are enzymes that elongate DNA molecules by adding nucleotides to the 3' end of a strand. The typical reaction involved is a nucleophilic attack. (Abeles, 1992
Polymerases in E. coli
The primary role of DNA Polymerase I (abreviated Pol I) in DNA replication is the removal of RNA primers present on the newly replicated DNA strand and the subsequent filling in of the resulting gaps between Okazaki fragments with nucleotides complementary to those present on the sequences of the template strand corresponding to the gap. Pol I does not possess a mechanism for linking these nucleotides with phosphodiester bonds, resulting in nicks along the sequence over which Pol I is active. (Lehman et al. 1958
) DNA polymerases rarely make errors, making fewer than one mistake per 10 million nucleotides added (Kunkel and McCulloch ,2008
). The nick left behind is connected by Ligase
DNA Polymerase II is responsible for repairing mutations in DNA molecules. When DNA polymerase II was replaced with a mutant version of Pol II, the mutation rate in the organism was more than 3 times that in an organism with a functioning Pol II. (Foster et al. 1995
)DNA Polymerase II is only used in DNA repair.
Pol III is composed of three sub units: α, є, and Ѳ. In addition to being a 5'-> 3' polymerase, it has both 3'→5' exonuclease activity and 5'→3' exonuclease activity. The 5'-> 3' exonuclease activity must start hydrolyzing the 5' end of a single-strand DNA polymer, but can continue hydrolysis into a double-stranded region (McHenry and Crow 1978
Discovery of DNA Polymerases
The enzyme DNA polymerase was discovered in the year 1955 Lehman 2003
. The first DNA polymerase, called DNA polymerase I or Pol I, was isolated from E. coli
by Arthur Kornberg during his time as the chairman of the Department of Microbiology at the Washington University School of Medicine. Subsequently, he and his colleages succeeded in elucidating a large portion of the mechanism of DNA replication in E. coli, for which Kornberg would be jointly awarded the 1959 Nobel Prize in Physiology or Medicine (Berg and Lehman 2007
) (Nicole et al. 2005
Kornberg and his team treated the E. coli
with streptomycin to obtain two different fractions of the cell extracts. The supernatant was free of any nucleic acids and the precipitate did contain nucleic acid. Each of these two fractions were divided further into two more fractions according to heat stability. The heat labile fraction of the precipitate was determined to be the DNA polymerase because of its capacity to catalyze the formation of phosphdiester bonds. (Lehman 2003
) DNA polymerase II, (Pol II), was discovered in 1970, 14 years after the original Pol I was isolated in E. coli
. Kornberg and Malcolm L. Gefter made the discovery while studying the role of Pol I in E. coli
DNA replication. Pol I was originally believed to be the only DNA polymerase taking part in DNA replication, but that was falsified through a 1969 study by British biologist John Cairns and his lab assistant Paula De Lucia. Cairns was able to isolate a distinctly different mutant form of Pol I. Treatment of this mutant form allowed Kornberg and Gefter to isolate Pol II
(Tessman and Kennedy 1994
DNA polymerase reaction
All DNA-dependent DNA polymerases operate in approximately the same way. The substrates are the 3'-hydroxyl end of the growing DNA strand, and a deoxyribonucleoside triphosphate or dNTP.
The oxygen in 3'-OH makes a nucleophilic attack on the alpha phosphate (the one closest to the sugar) of the dNTP. The oxygen has two orbitals full of electrons that aren't involved in bonding, and they are attracted to large nuclei with multiple protons, such as the phosphate nucleus. When these electrons move toward the phosphorus nucleus, there is the possibility of forming a covalent bond between the phosphorus and that oxygen. However, in order for this to happen, one of the oxygens already bound to the phosphorus must be displaced. Normally, this would be quite difficult, as the covalent bond between the oxygen and phosphorus is quite stable. In this case, though, one of those oxygens already bound to the phosphorus is also bound to another phosphate – see the figure above. In fact, it's bound to a group of two phosphates, called pyrophosphate (and also a Mg2+ ion, which is a cofactor for the enzyme). This group of two phosphates is an example of what is known as a "good leaving group." If the pyrophosphate group leaves the dNTP, it will quickly react with water to form two inorganic phosphates (Pi). Since pyrophosphate is so unstable and reactive, it doesn't last long at all in water; this means that its concentration is always low. Think of the reactions like this:
--> dNMP + pyrophosphate
---> dNMP + 2 Pi
Since the pyrophosphate concentration is always quite low, the reaction equilibrium is shifted forward; in other words, it's relatively likely that the pyrophosphate will come off the dNTP. That means that it's pretty easy to displace the oxygen in that pyrophosphate from the alpha phosphorus, so the nucleophilic attack succeeds. The result is that the dNMP (a deoxyribonucleoside monophosphate, or nucleotide) becomes covalently bound to the 3' carbon of the sugar at the end of the DNA strand, thus lengthening the strand by one nucleotide. Then the process repeats (Abeles, 1992
Why can't this happen at the other end of the strand?
Why 3' -> 5' won't work
Assume that the active site of DNA polymerase is going to orient the incoming dNTP so that a 5'-3' bond would form (rather than joining the 5' carbon of the dNTP to the 5' end of the DNA strand, upside down). So instead, the dNTP would be coming down toward the 5' end of the existing strand with its 3' OH down, to make a nucleophilic attack on the 5' phosphate of the DNA strand.
In order for the nucleophilic attack to succeed, an oxygen has to leave the phosphate on the 5' end of the DNA strand, but there's nothing else attached to it. It's not a good leaving group at all, so this reaction isn't favored. Therefore, DNA always grows from the 5' to 3' direction, by adding nucleotides onto the 3' end of the existing strand .
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Foster et al. (1995). Proofreading-defective DNA polymerase II increases adaptive mutation in Escherichia coli . Biochemisty, 92(17).
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