Central Dogma

DNA Replication

Messelson and Stahl

1. Did the two strands unwind and each act as a template for new strands? This is semiconservative replication, because each new strand is half comprised of molecules from the old strand.
2. Did the strands not unwind, but somehow generate a new double stranded DNA copy of entirely new molecules? This is conservative replication.

In order to determine which of these models was true, the following experiment was performed: The original DNA strand was labelled with the heavy isotope of nitrogen, N-15. This DNA was allowed to go through one round of replication with N-14, and then the mixture was centrifuged so that the heavier DNA would form a band lower in the tube, and the intermediate (one N-15 strand and one N-14 strand) and light DNA (all N-14) would appear as a band higher in the tube. The expected results for each model were:

The actual results were as expected for the semiconservative model and thus Watson and Crick's suspicion was borne out.

Biochemical Mechanism of DNA Replication

The Enzymes of DNA Replication

1. Topoisomerase is responsible for initiation of the unwinding of the DNA. The tension holding the helix in its coiled and supercoiled structure can be broken by nicking a single strand of DNA. Try this with string. Twist two strings together, holding both the top and the bottom. If you cut only one of the two strings, the tension of the twisting is released and the strings untwist.

2. Helicase accomplishes unwinding of the original double strand, once supercoiling has been eliminated by the topoisomerase. The two strands very much want to bind together because of their hydrogen bonding affinity for each other, so the helicase activity requires energy (in the form of ATP ) to break the strands apart.

3. DNA polymerase proceeds along a single-stranded molecule of DNA, recruiting free dNTP's (deoxy-nucleotide-triphosphates) to hydrogen bond with their appropriate complementary dNTP on the single strand (A with T and G with C), and to form a covalent phosphodiester bond with the previous nucleotide of the same strand. The energy stored in the triphosphate is used to covalently bind each new nucleotide to the growing second strand. There are different forms of DNA polymerase , but it is DNA polymerase III that is responsible for the processive synthesis of new DNA strands. DNA polymerase cannot start synthesizing de novo on a bare single strand. It needs a primer with a 3'OH group onto which it can attach a dNTP. DNA polymerase is actually an aggregate of several different protein subunits, so it is often called a holoenzyme. The holoenzyme also has proofreading activities, so that it can make sure that it inserted the right base, and nuclease (excision of nucleotides) activities so that it can cut away any mistakes it might have made.

4. Primase is actually part of an aggregate of proteins called the primeosome. This enzyme attaches a small RNA primer to the single-stranded DNA to act as a substitute 3'OH for DNA polymerase to begin synthesizing from. This RNA primer is eventually removed by RNase H and the gap is filled in by DNA polymerase I.

5. Ligase can catalyze the formation of a phosphodiester bond given an unattached but adjacent 3'OH and 5'phosphate. This can fill in the unattached gap left when the RNA primer is removed and filled in. The DNA polymerase can organize the bond on the 5' end of the primer, but ligase is needed to make the bond on the 3' end.

6. Single-stranded binding proteins are important to maintain the stability of the replication fork. Single-stranded DNA is very labile, or unstable, so these proteins bind to it while it remains single straded and keep it from being degraded.

The Replication Fork

Why can DNA polymerase only act from 5' to 3'? The reason is the relative stability of each end of DNA. A triphosphate is required to provide energy for the bond between a newly attached nucleotide and the growing DNA strand. However, this triphosphate is very unstable and can easily break into a monophosphate and an inorganic pyrophosphate, which floats away into cell. At the 5' end of the DNA, this triphosphate can easily break, so if a strand has been sitting in the cell for a while, it would not be able to attach new nucleotides to the 5' end once the phosphate had broken off. On the other hand, the 3' end only has a hydroxyl group, so as long as new nucleotide triphosphate are always brought by DNA polymerase, synthesis of a new strand can continue no matter how long the 3' end has remained free.

This presents a problem, since one strand of the double helix is 5' to 3' , and the other one is 3' to 5'. How can DNA polymerase synthesize new copies of the 5' to 3' strand, if it can only travel in one direction? This strand is called the lagging strand, and DNA polymerase makes a second copy of this strand in spurts, called Okazaki fragments, as shown in the diagram. The other strand can proceed with synthesis directly, from 5' to 3', as the helix unwinds. This is the leading strand.