DNA Replication: Prokaryotes and Yeast

Abstract

An organism's ability to pass on genetic information to further generations requires the accurate reproduction of its genomic DNA, a process termed DNA replication. Examination of the events that take place during DNA replication has revealed numerous well‐orchestrated protein–protein interactions and enzymatic reactions, which ultimately leads to synthesis of an essentially identical replica of the cell's complete genome.

Keywords: bacteria; bacteriophages; initiation; elongation

Figure 1.

The E. coli chromosomal DNA replication origin, oriC. The four boxes to the right, shaded blue, represent the 9 bp recognition sites for dnaA proteins, and the three red boxes to the left represent the 13 bp AT‐rich domains melted during the initiation of E. coli DNA replication.

Figure 2.

Initiation of chromosomal DNA replication in E. coli. dnaA binds to the 9 bp sequences of oriC in the presence of the protein HU and ATP, forming a complex of 20–40 dnaA molecules around which the DNA of the origin is wrapped and is referred to as the initial complex. In the presence of 5 mmol L1 ATP and at 38°C, the initial complex is converted to an open complex via the ATP‐dependent melting of the AT‐rich domain by dnaA. The dnaB–dnaC complex is then directed to the melted region of DNA through the interaction of dnaC with dnaA. dnaB is now able to recognize the single‐stranded DNA, further unwinding the DNA and leading to the formation of the prepriming complex.

Figure 3.

The E. coli replication fork. After initiation has occurred, two replication forks form at the points where single‐stranded DNA meets double‐stranded DNA. (a) Diagram of the proteins present at a replication fork in E. coli. The progression of the replication fork is directed through the helicase activity of dnaB followed by the synthesis of RNA primers by dnaG on both the leading strand (bottom strand) and lagging strand (top strand). The DNA polymerase III holoenzyme (DNA pol III HE) then synthesizes new strands of DNA, beginning with the RNA primers. The areas containing single‐stranded DNA are stabilized through the binding of single‐stranded DNA‐binding proteins (SSB). A more accurate representation of a replication fork is shown in (b). Here, the DNA is unwound by dnaB at the leading end of the replication fork, and a nascent strand of DNA is synthesized on the leading strand (top strand) by a DNA pol III HE. Tethered to the DNA by a β subunit dimer, the DNA pol III HE moves along the leading strand of DNA. Synthesis of the leading strand (top strand) is coordinated with synthesis of the lagging strand (bottom strand) through the dimerization of two DNA pol III HE complexes by the γ subunit. The DNA of the lagging strand is looped around one DNA pol III complex and, following primer synthesis and loading of a β dimer at the primer terminus by the γ complex, the DNA pol III HE synthesizes multiple short 1000–2000 bp Okazaki fragments on to the lagging strand. These Okazaki fragments will become ligated to one another, forming a long, contiguous strand of DNA. The direction of fork movement and leading and lagging strand movement are denoted with arrows.

Figure 4.

The chromosomal DNA replication terminus of E. coli. The replication terminus is located 180° around the E. coli chromosome from oriC and contains two sets of three directly repeated sequences called ter sites. At these ter sites, the Tus protein binds to the DNA and abrogates replication fork movement. However, the movement of one replication fork can only be stopped by a Tus protein binding to a single ter site that is in the proper orientation. This unidirectional or polar blockade of replication by Tus is represented by the sequences to the left and the leftward‐moving replication fork (top arrow), and the sequences to the right and the rightward‐moving replication fork (bottom arrow). When a replication fork encounters these sequences bound with Tus, an interaction between dnaB and Tus occurs, most likely resulting in inhibition of its helicase activity and cessation of replication fork movement.

Figure 5.

The DNA replication of phage λ. Upon infection, the λ DNA is injected into the bacterium as a linear molecule. The linear λ chromosome then cyclizes through the hybridization of cohesive termini (cos sites) at the ends of the molecule (a). Circle‐to‐circle DNA replication initiates at a single replication origin ori λ (b) by transcription of an RNA primer through the origin from one of two promoters (i.e. PR1 and PR2). Replication forks are formed, and circle‐to‐circle replication proceeds (c) producing two daughter molecules (d). After a few rounds of circle‐to‐circle replication, λ switches to an alternative mode of DNA replication called rolling‐circle replication (E–G). Rolling‐circle replication initiates by the nicking of one strand of the circular DNA, resulting in a free 3′‐OH terminus that serves as a primer for DNA synthesis (e). Polymerization of the new strand, rolling round and round the circular molecule, causes displacement of the old strand, forming long tandemly repeated λ chromosomes called concatemers (f). Single λ chromosomes are generated from the site‐specific cleavage (lightning bolt) of the λ concatemer at the cos sites (g).

Figure 6.

T7 DNA replication. DNA replication of the T7 phage initiates through the synthesis of an RNA transcript from one of two T7 promoters (PR1, PR2) located to the left of the T7 origin of replication (oriT7), displacing the complementary DNA strand forming an R loop (a). The 3′‐OH terminus of the RNA transcript then serves as a primer terminus for leading‐strand synthesis, and two replication forks are assembled at the R loop (b). DNA replication then proceeds bidirectionally, copying the T7 genome. The T7 genome, unlike λ, never cyclizes during its infection cycle and, because the DNA polymerases require a 3′‐OH terminus from which to elongate, single‐stranded DNA overhangs are generated after the removal of RNA primers from the 5′ ends (c). To circumvent the loss of the DNA overhang at the ends of the T7 genome, the T7 phage forms long DNA concatemers by the hybridization of the terminal repeats (TR) (d). Full‐length T7 genomes are then generated through cleavage of the concatemers at specific sequences within the TR called pac sites (e).

Figure 7.

T4 DNA replication: initiation from R loops and D loops. T4 DNA replication occurs in two stages. In stage 1, T4 replication initiates from an RNA primer synthesized through one of multiple origins (R loop‐dependent) scattered throughout the T4 genome. After a few rounds of origin‐initiated DNA replication, the resultant T4 genomes contain free 3′ single‐stranded DNA overhangs at their ends. In stage 2, these 3′ overhangs are then able to invade the DNA of an adjacent T4 chromosome, displacing a homologous region of DNA and forming a D‐loop. The 3′‐OH of the invading single‐stranded DNA is then able to serve as a primer terminus for leading strand synthesis, enabling replication to proceed.

Figure 8.

The mechanisms of DNA replication initiation from D loops during T4 DNA replication. (a) Unidirectional DNA replication results from the cleavage of the hybridized (solid green circle) strand of DNA followed by the formation of a single replication fork. (b) Bidirectional DNA replication occurs through the cleavage of the displaced (solid blue circle) strand of DNA followed by formation of two replication forks. (c) Tridirectional DNA replication is the result of the formation of three replication forks on an uncleaved template. Alternatively, tridirectional DNA replication could also occur if two of the DNA strands were cleaved (not shown).

Figure 9.

The primosome assembly during covalently closed single‐stranded RF DNA replication in the phage ΦX174. After infection, the single‐stranded DNA of ΦX174 is converted to double‐stranded replicative form (RF) DNA by the assembly of a primosome at a primosome assembly site (PAS). The cellular protein PriA first recognizes the secondary structure of the PAS and binds to the single‐stranded DNA. PriA then recruits PriB and PriC, followed by dnaT and dnaB. dnaB synthesizes an RNA primer on to the single‐stranded DNA of ΦX174, which is elongated, forming a double‐stranded RF DNA template.

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References

Bramhill D and Kornberg A (1988) Amodel for initiation at origins of DNA replication. Cell 54: 915–918.

Bussiere DE and Bastia D (1999) Termination of DNA replication ofbacterial and plasmid chromosomes. Molecular Microbiology 6: 1611–1618.

Kim DR and McHenry CS (1996) Identification of the β‐bindingdomain of the α subunit of Escherichia coli polymerase IIIholoenzyme. Journal of Biological Chemistry 271:20699–20704.

Kong XP, Onrust R, O'Donnell M and Kuriyan J (1992) Three‐dimensionalstructure of the β subunit of E. coli DNA polymerase IIIholoenzyme: a sliding clamp. Cell 69:425–437.

Krenzer KN (2000) Recombination‐dependent DNA replication in phageT4. Trends in Biochemical Sciences 25:165–173.

Leu FP, Hingorami MM, Turner J and O'Donnell M (2000) The δsubunit of DNA polymerase III holoenzyme serves as a sliding clamp unloader in Escherichia coli. Journal of Biological Chemistry 275: 34609–34618.

Marians KJ (1992) Prokaryotic DNA replication. Annual Reviews of Biochemistry 61: 673–719.

Marians KJ (2000) PriA: atthe crossroads of DNA replication and recombination. Progress inNucleic Acids Research 63: 39–65.

Ogawa T, Hirose S, Okazaki T and Okazaki R (1977) Mechanism of DNA chain growth XVI. Analyses ofRNA‐linked DNA pieces in Escherichia coli with polynucleotidekinase. Journal of Molecular Biology 112:121–140.

Olson MW, Dallmann HG and McHenry CS (1995) DnaX complex of Escherichiacoli DNA polymerase III holoenzyme, the χ. Ψ complex functions byincreasing the affinity of τ and γ for δ.δ′ to aphysiologically relevant range. Journal of BiologicalChemistry 270: 29570–29577.

Stillman B (1996) Cell cyclecontrol of DNA replication. Science 274(5293): 1659–1664.

Toyn JH, Toone WM, Morgan BA and Johnston LH (1995) The activation of DNAreplication in yeast. Trends in Biochemical Sciences 20(2): 70–73.

Wang TA and Li JJ (1995) Eukaryotic DNA replication. Current Opinion in Cell Biology 7(3):414–420.

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Hoelz, Derek J, Hickey, Robert J, and Malkas, Linda H(Mar 2004) DNA Replication: Prokaryotes and Yeast. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1038/npg.els.0003744]