protein in Xenopus egg extract was determined by fractionating extract by SDS gel electrophoresis in parallel with purified XlOrc2 protein and then immunoblotting with anti-XlOrc2 antiserum. Proteins were identified by their size and immunospecificity. (B) DNA synthesis in Xenopus sperm chromatin was measured in Xenopus egg extract diluted 10-fold with XlOrc2-depleted extract. SpOrc4 (125 kDa) was added to extract (0, 16, 32 and 64 ng, respectively) containing 4.5 ng XlOrc2 (65 kDa), to give molar ratios of SpOrc4/XlOrc2 of 0 (filled square), 1.8 (open square), 3.7 (open circle) and 7.4 (open triangle). Sperm chromatin was then added, and the mixture incubated for the times indicated before measuring acid-insoluble [³²P]DNA. In the absence of SpOrc4, 20–30% of template DNA was replicated by 90 min of incubation (+XlOrc2). DNA replication was not detected either in XlOrc2-depleted extract (open diamond) or in XlOrc2- depleted extract supplemented with SpORC (filled diamond). (C) Percent inhibition was calculated from the 90 min data in (B). Dashed lines indicate 40–70% inhibition. (D) SpOrc4 was mixed either with an equimolar amount of ORC-5 complex, or with high-speed supernatant from Xenopus egg extract (molar ratio SpOrc4/XlOrc2 = 1). Immunoprecipitation (IP) was then carried out with anti-SpOrc4 antibodies crosslinked to Protein A–agarose beads, and the proteins in the IP fractionated by SDS gel electrophoresis and detected by immunoblotting (Harlow and Lane, 1999). Lanes labeled ‘extract’ and SpOrc4 were the starting materials.

Fig. 4. Pre-RCs that were assembled prior to addition of SpOrc4 were not disrupted. (A) Sperm chromatin was incubated in egg extract either in the absence (filled square) or in the presence of SpOrc4 (open symbols). SpOrc4 was added at 0 (open circle), 10 (open triangle), 30 (open square) or 50 (open diamond) min after sperm chromatin. (B) Xenopus sperm chromatin was first incubated with the high-speed supernatant from an egg extract for 45 min. The sample was then divided into three portions. One portion was placed on ice (0′). One portion was incubated for an additional 10 min (10′). One portion was supplemented with SpOrc4 at a molar ratio with XlOrc2 of 3.7 and then incubated for an additional 10 min. Chromatin was isolated from each sample, fractionated by SDS gel electrophoresis, and the amounts of XlOrc2 and XlCdc6 determined by immunoblotting.

Fig. 5. Inhibition of Xenopus ORC-dependent DNA synthesis was dependent on sequence-specific binding of SpOrc4. Either SpOrc4, SpOrc4-C or MmTead2 was added to Xenopus egg extract at the indicated molar ratio to XlOrc2, and the mixture incubated with sperm chromatin for 90 min. SpOrc4-C lacks the AT-hook domains. MmTead2 binds to a different sequence than does SpOrc4.

Fig. 6. SpOrc4 specifically inhibited binding of XlORC to sperm chromatin and assembly of pre-RCs. Sperm chromatin was incubated in egg extract supplemented with the indicated amounts of SpOrc4, SpOrc4-C or MmTead2, as previously described for measuring DNA synthesis. After 20 min, chromatin was isolated and chromatin-bound proteins were fractionated by SDS–PAGE. (A) Proteins were stained with Coomassie Blue. (B–D) The indicated protein was detected by immunoblotting with the appropriate antibody. Antibody prepared against SpOrc4-C detected SpOrc4-C in western blots.

Fig. 7. SpOrc4 and XlORC bound preferentially to asymmetric A:T-sequences. (A) Xenopus egg extract supplemented with SpOrc4 at a molar ratio with XlOrc2 of 3.7 (Figure 6) was incubated with sperm chromatin for 10 min. The reaction was divided into eight portions. Chromatin was isolated from each fraction by sedimentation and eluted with the indicated amount of KCl. For comparison, purified SpOrc4 was adsorbed to the indicated DNA resin in 40 mM KCl, and then eluted sequentially with one column volume Buffer A containing the indicated concentration of KCl. Aliquots of each fraction (including the original sample) were subjected to SDS–PAGE and immunoblotted for the indicated protein. AA/TT is poly(dA)·poly(dT) cellulose. AT/TA is poly(dA-dT)·poly(dT-dA) cellulose. GC/CG is poly(dG-dC)·poly(dC- dG) cellulose. (B) Cdc6-depleted Xenopus egg extract was incubated with sperm chromatin for 10 min, and the proteins eluted sequentially with the indicated KCl concentration. Fractions of XlOrc2 eluted with low salt concentrations (a) and with high salt concentrations (b) are indicated. For comparison, complete Xenopus egg extract was diluted with Buffer A, adsorbed to the indicated DNA resin, and analyzed as in (A). Proteins were identified by their size and immunospecificity. In XlCdc6 assays, the upper band was XlOrc2 and the lower was XlCdc6. The two antibodies were applied sequentially to the same membrane.