Abstract
Chromosomal DNA replication is one of the central biological events occurring inside cells. Due to its large size, the replication of genomic DNA in eukaryotes initiates at hundreds to tens of thousands of sites called DNA origins so that the replication could be completed in a limited time. Further, eukaryotic DNA replication is sophisticatedly regulated, and this regulation guarantees that each origin fires once per S phase and each segment of DNA gets duplication also once per cell cycle. The first step of replication initiation is the assembly of pre-replication complex (pre-RC). Since 1973, four proteins, Cdc6/Cdc18, MCM, ORC and Cdt1, have been extensively studied and proved to be pre-RC components. Recently, a novel pre-RC component called Sap1/Girdin was identified. Sap1/Girdin is required for loading Cdc18/Cdc6 to origins for pre-RC assembly in the fission yeast and human cells, respectively. At the transition of G1 to S phase, pre-RC is activated by the two kinases, cyclindependent kinase (CDK) and Dbf4-dependent kinase (DDK), and subsequently, RPA, primase-polα, PCNA, topoisomerase, Cdc45, polδ, and polɛ are recruited to DNA origins for creating two bi-directional replication forks and initiating DNA replication. As replication forks move along chromatin DNA, they frequently stall due to the presence of a great number of replication barriers on chromatin DNA, such as secondary DNA structures, protein/DNA complexes, DNA lesions, gene transcription. Stalled forks must require checkpoint regulation for their stabilization. Otherwise, stalled forks will collapse, which results in incomplete DNA replication and genomic instability. This short review gives a concise introduction regarding the current understanding of replication initiation and replication fork stabilization.
Article PDF
Similar content being viewed by others
References
Bell SP, Dutta A. DNA replication in eukaryotic cells. Annu Rev Biochem, 2002, 71: 333–374
Marahrens Y, Stillman B. A yeast chromosomal origin of DNA replication defined by multiple functional elements. Science, 1992, 255: 817–823
Newlon CS, Theis JF. The structure and function of yeast ARS elements. Curr Opin Genet Dev, 1993, 3: 752–758
Palzkill TG, Newlon CS. A yeast replication origin consists of multiple copies of a small conserved sequence. Cell, 1988, 53: 441–450
Clyne RK, Kelly TJ. Genetic analysis of an ARS element from the fission yeast Schizosaccharomyces pombe. EMBO J, 1995, 14: 6348–6357
Okuno Y, Satoh H, Sekiguchi M, Masukata H. Clustered adenine/thymine stretches are essential for function of a fission yeast replication origin. Mol Cell Biol, 1999, 19: 6699–6709
Kong D, DePamphilis ML. Site-specific DNA binding of the Schizosaccharomyces pombe origin recognition complex is determined by the Orc4 subunit. Mol Cell Biol, 2001, 21: 8095–8103
Kong D, DePamphilis ML. Site-specific ORC binding, prereplication complex assembly and DNA synthesis at Schizosaccharomyces pombe replication origins. EMBO J, 2002, 21: 5567–5576
Arcangioli B, Copeland TD, Klar AJ. Sap1, a protein that binds to sequences required for mating-type switching, is essential for viability in Schizosaccharomyces pombe. Mol Cell Biol, 1994, 14: 2058–2065
Arcangioli B, Klar AJ. A novel switch-activating site (SAS1) and its cognate binding factor (SAP1) required for efficient mat1 switching in Schizosaccharomyces pombe. EMBO J, 1991, 10: 3025–3032
Aladjem MI, Rodewald LW, Kolman JL, Wahl GM. Genetic dissection of a mammalian replicator in the human beta-globin locus. Science, 1998, 281: 1005–1009
Dijkwel PA, Hamlin JL. The Chinese hamster dihydrofolate reductase origin consists of multiple potential nascent-strand start sites. Mol Cell Biol, 1995, 15: 3023–3031
Kong D, Coleman TR, DePamphilis ML. Xenopus origin recognition complex (ORC) initiates DNA replication preferentially at sequences targeted by Schizosaccharomyces pombe ORC. EMBO J, 2003, 22: 3441–3450
Austin RJ, Orr-Weaver TL, Bell SP. Drosophila ORC specifically binds to ACE3, an origin of DNA replication control element. Genes Dev, 1999, 13: 2639–2649
Aggarwal BD, Calvi BR. Chromatin regulates origin activity in Drosophila follicle cells. Nature, 2004, 430: 372–376
Kohzaki H, Murakami Y. Transcription factors and DNA replication origin selection. Bioessays, 2005, 27: 1107–1116
Mendez J, Stillman B. Perpetuating the double helix: molecular machines at eukaryotic DNA replication origins. Bioessays, 2003, 25: 1158–1167
Arias EE, Walter JC. Strength in numbers: preventing rereplication via multiple mechanisms in eukaryotic cells. Genes Dev, 2007, 21: 497–518
Diffley JF. Regulation of early events in chromosome replication. Curr Biol, 2004, 14: R778–786
Diffley JF, Cocker JH, Dowell SJ, Rowley A. Two steps in the assembly of complexes at yeast replication origins in vivo. Cell, 1994, 78: 303–316
Tsakraklides V, Bell SP. Dynamics of pre-replicative complex assembly. J Biol Chem, 2010, 285: 9437–9443
Remus D, Beuron F, Tolun G, Griffith JD, Morris EP, Diffley JF. Concerted loading of Mcm2-7 double hexamers around DNA during DNA replication origin licensing. Cell, 2009, 139: 719–730
Evrin C, Clarke P, Zech J, Lurz R, Sun J, Uhle S, Li H, Stillman B, Speck C. A double-hexameric MCM2-7 complex is loaded onto origin DNA during licensing of eukaryotic DNA replication. Proc Natl Acad Sci USA, 2009, 106: 20240–20245
Vijayraghavan S, Schwacha A. The eukaryotic Mcm2-7 replicative helicase. Subcell Biochem, 2012, 62: 113–134
Tanaka S, Araki H. Helicase activation and establishment of replication forks at chromosomal origins of replication. Cold Spring Harb Perspect Biol, 2013, 5: 1–14
Jallepalli PV, Kelly TJ. Rum1 and Cdc18 link inhibition of cyclin-dependent kinase to the initiation of DNA replication in Schizosaccharomyces pombe. Genes Dev, 1996, 10: 541–552
Muzi Falconi M, Brown GW, Kelly TJ. cdc18+ regulates initiation of DNA replication in Schizosaccharomyces pombe. Proc Natl Acad Sci USA, 1996, 93: 1566–1570
Nishitani H, Nurse P. p65cdc18 plays a major role controlling the initiation of DNA replication in fission yeast. Cell, 1995, 83: 397–405
Nishitani H, Lygerou Z, Nishimoto T, Nurse P. The Cdt1 protein is required to license DNA for replication in fission yeast. Nature, 2000, 404: 625–628
Piatti S, Lengauer C, Nasmyth K. Cdc6 is an unstable protein whose de novo synthesis in G1 is important for the onset of S phase and for preventing a ‘reductional’ anaphase in the budding yeast Saccharomyces cerevisiae. EMBO J, 1995, 14: 3788–3799
Kearsey SE, Labib K, Maiorano D. Cell cycle control of eukaryotic DNA replication. Curr Opin Genet Dev, 1996, 6: 208–214
Pelizon C, Madine MA, Romanowski P, Laskey RA. Unphosphorylatable mutants of Cdc6 disrupt its nuclear export but still support DNA replication once per cell cycle. Genes Dev, 2000, 14: 2526–2533
Nguyen VQ, Co C, Li JJ. Cyclin-dependent kinases prevent DNA re-replication through multiple mechanisms. Nature, 2001, 411: 1068–1073
Kiang L, Heichinger C, Watt S, Bahler J, Nurse P. Cyclin-dependent kinase inhibits reinitiation of a normal S-phase program during G2 in fission yeast. Mol Cell Biol, 2009, 29: 4025–4032
Cha RS, Kleckner N. ATR homolog Mec1 promotes fork progression, thus averting breaks in replication slow zones. Science, 2002, 297: 602–606
Branzei D, Foiani M. Maintaining genome stability at the replication fork. Nat Rev Mol Cell Biol, 2010, 11: 208–219
Postow L, Crisona NJ, Peter BJ, Hardy CD, Cozzarelli NR. Topological challenges to DNA replication: conformations at the fork. Proc Natl Acad Sci USA, 2001, 98: 8219–8226
Mirkin EV, Mirkin SM. Replication fork stalling at natural impediments. Microbiol Mol Biol Rev, 2007, 71: 13–35
Barlow JH, Faryabi RB, Callén E, Wong N, Malhowski A, Chen HT, Gutierrez-Cruz G, Sun HW, McKinnon P, Wright G, Casellas R, Robbiani DF, Staudt L, Fernandez-Capetillo O, Nussenzweig A. Identification of early replicating fragile sites that contribute to genome instability. Cell, 2013, 152: 620–632
Durkin SG, Glover TW. Chromosome fragile sites. Annu Rev Genet, 2007, 41: 169–192
Casper AM, Nghiem P, Arlt MF, Glover TW. ATR regulates fragile site stability. Cell, 2002, 111: 779–789
Azvolinsky A, Giresi PG, Lieb JD, Zakian VA. Highly transcribed RNA polymerase II genes are impediments to replication fork progression in Saccharomyces cerevisiae. Mol Cell, 2009, 34: 722–734
Wright JA, Chan AK, Choy BK, Hurta RA, McClarty GA, Tagger AY. Regulation and drug resistance mechanisms of mammalian ribonucleotide reductase, and the significance to DNA synthesis. Biochem Cell Biol, 1990, 68: 1364–1371
Wyatt MD, Pittman DL. Methylating agents and DNA repair responses: methylated bases and sources of strand breaks. Chem Res Toxicol, 2006, 19: 1580–1594
Sogo JM, Lopes M, Foiani M. Fork reversal and ssDNA accumulation at stalled replication forks owing to checkpoint defects. Science, 2002, 297: 599–602
Fersht N, Hermand D, Hayles J, Nurse P. Cdc18/CDC6 activates the Rad3-dependent checkpoint in the fission yeast. Nucleic Acids Res, 2007, 35: 5323–5337
Hermand D, Nurse P. Cdc18 enforces long-term maintenance of the S phase checkpoint by anchoring the Rad3-Rad26 complex to chromatin. Mol Cell, 2007, 26: 553–563
D’Urso G, Grallert B, Nurse P. DNA polymerase alpha, a component of the replication initiation complex, is essential for the checkpoint coupling S phase to mitosis in fission yeast. J Cell Sci, 1995, 108(Pt 9): 3109–3118
Zegerman P, Diffley JF. Checkpoint-dependent inhibition of DNA replication initiation by Sld3 and Dbf4 phosphorylation. Nature, 2010, 467: 474–478
Hu J, Sun L, Shen F, Chen Y, Hua Y, Liu Y, Zhang M, Hu Y, Wang Q, Xu W, Sun F, Ji J, Murray JM, Carr AM, Kong D. The intra-S phase checkpoint targets Dna2 to prevent stalled replication forks from reversing. Cell, 2012, 149: 1221–1232
Author information
Authors and Affiliations
Corresponding authors
Additional information
This article is published with open access at link.springer.com
Rights and permissions
Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (https://creativecommons.org/licenses/by/4.0), which permits use, duplication, adaptation, distribution, and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.
About this article
Cite this article
Wu, L., Liu, Y. & Kong, D. Mechanism of chromosomal DNA replication initiation and replication fork stabilization in eukaryotes. Sci. China Life Sci. 57, 482–487 (2014). https://doi.org/10.1007/s11427-014-4631-4
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11427-014-4631-4