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DNA replication or DNA synthesis is the process of copying a double-stranded DNA strand, prior to cell division (in eukaryotes, during the S phase of 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.
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 proceeds away from the origin, unwinding and replicating the DNA strands as they move.
The factors involved are collectively called the pre-replication complex. They are the following:
- A helicase, which unwinds the DNA ahead of the fork.
- A primase, which generates an RNA primer to be used in DNA replication.
- A DNA holoenzyme, which is actually a complex of enzymes that performs the actual replication.
After the helicase unwinds the DNA, single-strand binding protein is used to hold the DNA strands in place. RNA primase is then 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 a leading strand. The opposite original strand, from 5’ to 3’, is a lagging strand.
Since the DNA replication on the lagging strand is not continuous, a new DNA polymerase has to be added each time as the helicase unwinds more DNA. As a result, the replicated DNA is fragmented, called Okazaki fragments. Another enzyme, DNA ligase, is used to connect the fragments.
Coupled leading strand and lagging strand synthesis is achieved by the action of the polIII holoenzyme.
When the polymerase reaches the end of replication, there is another problem due to the antiparallel structure. The RNA primer on the leading strand occupies a small portion of the DNA, which is not exposed to polymerase and therefore is not copied.
As a result, there would be a gap on the newly duplicated DNA at the original leading strand on the 5’ end. The solution is quite simple. The sticking out 3’ end consists of noncoding DNA called the telomere, which can be simply cut off.
Before the DNA replication is finally complete, enzymes are used to proofread the sequences to make sure the nucleotides are paired up correctly. If mistake or damage occurs, an enzyme called 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 average human chromosome contains an enormous number of nucleotide pairs that are copied at about 50 base pairs per second. Yet, the entire replication process takes only about an hour. 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.
With multiple replication origin sites, a question is: how does the cell know which DNA has already been replicated and which still awaits replication? To date, two replication control mechanism 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 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. These mutants should incorporate nucleotides into DNA at 30° but not at 42°C. Protein synthesis should not be affected.
There are two outcomes for a graph of incorporation of labelled nucleotides into DNA vs time:
- Quick stop indicates the mutation is in a DNA synthesis factor.
- Slow stop indicates the mutation is possibly in an initiation factor. (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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