Discovery of polymerase

In the mid-1950s, A. Kornberg and his colleagues thought that DNA replication must be the catalyst of an enzyme, and determined to isolate the enzyme and study its structure and mechanism of action. To this end, they isolated the protein, and then added to the in vitro synthesis system that isotope-labeled dNTP, Mg2 + and template DNA, after a lot of work, in 1956 finally found DNA polymerase I. DNA polymerase I was originally called Kornberg enzyme. DNA polymerase II and DNA polymerase III were subsequently found. DNA polymerase I began to think that DNA is the main bacteria in bacteria replication, polo ralph lauren, DNA polymerase I was found mutants can still copy, it is clear that it is not the protagonist. It is now known that DNA polymerase III plays a dominant role in DNA replication, and the function of polymerase II is not well understood.

The common features of DNA polymerase

1、deoxynucleotide triphosphate (dNTP) as the precursor catalytic synthesis of DNA;

2、can not start a new DNA chain, there must be primers to provide 3'-OH;

3、The dNTPs were added to the 3'-OH end of the growing DNA strand and the direction of synthesis was 5 '→ 3'.

The functions of polymerase

[1] Polymerization: the primer RNA'-OH end to dNTP as substrate, according to the instructions on the template DNA polymerase I one by one to add the nucleotide is DNA polymerase I polymerization. The specificity of the enzyme mainly for the newly entered deoxynucleotides must be matched with the template DNA only when catalytic. DNTP into the binding site, the enzyme may change the conformation, and promote the 3'-OH and 5'-PO4 to form phosphodiester bond. If the wrong nucleotide into the binding site, it can not be paired with the template, can not change the enzyme conformation and 3'-5 'exonuclease active sites identified and excised.

[2] 3 '→ 5' exonuclease activity - proofreading: The main function of this enzyme activity is from 3 'to 5' direction to identify and excise the end of unpaired DNA growth chain nucleotides. When there is no reaction substrate dNTP in the reaction system, there is no polymerization phenomenon due to the temporary free phenomenon, which was 3 '→ 5' exonuclease activity degradation. Increasing the temperature of the reaction system can promote this effect, indicating that the temperature increases the chance of the 3 'end of the DNA growth chain to separate from the template and thus the degradation is enhanced. When the dNTPs are added to the reaction system, only the above-mentioned nucleotides complementary to the template are added to inhibit the exonuclease activity and the DNA synthesis is continued.

[3] 5 '→ 3' exonuclease activity - excision repair: 5 '→ 3' exonuclease activity is from 5 '→ 3' direction DNA hydrolysis of DNA in front of the chain, the main produce 5'-deoxy Nucleotides. This enzyme activity only on the mating part of the DNA (double-chain) phosphodiester bond cleavage activity in the direction of 5 '→ 3'. Each time to remove 10 nucleotides, and DNA polymerization can stimulate the 5 '→ 3' exonuclease activity of 10 times. Therefore, this enzyme activity may play an important role in the repair of DNA damage. The removal of the 5 & apos; -terminal RNA primer from the Okazaki fragment depends on this exonuclease activity.

[4] pyrophosphorolysis: DNA polymerase I this activity can catalyze 3 'end pyrophosphorylated DNA molecules. This action is the decomposition of inorganic pyrophosphate DNA growth chain, DNA polymerization can be considered the reverse reaction, and the role of DNA hydrolysis that requires the presence of template DNA.

[5] pyrophosphate exchange: catalytic dNTP end of the PPi with inorganic pyrophosphate exchange reaction.