Eukaryotic Replication Fork


In eukaryotic cells, DNA (deoxyribonucleic acid) synthesis occurs at specific sites that move through the genome called replication forks. Multiprotein complexes at these forks catalyse the synthesis of two new strands of DNA using parental strands as templates to produce two complete copies of the parental DNA. The eukaryotic replication fork machinery must deal with the chromatin and chromosome structure of eukaryotic genomes, be able to replicate DNA in the context of a complex cell cycle, and be able to deal with the constant threat of mutations that could arise due to replication of damaged DNA, all while trying to efficiently replicate the DNA with high fidelity. The resulting eukaryotic replication fork is a tightly controlled, yet incredibly efficient biological machine capable of synthesizing billions of base pairs of DNA in the span of hours.

Key Concepts:

  • Eukaryotic DNA replication requires the concerted action of multiple DNA polymerases and accessory factors that replicate the DNA with high efficiency and accuracy.

  • Eukaryotic chromosomes are replicated in a semidiscontinuous manner by replication forks that coordinate the simultaneous replication of the leading and lagging strands.

  • The eukaryotic replication machinery is equipped to displace histones that coat chromatin DNA and reconstitute fully functional nucleoprotein chromosomes after DNA replication.

Keywords: DNA replication; DNA polymerase; DNA synthesis; replication fork; checkpoint

Figure 1.

Schematic representation of a replication fork. Black lines – template DNA and red arrows – newly synthesized DNA. Positions of leading and lagging strands are indicated.

Figure 2.

Mechanism of Okazaki fragment synthesis and polymerase switching. Black lines – template DNA; red lines – newly synthesized DNA and green lines – RNA primers. A polymerase switch between DNA Pol α, RPA, RF‐C and DNA Pol δ, is shown in steps 1–4, starting with an RPA ‐coated ssDNA molecule that contains the 5′ end of a completed Okazaki fragment on the right. Even though proteins are shown freely diffusing, they probably remain transiently associated with the DNA as part of a replisome complex. Steps 5–7 indicate the flap displacement by Pol δ, and the RNA primer processing by Fen1 and DNA ligase 1.

Figure 3.

Model of a eukaryotic replication fork. Schematic representation of replication fork topology based on known protein–protein interactions and analogy to prokaryotic replication forks. Black lines – template DNA; red lines – newly synthesized DNA and green lines – RNA primers. Looping of the lagging strand template DNA allows the replication machinery to move in the direction of the leading strand while synthesizing the lagging strand in the opposite direction.



Aparicio OM, Weinstein DM and Bell SP (1997) Components and dynamics of DNA replication complexes in S. cerevisiae: redistribution of MCM proteins and Cdc45p during S phase. Cell 91(1): 59–69.

Bambara RA, Murante RS and Henricksen LA (1997) Enzymes and reactions at the eukaryotic DNA replication fork. Journal of Biological Chemistry 272(8): 4647–4650.

Bauerschmidt C, Pollok S, Kremmer E, Nasheuer HP and Grosse F (2007) Interactions of human Cdc45 with the Mcm2‐7 complex, the GINS complex, and DNA polymerases delta and epsilon during S phase. Genes to Cells 12(6): 745–758.

Bell SP and Dutta A (2002) DNA replication in eukaryotic cells. Annual Review of Biochemistry 71: 333–374.

Bochman ML and Schwacha A (2008) The Mcm2‐7 complex has in vitro helicase activity. Molecular Cell 31(2): 287–293.

Brush GS and Kelly TJ (1996) Mechanisms for Replicating DNA. In: DePamphilis ML (ed.) DNA Replication in Eukaryotic Cells, pp. 1–43. Plainview, NY: Cold Spring Harbor Laboratory Press.

Burgers PM (2009) Polymerase dynamics at the eukaryotic DNA replication fork. Journal of Biological Chemistry 284(7): 4041–4045.

Chastain PD, Makhov AM 2nd, Nossal NG and Griffith J (2003) Architecture of the replication complex and DNA loops at the fork generated by the bacteriophage t4 proteins. Journal of Biological Chemistry 278(23): 21276–21285.

Corpet A and Almouzni G (2009) Making copies of chromatin: the challenge of nucleosomal organization and epigenetic information. Trends in Cell Biology 19(1): 29–41.

Dalgaard JZ, Eydmann T, Koulintchenko M et al. (2009) Random and site‐specific replication termination. Methods in Molecular Biology 521: 35–53.

Feng W and D'Urso G (2001) Schizosaccharomyces pombe cells lacking the amino‐terminal catalytic domains of DNA polymerase epsilon are viable but require the DNA damage checkpoint control. Molecular and Cellular Biology 21(14): 4495–4504.

Fien K, Cho YS, Lee JK et al. (2004) Primer utilization by DNA polymerase alpha‐primase is influenced by its interaction with Mcm10p. Journal of Biological Chemistry 279(16): 16144–16153.

Formosa T (2008) FACT and the reorganized nucleosome. Molecular Biosystems 4(11): 1085–1093.

Gambus A, Jones RC, Sanchez‐Diaz A et al. (2006) GINS maintains association of Cdc45 with MCM in replisome progression complexes at eukaryotic DNA replication forks. Nature Cell Biology 8(4): 358–366.

Garg P and Burgers PM (2005) DNA polymerases that propagate the eukaryotic DNA replication fork. Critical Reviews in Biochemistry and Molecular Biology 40(2): 115–128.

Hoege C, Pfander B, Moldovan GL, Pyrowolakis G and Jentsch S (2002) RAD6‐dependent DNA repair is linked to modification of PCNA by ubiquitin and SUMO. Nature 419(6903): 135–141.

Hubscher U, Maga G and Spadari S (2002) Eukaryotic DNA polymerases. Annual Review of Biochemistry 71: 133–163.

Jackson DA and Pombo A (1998) Replicon clusters are stable units of chromosome structure: evidence that nuclear organization contributes to the efficient activation and propagation of S phase in human cells. Journal of Cell Biology 140(6): 1285–1295.

Kitamura E, Blow JJ and Tanaka TU (2006) Live‐cell imaging reveals replication of individual replicons in eukaryotic replication factories. Cell 125(7): 1297–1308.

Kubota Y, Takase Y, Komori Y et al. (2003) A novel ring‐like complex of Xenopus proteins essential for the initiation of DNA replication. Genes & Development 17(9): 1141–1152.

Lambert S and Carr AM (2005) Checkpoint responses to replication fork barriers. Biochimie 87(7): 591–602.

Li JJ and Kelly TJ (1984) Simian virus 40 DNA replication in vitro. Proceedings of the National Academy of Sciences of the USA 81(22): 6973–6977.

Lopes M, Cotta‐Ramusino C, Pellicioli A et al. (2001) The DNA replication checkpoint response stabilizes stalled replication forks. Nature 412(6846): 557–561.

Maga G and Hubscher U (1996) DNA replication machinery: functional characterization of a complex containing DNA polymerase alpha, DNA polymerase delta, and replication factor C suggests an asymmetric DNA polymerase dimer. Biochemistry 35(18): 5764–5777.

Moldovan GL, Pfander B and Jentsch S (2007) PCNA, the maestro of the replication fork. Cell 129(4): 665–679.

Moyer SE, Lewis PW and Botchan MR (2006) Isolation of the Cdc45/Mcm2‐7/GINS (CMG) complex, a candidate for the eukaryotic DNA replication fork helicase. Proceedings of the National Academy of Sciences of the USA 103(27): 10236–10241.

Nick McElhinny SA, Gordenin DA, Stith CM, Burgers PM and Kunkel TA (2008) Division of labor at the eukaryotic replication fork. Molecular Cell 30(2): 137–144.

Niida H and Nakanishi M (2006) DNA damage checkpoints in mammals. Mutagenesis 21(1): 3–9.

Ohya T, Kawasaki Y, Hiraga S et al. (2002) The DNA polymerase domain of pol(epsilon) is required for rapid, efficient, and highly accurate chromosomal DNA replication, telomere length maintenance, and normal cell senescence in Saccharomyces cerevisiae. Journal of Biological Chemistry 277(31): 28099–28108.

Pacek M, Tutter AV, Kubota Y, Takisawa H and Walter JC (2006) Localization of MCM2‐7, Cdc45, and GINS to the site of DNA unwinding during eukaryotic DNA replication. Molecular Cell 21(4): 581–587.

Pursell ZF, Isoz I, Lundstrom EB, Johansson E and Kunkel TA (2007) Yeast DNA polymerase epsilon participates in leading‐strand DNA replication. Science 317(5834): 127–130.

Rampakakis E, Arvanitis DN, Di Paola D and Zannis‐Hadjopoulos M (2009) Metazoan origins of DNA replication: regulation through dynamic chromatin structure. Journal of Cellular Biochemistry 106(4): 512–520.

Rossi ML and Bambara RA (2006) Reconstituted Okazaki fragment processing indicates two pathways of primer removal. Journal of Biological Chemistry 281(36): 26051–26061.

Sancar A, Lindsey‐Boltz LA, Unsal‐Kacmaz K and Linn S (2004) Molecular mechanisms of mammalian DNA repair and the DNA damage checkpoints. Annual Review of Biochemistry 73: 39–85.

Sclafani RA and Holzen TM (2007) Cell cycle regulation of DNA replication. Annual Review of Genetics 41: 237–280.

Shibahara K and Stillman B (1999) Replication‐dependent marking of DNA by PCNA facilitates CAF‐1‐coupled inheritance of chromatin. Cell 96(4): 575–585.

Shikata K, Sasa‐Masuda T, Okuno Y, Waga S and Sugino A (2006) The DNA polymerase activity of Pol epsilon holoenzyme is required for rapid and efficient chromosomal DNA replication in Xenopus egg extracts. BMC Biochemistry 7: 21.

Stelter P and Ulrich HD (2003) Control of spontaneous and damage‐induced mutagenesis by SUMO and ubiquitin conjugation. Nature 425(6954): 188–191.

Stillman B (2008) DNA polymerases at the replication fork in eukaryotes. Molecular Cell 30(3): 259–260.

Tabancay AP Jr. and Forsburg SL (2006) Eukaryotic DNA replication in a chromatin context. Current Topics in Developmental Biology 76: 129–184.

Takayama Y, Kamimura Y, Okawa M et al. (2003) GINS, a novel multiprotein complex required for chromosomal DNA replication in budding yeast. Genes & Development 17(9): 1153–1165.

Tercero JA and Diffley JF (2001) Regulation of DNA replication fork progression through damaged DNA by the Mec1/Rad53 checkpoint. Nature 412(6846): 553–557.

Tercero JA, Labib K and Diffley JF (2000) DNA synthesis at individual replication forks requires the essential initiation factor Cdc45p. EMBO Journal 19(9): 2082–2093.

Tutter AV and Walter JC (2006) Chromosomal DNA replication in a soluble cell‐free system derived from Xenopus eggs. Methods in Molecular Biology 322: 121–137.

Unsal‐Kacmaz K, Chastain PD, Qu PP et al. (2007) The human Tim/Tipin complex coordinates an Intra‐S checkpoint response to UV that slows replication fork displacement. Molecular and Cellular Biology 27(8): 3131–3142.

Waga S and Stillman B (1994) Anatomy of a DNA replication fork revealed by reconstitution of SV40 DNA replication in vitro. Nature 369(6477): 207–212.

Waga S and Stillman B (1998) The DNA replication fork in eukaryotic cells. Annual Review of Biochemistry 67: 721–751.

Walther AP, Bjerke MP and Wold MS (1999) A novel assay for examining the molecular reactions at the eukaryotic replication fork: activities of replication protein A: required during elongation. Nucleic Acids Research 27(2): 656–664.

Wold MS (1997) Replication protein A: a heterotrimeric, single‐stranded DNA‐binding protein required for eukaryotic DNA metabolism. Annual Review of Biochemistry 66: 61–92.

Zhu W, Ukomadu C, Jha S et al. (2007) Mcm10 and And‐1/CTF4 recruit DNA polymerase alpha to chromatin for initiation of DNA replication. Genes & Development 21(18): 2288–2299.

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Walther, André P(Mar 2010) Eukaryotic Replication Fork. In: eLS. John Wiley & Sons Ltd, Chichester. [doi: 10.1002/9780470015902.a0001050.pub2]