DNA Replication: Prokaryotes

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DNA Replication: Prokaryotes

Shanna J Smith, City of Hope Beckman Research Institute, Duarte, California, USA
Robert G Lingeman, City of Hope Beckman Research Institute, Duarte, California, USA
Caroline M Li, City of Hope Beckman Research Institute, Duarte, California, USA
Mustafa Raoof, City of Hope National Medical Center, Duarte, California, USA
Robert J Hickey, City of Hope Beckman Research Institute, Duarte, California, USA
Linda H Malkas, City of Hope Beckman Research Institute, Duarte, California, USA

Published online: August 2018

DOI: 10.1002/9780470015902.a0003744.pub2

Full Article on Wiley Online Library

Abstract
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Abstract

The accurate replication of deoxyribonucleic acid (DNA) is necessary to all biological life. DNA replication involves three distinct phases: initiation, elongation and termination, and the commonality runs through all types of living cellular‐based organisms. While all forms of life replicate DNA in a similar manner, higher order organisms tend to have more proteins and enzymes involved in the process, with complex mechanisms poorly understood. For this reason, bacteria and archaeans have proven to be valuable tools to be used to further our understanding of mammalian life. The most commonly characterised of the prokaryotes is the bacteria Escherichia coli (E. coli) and its infecting phages (bacteriophages), with each having a mechanism unique to the process.

Key Concepts

The study of replication in prokaryotes has formed the basis of our understanding of replicative mechanisms common to all organisms.
Escherichia coli (E. coli) is an excellent bacterial system that can be used to study the three distinct stages of DNA replication: initiation, elongation and termination.
Bacteriophages represent a special group of prokaryote‐like systems that provide a platform to study specific mechanisms of replication in a more isolated and simplified manner.
Circular single‐stranded DNA bacteriophages, such as ΦX174, replicate their genome through an intermediate double‐stranded replicative form.
Double‐stranded DNA bacteriophages, such as λ, cyclise upon infection and replicate similar to their host.
T series bacteriophage systems do not cyclise upon infection, and have developed clever strategies to terminate DNA replication without loss of genetic information (3′ overhang problem).

Keywords: bacteria; bacteriophages; Escherichia coli; initiation; elongation; replication fork; replicative form; termination

	Figure 1. E. coli DNA replication initiation. (a) The E. coli oriC is composed of the DUE and the DAR regions. The DUE region is composed of three 13‐mer AT‐rich repeats. The DAR region is composed of three dnaA boxes that bind dnaA‐ATP with high affinity (R1, R4 and R2) and 10 dnaA boxes that bind dnaA‐ATP with lower affinity (R3, R5, I1, I2, I3, C1, C2, C3, τ1 and τ2). The DAR region also contains binding sites for Fis and IHF. (b) Replication is initiated when the amount of dnaA‐ATP bound to the dnaA boxes in the DAR region exceeds a certain threshold. dnaA‐ATP binds the high‐affinity R4 box and forms a dnaA‐ATP filament that extends into the adjacent low‐affinity boxes and dislodges Fis from its DNA‐binding site. (c) The removal of Fis allows IHF to bind to its DNA‐binding site, which in turn bends the DNA so that the R5 and R1 boxes are in close proximity. This allows dnaA‐ATP filaments to extend into the DUE region and separate the AT‐rich duplex DNA. dnaA‐ATP then recruits the dnaB helicase/dnaC helicase loader dimer to the opened DNA in preparation of the elongation stage.
	Figure 2. 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 synthesises new strands of DNA, beginning with the RNA primers. The areas containing single‐stranded DNA are stabilised through the binding of single‐stranded DNA‐binding proteins (SSB). (b) Illustration of the replication fork movement and the coordination of DNA replication along the leading and lagging strand. The DNA is unwound by dnaB at the leading end of the replication fork, and a nascent strand of DNA is synthesised 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 (bottom strand) is coordinated with synthesis of the lagging strand (top strand) through the dimerisation 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 synthesises multiple short 1000–2000‐bp Okazaki fragments on to the lagging strand template. These Okazaki fragments will become ligated to one another after primer removal and filling in by DNA polymerase I. The directions of fork movement and leading and lagging strand movement are denoted with arrows.
	Figure 3. E. coli elongation and termination. Two replication forks with individual replisomes travel bidirectionally from the oriC synthesising new DNA (grey) from the parental template (black). The characteristic theta (θ) structure is apparent as replication proceeds around the circular chromosome and the two daughter chromosomes are formed. As replication continues, the forks will encounter Tus‐ter units (terA through terJ) and pass through the permissive end of the first five units encountered, but will be prevented from passing through the nonpermissive side of the sixth unit that is oriented in the opposite direction. Replication ends when the forks converge between terC and terA.
	Figure 4. The DNA replication of phage λ. (a) Upon infection, the λ DNA is injected into the bacterium as a linear molecule. The linear λ chromosome then cyclises through the hybridisation of cohesive termini (cos sites) at the ends of the molecule. (b) Circle‐to‐circle DNA replication initiates at a single replication origin ori λ by transcription of an RNA primer through the origin from one of the two promoters (i.e. P_R1 and P_R2). (c) Replication forks are formed, and circle‐to‐circle replication proceeds. (d) Identical daughter strands produced. After a few rounds of circle‐to‐circle replication, λ switches to an alternative mode of DNA replication called rolling‐circle replication. (e) 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. (f) Polymerisation 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. (g) Single λ chromosomes are generated from the site‐specific cleavage (lightning bolt) of the λ concatemer at the cos sites.
	Figure 5. The first stage ΦX174 replication: the creation of the replicative form (RF). (a) Pri‐A, Pri‐B, Pri‐C and dnaT are loaded onto the stem loops formed as a result of super coiling of the virally injected (+) strand. The Pri‐A complex then recruits the dnaB helicase/dnaC helicase loader dimer. (b) Recruitment of dnaB–dnaC results in the formation of the preprimosome which then recruits dnaG. (c) The addition of the RNA primase, dnaG, completes assembly of the primosome. (d) The primosome lays complementary RNA strands at each 5′GTC'3 encountered on the (+) strand. (e) DNA pol III HE synthesises DNA using the 3′OH of the RNA primers. DNA pol I removes the RNA primers while filling the gap with DNA. DNA ligase then seals the remaining nick to complete synthesis of the (−) strand. (f) The double‐stranded product is called the replicative form (RF). The RF provides a stable (−) strand template and essential genes required for production of the many (+) strands that will be created in the second stage of ΦX174 replication. These (+) strands will be packaged into capsids to propagate the virus.
	Figure 6. 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 synthesised through one of the 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 7. The mechanisms of DNA replication initiation from D loops during T4 DNA replication. (a) Unidirectional DNA replication results from the cleavage of the hybridised (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 8. T7 DNA replication. (a) DNA replication of the T7 phage initiates through the synthesis of an RNA transcript from one of the two T7 promoters (P_R1 and P_R2) located to the left of the T7 origin of replication (oriT7), displacing the complementary DNA strand forming an R loop. (b) 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. DNA replication then proceeds bidirectionally, copying the T7 genome. (c) The T7 genome, unlike λ, never cyclises 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. (d) To circumvent the loss of the DNA overhang at the ends of the T7 genome, the T7 phage forms long DNA concatemers by the hybridisation of the terminal repeats (TR). (e) Full‐length T7 genomes are then generated through cleavage of the concatemers at specific sequences within the TR called pac sites.

Article Title:	DNA Replication: Prokaryotes
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