DNA Replication

DNA replicates by the semiconservative model of replication. This model states that during replication of double stranded DNA, the two strands separate and each separate strand is then used as a template to make a new strand. In this manner, the cell ends up with two identical molecules of the original DNA, with each molecule containing one strand from the original piece of DNA and one newly synthesized strand. This model was first proposed by Watson and Crick and was confirmed by the experiments of Meselson and Stahl. Be sure to read about these experiments in your text!

DNA Polymerase

The enzyme that catalyzes DNA replication is DNA Polymerase. Don't confuse this with any other enzyme (meaning RNA polymerase - DNA polymerase is used to replicate DNA when the cell is undergoing mitosis or meiosis, RNA polymerase catalyzes transcription).

DNA polymerase has the following properties:

  1. requires the four nucleotides as the triphosphate forms, ATP, GTP, TTP and CTP
  2. requires single-stranded DNA as a template
  3. always adds nucleotides to the free 3' end of an existing strand
  4. requires a primer, an existing strand of nucleic acids

Replication

The replication of DNA requires more than just DNA polymerase. For example, the two strands of DNA must be separated before DNA polymerase can replicate the strands. The enzyme that separates the strands is known as helicase. This enzyme first binds to a specific site on the DNA known as the origin of replication (prokaryotes have only a single origin on their chromosome while eukaryotes have several on each of their chromosomes) and separates the strands - this allows the DNA polymerase to bind. As the replication process continues, the helicase moves down the DNA and to continue separating the DNA strands. Once the stands are separated, there are at least three problems that now must be addressed: First of all, the separation of the strands of DNA induces supercoiling in the remaining part of the DNA. This is similar to what happens when your coiled telephone cord gets overwound and forms knots. An enzyme known as topoisomerase (a.k.a. gyrase) relieves the supercoiling. In addition, single-stranded DNA is not very stable and is susceptable to degradation. To prevent this, there are a set of single-stranded binding proteins that are needed to stabilize the separated strands during replication. Finally, DNA polymerase requires a primer to operate. That is, the polymerase can only add nucleotides to an existing strand (the primer!). This existing strand is laid down by the enzyme primase. Primase lays down a strand of about 8-10 nucleotides that is complementary to the replicating strand. DNA polymerase can then add the remaining nucleotides to the 3' end of the primer. One more thing, the primer is composed of RNA nucleotides!

Replication starts at the origin and progresses in both directions. As the strands are separated, they form replication forks which are the sites of the replication. Because DNA polymerase can only add to the 3' end, the replication of oned of the strands is continuous while the other is replicated in small fragments known as Okazaki fragments. This is shown in the following figure.

There are now two more problems: How do the Okazaki fragments get joined together? and how does cell remove the RNA primer. An enzyme known as ligase connects the various fragments of the newly synthesized DNA. The RNA primers are replaced with DNA by a special form of DNA polymerase.

 

 

 

 

This figure was obtained from http://esg-www.mit.edu:8001/esgbio/dogma/repl.html

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