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Regulated eukaryotic DNA replication origin firing with purified proteins

Abstract

Eukaryotic cells initiate DNA replication from multiple origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric minichromosome maintenance (MCM) complex is first loaded at replication origins during G1 phase, and then converted to the active CMG (Cdc45–MCM–GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA replication initiation with 16 purified replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo. Cyclin-dependent kinase (CDK) inhibits MCM loading by phosphorylating the origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4-dependent kinase (DDK) promotes replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated eukaryotic DNA replication.

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Figure 1: DDK- and CDK-dependent firing-factor recruitment with purified proteins.
Figure 2: The purified firing factors are functional.
Figure 3: The initiation of DNA synthesis with purified proteins.
Figure 4: Requirements for origin firing.
Figure 5: Regulation of MCM loading and origin firing by S-CDK.
Figure 6: DDK phosphorylation of MCM promotes origin firing either before or after S-CDK.

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Acknowledgements

We are grateful to B. Pfander and M. Douglas for the Mcm10 expression plasmid and advice on purification, C. Kurat for construction of plasmids used to generate ARS1 linear templates, K. On and D. Boos for Sic1 and A-Cdk2 and K. Labib for Psf1 antibodies and the E. coli GINS expression strain. We thank A. Alidoust and N. Patel for growing yeast cultures. This work was supported by Cancer Research UK, a FEBS Return-to-Europe fellowship to J.T.P.Y., a Boehringer Ingelheim Fonds PhD fellowship to A.J. and an ERC grant (249883 – EUKDNAREP) to J.F.X.D.

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Contributions

J.F.X.D. and J.T.P.Y. designed the experiments and wrote the manuscript. J.T.P.Y. performed the experiments. T.D.D., A.J. and A.E. generated protein expression constructs and strains, and established protein purification protocols.

Corresponding author

Correspondence to John F. X. Diffley.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Coomassie-stained SDS–PAGE analysis of multi-subunit complexes required for DNA replication.

a, Annotation of the polyacrylamide gels in Fig. 1a in which individual protein subunits have been labelled. b, Analysis of A-Cdk2. The protein complex consists of human Cdk2 and the bovine cyclin A-3 fragment32.

Extended Data Figure 2 Analysis of firing factor recruitment.

a, Immunoblots of protein recruitment conducted as in Fig. 1b but with 0.3 M KCl washes. b, Stability of recruited firing factors following washes of varying strength (lanes 1 and 5, 0.3 M K-glu; lanes 2 and 6, 0.3 M KCl; lanes 3 and 7, 0.45 M KCl; lanes 4 and 8, 0.6 M KCl). c, Psf2–Flag was depleted from a yJY18 S phase extract by two rounds of incubation with anti-Flag M2 magnetic beads. Levels of Psf2–Flag were determined by immunoblotting with the Flag-M2 antibody. Soluble and bead bound protein fractions are illustrated. d, Extract-based replication reaction schemes. In the left pathway (i), loaded MCM is treated with DDK and added to a KO3 extract (Sld3, Sld7, Cdc45, Dpb11, Sld2 overexpression). In the right pathway (ii), firing factors are recruited to MCM as illustrated in Fig. 1b and the complex is added to a yJY18 extract (no firing factor overexpression) in which Psf2 (GINS complex) has been depleted. e, Replication reactions as described in d using A-Cdk2 for firing factor recruitment. Where indicated, Sic1 was added to the extract 20 min before replication.

Extended Data Figure 3 Characterization of the in vitro DNA replication reaction.

a, b, Replication reactions conducted as in Fig. 3a with A-Cdk2 on ARS1 circular DNA templates for 1 h. c, Time course of a standard replication reaction using A-Cdk2 on the ARS1 linear DNA template. d, Quantitation of a time course conducted as in c. DNA synthesis was normalized to the total DNA synthesis at 90 min. Small and large replication products were not quantified separately at 5 min as they are not well resolved at this time point. e, Pulse chase experiment conducted with A-Cdk2 on the ARS1 linear DNA template. For the pulse the dCTP concentration was reduced to 4 μM. Following a 10 min incubation unlabelled dCTP was added to 100 μM.

Extended Data Figure 4 Characterization of RPA recruitment.

Unless stated reactions were conducted on ARS1 circular templates. a, Vaccinia virus topoisomerase I supports DNA replication with purified proteins. Replication reactions with either Topo II (25 nM) or vaccinia virus topoisomerase I (0.125 units per μl). Two different Topo II fractions (Fr1 and Fr2) were used for comparison. b, Nucleotide dependence of RPA recruitment in a complete replication reaction with Topo II. c, RPA recruitment reactions were conducted on ARS1 circular template in the presence of vaccinia virus topoisomerase I (0.125 units per μl), or on a linear template in the absence of a topoisomerase. dNTPs, C/G/UTP, pol α and Ctf4 were omitted from the final step of the reaction.

Extended Data Figure 5 Regulation of MCM loading and origin firing by S-CDK is ATP dependent.

a, Replication reaction where ORC was pre-incubated with S-CDK before MCM loading. When Sic1 was added before ORC the mix was incubated for 5 min and ORC was then added for 10 min. b, Pre-incubation of ORC with S-CDK in the presence or absence of ATP. After incubation with Sic1, ATP was added to the reaction lacking ATP. c, Sld3/7 and Sld2 were pre-incubated with S-CDK as illustrated in Fig. 5d in the presence or absence of ATP. Following incubation with Sic1, samples that did not contain ATP for the pre-incubation step were supplemented with ATP.

Extended Data Figure 6 Cartoon illustrating protein kinase regulated eukaryotic DNA replication origin firing with purified proteins.

Firing factors are recruited to loaded MCM in a DDK- and CDK-dependent manner. DNA synthesis is initiated once the DNA template has been unwound. CDK also functions to inhibit MCM loading by phosphorylating ORC.

Extended Data Figure 7 Internally Flag-tagged Cdc45 supports normal DNA replication in S phase extracts.

a, The previously reported interaction between Sld3 and Cdc45 (ref. 33) was exploited to co-immunoprecipitate Cdc45 from yJY16 extracts (Dpb11 and Sld2 overexpression) by incubation with Flag–Sld3/7 that was pre-coupled to anti-Flag M2 magnetic beads. b, In vitro extract-based replication reaction on soluble ARS1 circular template using yJY16 extracts where endogenous Cdc45 was depleted as indicated. The extract was supplemented with purified Sld3/7 as the complex is not overexpressed in yJY16. The experiment was conducted for 30 min as described previously6 and products were separated through a 1% native agarose gel. Internally Flag-tagged Cdc45 (52 nM) was added back as indicated. The locations of the different replication products are illustrated.

Extended Data Table 1 Affinity tag strategies for protein purification
Extended Data Table 2 Saccharomyces cerevisiae strains
Extended Data Table 3 Plasmids used to generate yeast expression strains

Supplementary information

Supplementary Information

This file contains the DNA sequences used for expression in S.cerevisiae. (PDF 292 kb)

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Yeeles, J., Deegan, T., Janska, A. et al. Regulated eukaryotic DNA replication origin firing with purified proteins. Nature 519, 431–435 (2015). https://doi.org/10.1038/nature14285

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