In Prokaryotes
The search for an enzyme that could synthesize DNA began in 1955. Work by Arthur Kornberg and colleagues led to the characterization and purification of DNA polymerase from E. coli cells, a single polypeptide enzyme now called DNA polemerase I (Mr 103,000). Later on investigators found that E. cloi cells contain at least four different DNA polymerases.
Features of DNA synthethic process common to all DNA polymerases:
1. The fundamental reaction is a nucleophilic attack by the 3�-hydroxyl group of the nucleotide at the 3� end of the growing strand on the 5� a phosphorous of the incoming deoxynucleoside 5� triphosphate.
2. Two central requirements of DNA polymerase I- require a template and requirement of primer.
Soon after the isolation of DNA polymerase I evidences began to accumulate that it is not suited for replication of large E. coli chromosome.
a) The rate at which it adds nucleotides (600 nucleotides/min) is too slow by a factor of 100 or more to account for the rate at which the replication fork is observed to move in the bacterial cell.
b) DNA polymerase I has a relatively slow processivity.
c) Genetic studies have demonstrated that many genes, and therefore many proteins, are involved in replication: DNA polymerase I do not act alone.
d) In 1969, John Cairns isolated a bacterial strain with an altered gen for DNA polymerase I that produced an inactive enzyme.�
In 1970s other DNA polymerases were discovered DNA polymerase II and DNA polymerase III. DNA polymerase IV and DNA polymerase V were identified in 1999, are involved in an unusual form of DNA repair.
DNA polymerase I is not the primary enzyme of replication; instead it performs a host of clean up functions during replication, recombination and repair. This special function is enhanced by its 5� to 3� activity. Because of this exonuclease activity, DNA polymerase I can replace a segment of DNA (or RNA) paired to the template strand in a process of nick translation. This activity is distinct from the 3� to 5� proofreading exonuclease. When the 5� to 3� exonuclease domain is removed, the remaining (Mr 68,000), called the large or Klenow fragment, retains the polymerization and proofreading activities.
DNA polymerase III is a very complex having ten types subunits. Its polymerizing and proofreading activities reside in subunits a and e. b The q subunit associated with a and e to form core polymerase, which can polymerize DNA but with limited processivity. Two core polymerases can be linked in a complex by a dimmer of t (tau)subunits. This dimeric polymerase complex can then associate with a clamp-loading complex, which consists of six subunits of five types, g2 dd� cy. This assembly of 14 protein subunits (9 different types) is called DNA polymerase III.
DNA polymerase II is an enzyme involved in DNA repair.
In Eukaryotes
Eukaryotes have several types of DNA polymerases.
The replication of nuclear chromosomes involves DNA polymerase a, in association with DNA polymerase d. DNA polymerase a is typically a multi subunit enzyme with similar structure and properties in all eukaryotic cells. One subunit has a primase activity and the largest subunit (Mr ~ 180,000) contains polymerization activity. However, this has no proof reading activity, making it unsuitable for high fidelity DNA replication. DNA polymerase a is believed to function only in the synthesis of short primers for Okazaki fragments on the lagging strand. Multi subunit DNA polymerase extends these primers d.
DNA polymerase e replaces DNA polymerase d in some situations, like in DNA repair. DNA polymerase e may also function at the replication fork, perhaps playing a role analogous to that of the bacterial DNA polymerase I.
