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DNA replication

From Molecular Biology Wiki

Contents

Steps

The following are the steps in the replication of DNA.


This video is a summary of DNA Replication.

Initiation

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.

The factors involved are collectively called the pre-replication complex. It consists of the following:

  • 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.

Replication

After the helicase unwinds the DNA, single-strand binding protein is used to hold the DNA strands apart. 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 the leading strand. The complement of the leading strand, from 5’ to 3’, is the lagging strand.

Molecular Visualizations of DNA Replication.


Each time the helicase unwinds additional DNA, a (potentially) new DNA polymerase needs to be added. 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.

Coupled leading strand and lagging strand synthesis is achieved by the action of the polIII holoenzyme.

Termination

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 terefore 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 of non-circular (viz. eukaryotic) chromosomes. 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.

Equation

A chemical equation can be written that represents the process: (DNA)n + dNTP ↔ (DNA)n+1 + PPi

Summary

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

Conditional mutants

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:

  1. Quick stop indicates the mutation is in a DNA synthesis factor.
  2. 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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