Competition for the Nascent Leading Strand Shapes the Requirements for PCNA Loading in the Replisome

Overview

Journal EMBO J

Specialties Biotechnology
Molecular Biology

Date 2025 Feb 28

PMID 40021844

Authors

Emma E Fletcher

Morgan L Jones

Joseph T P Yeeles

Affiliations

Soon will be listed here.

Abstract

During DNA replication, the DNA polymerases Pol δ and Pol ε utilise the ring-shaped sliding clamp PCNA to enhance their processivity. PCNA loading onto DNA is accomplished by the clamp loaders RFC and Ctf18-RFC, which function primarily on the lagging and the leading strand, respectively. RFC activity is essential for lagging-strand replication by Pol δ, but it is unclear why Ctf18-RFC is required for leading-strand PCNA loading and why RFC cannot fulfil this function. Here, we show that RFC cannot load PCNA once Pol ε has been incorporated into the budding yeast replisome and commenced leading-strand synthesis, and this state is maintained during replisome progression. By contrast, we find that Ctf18-RFC is uniquely equipped to load PCNA onto the leading strand and show that this activity requires a direct interaction between Ctf18 and the CMG (Cdc45-MCM-GINS) helicase. Our work uncovers a mechanistic basis for why replisomes require a dedicated leading-strand clamp loader.

References

Abramson J, Adler J, Dunger J, Evans R, Green T, Pritzel A . Accurate structure prediction of biomolecular interactions with AlphaFold 3. Nature. 2024; 630(8016):493-500. PMC: 11168924. DOI: 10.1038/s41586-024-07487-w. View

Aria V, Yeeles J . Mechanism of Bidirectional Leading-Strand Synthesis Establishment at Eukaryotic DNA Replication Origins. Mol Cell. 2018; . PMC: 6344338. DOI: 10.1016/j.molcel.2018.10.019. View

Bambara R, Murante R, Henricksen L . Enzymes and reactions at the eukaryotic DNA replication fork. J Biol Chem. 1997; 272(8):4647-50. DOI: 10.1074/jbc.272.8.4647. View

Baretic D, Jenkyn-Bedford M, Aria V, Cannone G, Skehel M, Yeeles J . Cryo-EM Structure of the Fork Protection Complex Bound to CMG at a Replication Fork. Mol Cell. 2020; 78(5):926-940.e13. PMC: 7276988. DOI: 10.1016/j.molcel.2020.04.012. View

Baris Y, Taylor M, Aria V, Yeeles J . Fast and efficient DNA replication with purified human proteins. Nature. 2022; 606(7912):204-210. PMC: 7613936. DOI: 10.1038/s41586-022-04759-1. View

Bermudez V, Maniwa Y, Tappin I, Ozato K, Yokomori K, Hurwitz J . The alternative Ctf18-Dcc1-Ctf8-replication factor C complex required for sister chromatid cohesion loads proliferating cell nuclear antigen onto DNA. Proc Natl Acad Sci U S A. 2003; 100(18):10237-42. PMC: 193545. DOI: 10.1073/pnas.1434308100. View

Boehm E, Gildenberg M, Washington M . The Many Roles of PCNA in Eukaryotic DNA Replication. Enzymes. 2016; 39:231-54. PMC: 4890617. DOI: 10.1016/bs.enz.2016.03.003. View

Bylund G, Burgers P . Replication protein A-directed unloading of PCNA by the Ctf18 cohesion establishment complex. Mol Cell Biol. 2005; 25(13):5445-55. PMC: 1156988. DOI: 10.1128/MCB.25.13.5445-5455.2005. View

Casas-Delucchi C, Daza-Martin M, Williams S, Coster G . The mechanism of replication stalling and recovery within repetitive DNA. Nat Commun. 2022; 13(1):3953. PMC: 9296464. DOI: 10.1038/s41467-022-31657-x. View

10.

Chilkova O, Stenlund P, Isoz I, Stith C, Grabowski P, Lundstrom E . The eukaryotic leading and lagging strand DNA polymerases are loaded onto primer-ends via separate mechanisms but have comparable processivity in the presence of PCNA. Nucleic Acids Res. 2007; 35(19):6588-97. PMC: 2095795. DOI: 10.1093/nar/gkm741. View

11.

Choe K, Moldovan G . Forging Ahead through Darkness: PCNA, Still the Principal Conductor at the Replication Fork. Mol Cell. 2017; 65(3):380-392. PMC: 5302417. DOI: 10.1016/j.molcel.2016.12.020. View

12.

Coster G, Frigola J, Beuron F, Morris E, Diffley J . Origin licensing requires ATP binding and hydrolysis by the MCM replicative helicase. Mol Cell. 2014; 55(5):666-77. PMC: 4157578. DOI: 10.1016/j.molcel.2014.06.034. View

13.

Devbhandari S, Remus D . Rad53 limits CMG helicase uncoupling from DNA synthesis at replication forks. Nat Struct Mol Biol. 2020; 27(5):461-471. PMC: 7225081. DOI: 10.1038/s41594-020-0407-7. View

14.

Douglas M, Abid Ali F, Costa A, Diffley J . The mechanism of eukaryotic CMG helicase activation. Nature. 2018; 555(7695):265-268. PMC: 6847044. DOI: 10.1038/nature25787. View

15.

Eickhoff P, Kose H, Martino F, Petojevic T, Abid Ali F, Locke J . Molecular Basis for ATP-Hydrolysis-Driven DNA Translocation by the CMG Helicase of the Eukaryotic Replisome. Cell Rep. 2019; 28(10):2673-2688.e8. PMC: 6737378. DOI: 10.1016/j.celrep.2019.07.104. View

16.

Frigola J, Remus D, Mehanna A, Diffley J . ATPase-dependent quality control of DNA replication origin licensing. Nature. 2013; 495(7441):339-43. PMC: 4825857. DOI: 10.1038/nature11920. View

17.

Fu Y, Yardimci H, Long D, Ho T, Guainazzi A, Bermudez V . Selective bypass of a lagging strand roadblock by the eukaryotic replicative DNA helicase. Cell. 2011; 146(6):931-41. PMC: 3209622. DOI: 10.1016/j.cell.2011.07.045. View

18.

Fujisawa R, Ohashi E, Hirota K, Tsurimoto T . Human CTF18-RFC clamp-loader complexed with non-synthesising DNA polymerase ε efficiently loads the PCNA sliding clamp. Nucleic Acids Res. 2017; 45(8):4550-4563. PMC: 5416766. DOI: 10.1093/nar/gkx096. View

19.

Garbacz M, Lujan S, Burkholder A, Cox P, Wu Q, Zhou Z . Evidence that DNA polymerase δ contributes to initiating leading strand DNA replication in Saccharomyces cerevisiae. Nat Commun. 2018; 9(1):858. PMC: 5829166. DOI: 10.1038/s41467-018-03270-4. View

20.

Garcia-Rodriguez L, Piccoli G, Marchesi V, Jones R, Edmondson R, Labib K . A conserved Polϵ binding module in Ctf18-RFC is required for S-phase checkpoint activation downstream of Mec1. Nucleic Acids Res. 2015; 43(18):8830-8. PMC: 4605302. DOI: 10.1093/nar/gkv799. View