REPLICATION OF DNA How is cellular DNA copied?
DNA replication begins with a partial unwinding of the double helix at an area known as the replication fork. This unwinding is accomplished by an enzyme known as DNA helicase. This unwound section appears under electron microscopes as a "
bubble" and is thus known as a replication bubble.
As the two DNA strands separate
the enzyme
DNA polymerase moves into position at the point where synthesis will begin.
But where does the DNA polymerase enzyme know where to begin synthesis? Is there some sort of marker, a start point?
YES; the start point for DNA polymerase is a short segment of RNA known as an RNA primer. The very term "primer" is indicative of its role which is to "prime" or start DNA synthesis at certain points. The primer is "laid down" complementary to the DNA template by an enzyme known as RNA polymerase or Primase.
The DNA polymerase (once it has reached its starting point as indicated by the primer) then adds nucleotides one by one in an exactly complementary manner, A to T and G to C.
How does the polymerase "know" which base to add?
DNA polymerase is described as being "template dependent" in that it will
"read" the sequence of bases on the template strand and then
"synthesize" the complementary strand. The template strand is ALWAYS read in the 3' to 5' direction (that is, starting from the 3' end of the template and reading the nucleotides in order toward the 5' end of the template). The new DNA strand (since it is complementary)
MUST BE SYNTHESIZED in the 5' to 3' direction (remember that both strands of a DNA molecule are described as being
antiparallel. DNA polymerase catalyzes the formation of the hydrogen bonds between each arriving nucleotide and the nucleotides on the template strand.
In addition to catalyzing the formation of Hydrogen bonds between complementary bases on the template and newly synthesized strands, DNA polymerase also catalyzes the reaction between the 5' phosphate on an incoming nucleotide and the free 3' OH on the growing polynucleotide (what we know is called a phosphodiester bond!). As a result, the new DNA strands can grow only in the 5' to 3' direction, and strand growth must begin at the 3' end of the template, right? Again, note that a phosphodiester bond is formed between the 3' OH group of the sugar and the 5' phosphate group of the incoming nucleotide.
Because the original DNA strands are complementary and run antiparallel, only one new strand can begin at the 3' end of the template DNA and grow continuously as the point of replication (the replication fork) moves along the template DNA. The other strand must grow in the opposite direction because it is complementary, not identical to the template strand. The result of this side's discontiguous replication is the production of a series of short sections of new DNA called Okazaki fragments (after their discoverer, a Japanese researcher). To make sure that this new strand of short segments is made into a continuous strand, the sections are joined by the action of an enzyme called DNA ligase which LIGATES the pieces together by forming the missing phosphodiester bonds!
The last step is for an enzyme to come along and remove the existing RNA primers (you don't want RNA in your DNA now that the primers have served their purpose, do you?) and then fill in the gaps with DNA. This is the job of yet another type of DNA polymerase which has the ability to chew up the primers (dismantle them) and replace them with the deoxynucleotides that make up DNA. Here is a link with a diagram of the overall process of DNA replication of Okazaki Fragments.
Since each new strand is complementary to its old template strand, two identical new copies of the DNA double helix are produced during replication. In each new helix, one strand is the old template and the other is newly synthesized, a result described by saying that the replication is semi-conservative. This process is shown schematically below. Crick described the DNA replication process and the fitting together of two DNA strands as being like a hand in a glove. The hand and glove separate, a new hand forms inside the old glove, and a new glove forms around the old hand. As a result, two identical copies now exist.
The process of DNA replication in all organisms is amazing, but in humans it seems particularly difficult to conceive. The sum of all genes in a human cell-the human
genome-is estimated to be approximately
3 billion base pairs, and a single DNA chain might contain up to 250 million pairs of bases. What's even more incredible is how few mistakes are made in this process despite the immense size of human DNA! An error occurs only about once in each 10-100 billion bases. As you would probably expect, the complete process of DNA replication in human cells takes several hours. To replicate such huge molecules as human DNA at this speed requires not one, but many replication forks, forming replication bubbles and producing many segments of DNA strands that eventually meet up together and are joined to form the newly synthesized double helix.
