DNA replication

Readers should first familiarize themselves with the structure and polarity of DNA; see the article: DNA

DNA replication is the process of synthesising an identical copy of DNA from a template parent molecule. Replication is essential in maintaining genetic continuity from one generation of cells (or viruses) to the next. Cellular proofreading and DNA repair mechanisms ensure that DNA is replicated with high fidelity. However there may occasionally be errors in the replication process leading to mutations in the sequence of nucleotides. In a cell, DNA replication begins at either a single (in bacteria) or multiple (in eukaryotes) position(s) in the genome called the origin(s) of replication.

DNA replication is naturally performed in vivo, however it may also be performed in vitro. In vitro DNA replication is an important procedure in molecular biology; it forms the basis of the polymerase chain reaction (PCR).

DNA replication is described as semi-conservative, meaning that each daughter DNA helix contains one strand donated by its parent, and one strand newly synthesised as a result of replication (so the parent helix is semi-conserved in each daughter).

There are notable differences in DNA replication between prokaryotes and eukaryotes; these are outlined in separate sections of this article.

Prokaryotic DNA replication:

In bacteria, DNA replication is bi-directional and begins at a single origin of replication, called OriC.

DNA replication is initiated by the binding of an initiator protein to a region called the dnaA box, of which there are five in E. coli, each containing the 9bp consensus sequence 5' TTATCCACA 3'. When the initiator protein binds to the dnaA box, the DNA helix is negatively supercoiled. Following this, a region of dnaB boxes further upstream of dnaA causes the melting apart of the two strands. This is possible because the dnaB boxes are AT-rich and it takes a lot less energy to break the hydrogen bonds in AT base pairs than in GC base pairs. ATP hydrolysis by the dnaA box facilitates melting at the dnaB box.

DnaA then recruits an enzyme called DNA helicase to opposite ends of the melted DNA; this is where the replication fork will form. The replication fork is the area where DNA has been melted to bear two 'prongs' of single-stranded DNA, each will later serve as a template for new DNA synthesis. SSB (single strand-binding) proteins are then recruited to prevent the now single-stranded DNA from either forming a secondary structure or re-annealing in such a way that would block the process of replication. An enzyme called DNA gyrase (also DNA topoisomerase) is finally needed to relieve the stress on the molecule that is created by DNA helicase (negative supercoiling leads to the build up of tension further down in the helix; this could inhibit replication). Once unwound, the enzyme dnaG (also primase, a type of RNA polymerase) arrives to prime each DNA template with an RNA primer so that DNA replication can begin.

Once the DNA is primed, the DNA polymerase III holoenzyme runs the length of each strand, polymerising free deoxynucleotides that are complementary to the sequence of the template strand. The enzyme has proof-reading ability because of a pocket within the enzyme structure that is complementary to the shape of correctly-bound base pairs. The new DNA molecule is synthesised in the 5' to 3' direction. This means that synthesis on the 3' to 5' template strand is straightforward; however, it is more complex on the 5' to 3' template strand. On this template strand, the nascent DNA is synthesised in many short 5'-3' fragments called Okazaki fragments. These strands are called the leading and lagging strands, respectively. While the leading strand requires only one RNA primer for DNA synthesis, the lagging strand requires several (one for each new Okazaki fragment). The primers are subsequently degraded by DNA polymerase I and the gaps (or nicks) between the fragments are filled by DNA ligase.

DNA replication is then terminated by interaction between termination sequences and a Tus protein. Because DNA in bacteria is usually circular, the two catenated (interlinked) DNA helices (each helix comprising one parent and one daughter strand, as is the nature of semiconservative replication) are separated; in E. coli this is performed by the enzyme topoisomerase IV.

Eukaryotic DNA replication:

In the eukaryotic cell cycle, DNA replication is a key event of the synthesis (S) phase. However, pre-initiation (preparation) for DNA synthesis is an event of the G1 phase. This ensures that DNA replication occurs only once per cell cycle. Due to size of the chromosomes, DNA replication in eukaryotes typically has multiple origins of replication.

The first step in DNA replication is the formation of the pre-initiation replication complex (the pre-RC). The formation of this complex occurs in two stages. The first stage requires that there is no CDK (cyclic-dependent kinase) activity. This can only occur in early G1. The formation of the pre-RC is known as licensing, but a licensed pre-RC cannot initiate replication in the G1 phase.

Current models hold that replication initiation begins with the binding of the origin recognition complex (ORC) to the origin. This complex is a hexamer of related proteins and remains bound to the origin, even after DNA replication occurs. Furthermore, the ORC is the functional analogue of prokaryotic dnaA (see above). Following the binding of ORC to the origin, various proteins coordinate the loading of the MCM (Mini Chromosome Maintenance) complex to the origin by first binding to ORC and then binding to the MCM complex (i.e. the proteins act as a bridge between them). The MCM complex is thought to be the major DNA helicase in eukaryotic organisms. Once binding of MCM occurs, a fully licensed pre-RC exists that is ready for DNA replication.

Activation of the complex occurs in S phase and requires various cyclin-dependent kinases that are activated during this phase of the cell cycle. The activation process begins with the addition of the protein Mcm10 to the pre-RC, which displaces another protein. Following this, a phosphorylation event activates the helicase. This phosphorylation recruits another protein to the complex, which itself recruits all of the DNA replication proteins to the replication fork (the site where the DNA has been melted into two single strands).

At this stage the origin fires and DNA synthesis begins. As with prokaryotic DNA replication, the 3' to 5' parent strand has a single primer and is replicated in a continuous fashion, while the 5' to 3' parent strand is replicated in Okazaki fragments with multiple primers that are later degraded and annealed by DNA ligase.

At least three different types of eukaryotic DNA polymerases are involved in the replication of DNA in animal cells (Pol α, Pol δ and POL ε).
  • Pol α forms a complex with a small catalytic (PriS) and a large noncatalytic (PriL) subunit, with the Pri subunits acting as a primase (synthesizing an RNA primer), and then with DNA Pol α elongating that primer with DNA nucleotides. After around 20 nucleotides elongation is taken over by Pol ε (on the leading strand) and δ (on the lagging strand).
  • Pol δ: Highly processive and has proofreading 3'->5' exonuclease activity. Thought to be the main polymerase involved in lagging strand synthesis, though there is still debate about its role.
  • Pol ε: Also highly processive and has proofreading 3'->5' exonuclease activity. Highly related to pol δ, and thought to be the main polymerase involved in leading strand synthesis, though there is again still debate about its role


DISCOVERING THAT DNA REPLICATION IS SEMI-CONSERVATIVE

The discovery that DNA replication is semi-conservative involved first growing a colony of E. coli cells in a medium containing a heavy isotope of nitrogen, 15N. While in this medium, any nitrogenous bases synthesized by the bacteria would be labelled with heavy nitrogen, 15N. The cells were then transferred to a medium containing regular nitrogen, 14N. Any bases incorporated while in this medium would contain only normal nitrogen.

The DNA was isolated from bacteria after living in these two media and centrifuged at 40,000 revolutions per hour in a caesium chloride solution. When left to disperse, the 'denser' nitrogen (15N) and normal nitrogen (14N) should appear as different bands in the solution. According to the semi-conservative theory, any DNA replication that occurred in the second medium would contain a mixture of one strand 15N to one strand 14N (as one parental strand was conserved from the medium containing the heavy nitrogen isotope). This would show in the solution as a single band. According the opposing, conservative theory, two bands should appear as some DNA would contain exclusively 15N and some exclusively 14N. One band appeared, supporting the semi-conservative theory.

Furthermore, after another round of replication in the second (normal nitrogen) medium, there should be some DNA containing the 15N/14N hybrid DNA and some containing exclusively 14N DNA. This would then generate two bands of DNA in the caesium chloride gradient and it indeed did. This further corroborated the semi-conservative theory.