DNA Polymerase , Definition,Structure and Function

DNA polymerase is an enzyme that is capable of synthesizing DNA molecules from deoxyribonucleotides (dNTP’s), which are the building blocks of a DNA molecule. It allows the strands of DNA to be duplicated. These enzymes store information present in DNA, allowing the new cell to contain the same information. These enzymes can use strands of DNA as a guide, or template, and assist in synthesizing the duplicate strands. They are also useful in the process of DNA repair. Mainly, these enzymes are vital for DNA replication because they guarantee the faithful duplication of DNA.


DNA Polymerase Definition, Structure, and Reaction



Structure of DNA Polymerase:

So far, the crystal structures that have been obtained for many DNA polymerases from all kingdoms of life have revealed a quite common structure. The overall architecture of a DNA pol resembles a human right hand and consists of three domains: palm, fingers and thumb. Upon binding of the DNA pol to the template/primer and the dNTP, conformational changes in the DNA pol as well as in the DNA itself lead to the assembly of a DNA polymerase active site. These changes allow a catalytically competent DNA polymerase to exert its function. The finger domain interacts with the incoming dNTP and single-stranded DNA. The thumb on the other hand binds to double-stranded DNA. This double-stranded DNA is either a primer hybridized to a template or represents the newly synthesized DNA with its templating strand. The palm domain harbors the catalytic residues that bind magnesium ions which are required for the phosphoryl transfer reaction. The phosphoryl transfer reaction includes the attachment of the dNMP to the 3 prime -OH group of the primer or the growing DNA chain and the release of pyrophosphate. The palm domain, where, as indicated, the catalytic reaction occurs, is extremely conserved in most DNA polymerases analyzed so far. Exceptions are:

(I)    The pol β-like nucleotidyltransferase superfamily X enzymes such as DNA pol β.

(ii)  The DNA pols III from E. coli and Thermus aquaticus and the DNA pol X from Deinococcus radiodurans.

 Despite these differences, all DNA pols utilize the same two-metal (mostly magnesium)-ion mechanism for dNTP addition. In contrast to the widely conserved palm domain, the fingers and the thumbs differ in the various DNA pol families.

 

DNA Polymerase Reaction:

Requirements:

A DNA pol needs:

(i)       The four bases in their activated form as deoxyribonucleoside triphosphates (dATP, dTTP, dCTP and dGTP) as substrate,

(ii)     A DNA template that instructs the DNA pol which bases to incorporate (A to T and C to G and vice versa),

(iii)    A primer containing a 3 prime -OH group at its end which is hybridized to a template (there are only a few exceptions to this, e.g. the so-called protein priming in bacteriophage φ29 and in adenovirus),

(iv)    The divalent cation magnesium as a cofactor,

      (v)     In some cases, other proteins called DNA pol auxiliary proteins.


DNA Polymerase Reaction


Mechanism:

A base is incorporated by the DNA Polymerase in the following 4 to 5 steps;

In the first step, the incoming dNTP is bonded to the templating base with the help of hydrogen bond (A to T and G to C and vice versa);

In the second step, water is excluded from the active site of DNA Polymerase;

In the third step, the geometric selection in the active site of DNA Polymerase takes place;

In the fourth step, the dNTP binding affinity leads to an induced conformational change in the active site and in the fifth step, the formation of a phosphodiester bond between the last base of the primer and the new nucleotide takes place by the release of pyrophosphate (PPi).

 DNA pols, as everything else in nature, can make mistakes and occasionally incorporate wrong bases. To reduce the amount of those mistakes many, but not all DNA pols, contain a second activity, the 3 → 5 exonuclease. This so-called proofreading exonuclease activity removes non-properly paired bases. Such incorrectly base paired nucleotides can either derive from a wrong pairing of two normal nucleotides or be caused by altered or missing bases.

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