DNA replication or DNA synthesis is the process of copying a double-stranded DNA strand in a cell, prior to cell division. In eukaryotes, this is during the S phase of the cell cycle, preceding mitosis and meiosis. The two resulting double strands are identical (if the replication went well), and each of them consists of one original and one newly synthesized strand. This is called semiconservative replication. The process of replication consists of three steps, initiation, replication and termination. Artificial DNA replication is carried out through polymerase chain reaction.
DNA replication. In the first step, the double helix shown above in blue is unwound by a helicase. Next, a molecule of DNA polymerase shown in green binds to one strand of the DNA. It moves along the strand, using it as a template for assembling a leading strand shown above in red of nucleotides and reforming a double helix. A second DNA polymerase molecule (also green) is used to bind to the other template strand as the double helix opens. This molecule must synthesize discontinuous segments of polynucleotides (called Okazaki fragments). Another enzyme, DNA ligase shown in violet, then stitches these together into the lagging strand.
In the initiation step, several key factors are recruited to an origin of replication. This origin of replication is unwound, and the partially unwound strands form a "replication bubble", with one replication fork on either end. Each group of enzymes at the replication fork moves away from the origin, unwinding and replicating the original DNA strands as they proceed. Primers mark the individual sequences and their start and end points, to be replicated.
The factors involved are collectively called the pre-replication complex. It consists of the following:
* A topoisomerase, which introduces negative supercoils into the DNA in order to minimize tortional strain induced by the unwinding of the DNA by helicase. This prevents the DNA from knotting up.
* A helicase, which unwinds and splits the DNA ahead of the fork. Thereafter, single-strand binding proteins (SSB) swiftly bind to the separated DNA, thus preventing the strands from reuniting.
* A primase, which generates an RNA primer to be used in DNA replication.
* A DNA holoenzyme, which in reality is a complex of enzymes that together perform the actual replication.
After the helicase unwinds the DNA, RNA primase is bound to the starting DNA site.
At the beginning of replication, an enzyme called DNA polymerase binds to the RNA primase, which indicates the starting point for the replication. DNA polymerase can only synthesize new DNA from the 5’ to 3’ (of the new DNA). Because of this, the DNA polymerase can only travel on one side of the original strand without any interruption. This original strand, which goes from 3’ to 5’, is called the leading strand. The complement of the leading strand, from 5’ to 3’, is the lagging strand.
Each time the helicase unwinds additional DNA, new DNA polymerase needs to be added to ensure there remains enough. As a result, the DNA of the lagging strand is replicated in a piecemeal fashion. Another enzyme, DNA ligase, is used to connect the so-called Okazaki fragments.
In prokaryotes, coupled leading strand and lagging strand synthesis is achieved by the action of the DNA polymerase III holoenzyme.
In eukaryotes, there are a number of DNA polymerases with exonuclease and proof-reading abilities to carry out replication.
Termination occurs when DNA replication forks meet one another or run to the end of a linear DNA molecule. Also, termination may occur when a replication fork is deliberately stopped by a special protein, called a replication terminator protein, that binds to specific sites on a DNA molecule.
When the polymerase reaches the end of a length of DNA, there is a potential problem due to the antiparallel structure of DNA. Because an RNA primer must be regularly laid down on the lagging strand, the last section of the lagging-strand DNA cannot be replicated because there is no DNA template for the primer to be synthesized on. To solve this problem, the ends of most chromosomes consist of noncoding DNA that contains repeat sequences. The end of a linear chromosome is called the telomere.
The repeat DNA in the telomere is not essential for survival, because it does not contain genes, so cells can endure the shortening of the chromosome at the telomere. Many cells use an enzyme called telomerase that adds the repeat units to the end of the chromosome so the ends do not become too short after multiple rounds of DNA replication. Many simple, single-celled organisms overcome the whole problem by having circular chromosomes.
Before the DNA replication is finally complete, enzymes are used to proofread the sequences to make sure the nucleotides are paired up correctly in a process called DNA repair. If mistake or damage occurs, enzymes such as a nuclease will remove the incorrect DNA. DNA polymerase will then fill in the gap.
A chemical equation can be written that represents the process:
(DNA)n + dNTP <-->(DNA)n+1 + PPi
The human genome contains 6 billion nucleotide pairs (arrayed in 46 linear chromosomes) that are copied at about 50 base pairs per second by each replication fork. Yet, in a typical cell the entire replication process takes only about 8 hours. This is because there are many replication origin sites on a eukaryotic chromosome. Therefore, replication can begin at some origins earlier than at others. As replication nears completion, "bubbles" of newly replicated DNA meet and fuse, forming two new molecules.
There must be some form of regulation and organisation of these multiple replication sites to prevent conflict. To date, two replication control mechanisms have been identified: one positive and one negative. For DNA to be replicated, each replication origin site must be bound by a set of proteins called the origin recognition complex. These remain attached to the DNA throughout the replication process. Specific accessory proteins, called licensing factors, must also be present for initiation of replication. Destruction of these proteins after initiation of replication prevents further replication cycles from occurring. This is because licensing factors are only produced when the nuclear membrane of a cell breaks down the during mitosis.
Measurement of DNA replication can be done using conditional mutants. Mutants that grow at 30°C but not at 42°C are collected. At this temperature these mutants should incorporate nucleotides into DNA. Protein synthesis should not be affected.
There are two outcomes for a graph of incorporation of labelled nucleotides into DNA vs time:
1. Quick stop indicates the mutation is in a DNA synthesis factor.
2. Slow stop indicates the mutation is possibly in an initiation factor such as dnaA.
The assay can measure the incorporation of deoxyribonucleotides into acid or ethanol insoluble forms. Gel filtration chromatography or ion exchange chromatography is used to get all protein fractions and is followed by assay for DNA polymerase.
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