The coordination of DNA unwinding and synthesis at replication forks promotes efficient and faithful replication of chromosomal DNA. Disruption of the balance between helicase and polymerase activities during replication stress leads to fork progression defects and activation of the Rad53 checkpoint kinase, which is essential for the functional maintenance of stalled replication forks. The mechanism of Rad53-dependent fork stabilization is not known. Using reconstituted budding yeast replisomes, we show that mutational inactivation of the leading strand DNA polymerase, Pol ε, dNTP depletion, and chemical inhibition of DNA polymerases cause excessive DNA unwinding by the replicative DNA helicase, CMG, demonstrating that budding yeast replisomes lack intrinsic mechanisms that control helicase–polymerase coupling at the fork. Importantly, we find that the Rad53 kinase restricts excessive DNA unwinding at replication forks by limiting CMG helicase activity, suggesting a mechanism for fork stabilization by the replication checkpoint.
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This work was supported by NIH grant no. R01GM107239. We thank X. Zhao for yeast strains.
The authors declare no competing interests.
Peer review information Beth Moorefield was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
a, Standard replication reactions were performed at various final salt concentrations as indicated. Template: pARS305. b, Titration of Pol δ into standard replication reactions. Template: pARS1. Reaction products were analyzed by 0.8 % alkaline agarose gel-electrophoresis and autoradiography. Uncropped gel images are available as source data.
Leading strands are in red, lagging strands in blue. ERI: early replication intermediate; LRI: Late replication intermediate.
a, Standard replication reactions with pARS1 were carried out in the absence or presence of Top1 or Top2, as indicated. Reactions were stopped 60 minutes after origin activation, and replication products analyzed by native agarose gel-electrophoresis and autoradiography after linearization with the unique cutter Nde I, which cuts near the origin (fully replicated DNA molecules will resolve into linear monomers after linearization, whereas replication products containing unreplicated regions will resolve into double Y-shaped intermediates. Representative gel is shown on the left. The bar diagram depicts the average dissolution efficiency (fraction of linear full-length molecules per reaction) and s.d. of three independent experiments. b, Replication products from experiment in Fig. 1b were analyzed by alkaline agarose gel-electrophoresis and autoradiography. c, Pulse-chase experiment demonstrating that the ERI is a precursor of the LRI. Replisomes were formed on chromatin templates in the presence of α-32P-dCTP and stalled by omission of topoisomerase from the reaction (lane 1). After 15 minutes, Top1 was added to the reaction to release the stalled replisomes and intermediates chased by simultaneous addition of excess cold dCTP. At the indicated time points (lanes 2 and 3) replication products were isolated and analyzed by native (left) or denaturing agarose gel-electrophoresis and autoradiography. d, Standard replication reactions carried out in the presence of either Top1, Top2, or both. Replication products were analyzed by native agarose gel-electrophoresis and autoradiography. Uncropped gel images and data for graph in panel a are available as source data.
Extended Data Fig. 4 Reduced DNA synthesis and excess DNA unwinding in the presence of Pol ε polymerase mutants.
a, Total relative DNA synthesis in reactions of Fig. 1e were measured using ImageJ and plotted over time. b, Standard DNA replication reactions were carried out in the presence of wild-type or the indicated Pol ε variants (60 nM). 45 minutes after origin activation reactions were stopped and replication products analyzed by alkaline (left) or native (right) agarose gel-electrophoresis and autoradiography. Template: pARS1. c, Model for formation of θ and U* replication intermediates during plasmid replication in vitro. In normal DNA replication, the origin is initially unwound upon CMG activation (top left), followed shortly thereafter by the commencement of DNA synthesis and the coupling of leading strand synthesis to DNA unwinding by CMG (top center). Compensatory positive supercoils formed in the template during unwinding and fork progression are removed by Top1 and/or Top2. After deproteinization, the resulting θ structure is maintained (top right). In contrast, under conditions that slow-down DNA synthesis after origin unwinding the CMG helicase progresses along the template (bottom left) in advance of DNA synthesis; compensatory positive supercoils generated during DNA unwinding are removed by Top1 and/or Top2, and the unwound single-stranded DNA is stabilized by RPA binding (bottom center). Upon deproteinization, unwound complementary DNA strands reanneal, causing compensatory negative supercoils and thus resulting in a partially replicated, negatively supercoiled replication intermediate, U* (bottom right). Data for graph in panel a and uncropped gel images for b are available as source data.
Pol εwt was titrated into standard replication reactions, reactions stopped 45 minutes after origin activation, and replication products analyzed by denaturing (left) or native (right) agarose gel-electrophoresis and autoradiography as indicated. Template: pARS1. Uncropped gel images are available as source data.
a, Reaction scheme: Singly primed single-stranded M13mp18 DNA was pre-incubated with RPA, RFC/PCNA, three nucleotides, and Pol δ to initiate primer extension; Pol ε was subsequently added along with the remaining fourth nucleotide and incubation continued for 3 minutes. b, Denaturing agarose gel analysis of primer extension products obtained according to reaction scheme in a, but with either Pol δ or Pol ε omitted from the reaction. Uncropped gel images are available as source data.
Extended Data Fig. 7 Excessive DNA unwinding under limiting dNTP conditions in the absence of Csm3-Tof1-Mrc1 and presence of Pol εwt.
Pulse-chase experiment of standard replication reaction performed at 0.25 μM each dNTP as in Fig. 5b, with the following changes: 1) Pol εwt was used instead of Pol εexo-; 2) Csm3-Tof1 and Mrc1 were omitted from the reaction; 3) pARS305 instead of pARS1 served as a template. Time indicates minutes after origin activation. The reaction was chased with 500 μM cold dATP 5 minutes after origin activation. Reaction products were analyzed by agarose gel-electrophoresis and autoradiography. Uncropped gel images are available as source data.
Extended Data Fig. 8 Inhibition of DNA unwinding after CMG uncoupling from DNA synthesis is dependent on the kinase activity of Rad53.
a, Experiment demonstrating that U* DNA obtained in the presence of aphidicolin is negatively supercoiled. DNA isolated from the reaction analyzed in Fig. 6c, lane 5, was either mock-treated (lane 1) or treated with E. coli Topo I (lane 2) and analyzed by native agarose gel-electrophoresis. b, Purified wild-type and kinase-dead Rad53 (Rad53kd). Rad53-P: Autophosphorylated forms of Rad53. c, Effect of Rad53 kinase activity on U* formation after fork release from topological block in the presence of aphidicolin. Reactions were carried out as in Fig. 6c, except that wild-type (lanes 1-5) or kinase-dead (lanes 6-10) Rad53 was added to the reaction prior to fork release. Uncropped gel images are available as source data.
Extended Data Fig. 10 Models for modes of fork progression in the presence of wild-type and catalytically dead Pol ε.
N and C indicate the N- or C-terminal exo-pol domain of Pol2. X indicates inactive mutant N-terminal Pol2 polymerase domain.
Source data for graphs.
Source data for graphs.
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Devbhandari, S., Remus, D. Rad53 limits CMG helicase uncoupling from DNA synthesis at replication forks. Nat Struct Mol Biol 27, 461–471 (2020). https://doi.org/10.1038/s41594-020-0407-7