DNA polymerization reactions and DNA polymerases.

Arthur kornberg first discovered DNA polymerase I. (DNa polymerase I., short-story DNA polI.) and later DNA polymerase II. and DNA polymerase III. (DNa polymerase II., III., short-write DNA pol II., DNA pol III.) Experiments have shown that the primary process of DNA replication in E. coli works on DNa pol III., while DNA polI. and DNA polII. play a role in the correction and repair of DNA misalmicks.

common to this enzyme is that

(1) requires a DNA template, so these enzymes are also known as DNA-dependent DNA polymerases (DNa dependent DNA polymerase, DDDP).

(2) requires RNA or DNA as primer( primer), i.e. DNA polymerases cannot catalyz the beginning of DNA from the beginning.

(3) catalytic dNTP is added to the 3' H end of the quotation, so the direction of DNA synthesis is 5' →3'.

(4) all three DNA polymerases are multi-functional enzymes that function at different stages of the DNA replication and repair process. Since DNA polymerase I. is the clearest studied and represents the basic characteristics of other DNA polymerases, we highlight the role of DNa pol I. and point out the specificity of two other DNA pols:

1.DNA polymerase I.:

DNA polI. is composed of achain with a molecular weight of 109KD. Enzyme molecules contain a Zn plus, which is necessary for polymerization activity.

E. coli has about 400 enzyme molecules per cell, each of which catalyzes 667nucleotides into DNA at 37 degrees C per minute. In the chain, the enzyme water can be mediated into two fragments with > a large molecular weight of 76KD, commonly referred to as a klenow fragment, and a small fragment of 34KD, using the dead herbSize fragments have different enzyme activity.

(1) polymerization activity of DNA polymerase 5' →3':

This is the primary activity of DNA polymerase, which adds complementary dNTP to the end of the quote RNA3'H one by one, in the order of nucleotides on the template DNA, and promotes 3 H and dNTP's 5' O4 form a phosphate phosphate bond, the enzyme's specificity is reflected in the newly entered dNTP must be paired with the template DNA base to have catalytic action, 5' →3' polymerization activity is present in the klenow fragment.

(2) DNA polymerase 3' →5' exochesnucleic acidase activity:

the main function of this enzyme activity is from 3 →' The 5' direction identifies and removes nucleotides that are free from the end of the DNA growth chain and are not paired with the template DNA, a function known as proofreading, which is essential to ensure the correctness of its polymerization and is therefore essential for the high fidelity of DNA replication.

(3) DNA polymerase 5' →3" excision ef activity:

this enzyme activity is from the 5' end of the DNA chain to the end of 3' hydrolyzed nucleotides, essentially by cutting off the phosphate phosphate key, can remove 10 nucleotides at a time. Therefore, this enzyme activity may play an important role in the repair of DNA damage and is necessary for the removal of RNA quotations at the 5' end of the completed DNA fragment.

the synergy between the polymerization activity of 5' →3' and the activity of 5' →3' exoceptase, which allows the inlet on a chain of DNA to move from the direction of 5' →3', a reaction called nick translation, which uses this reaction In-body dna fragments can be labeled with radioactive phosphorus (α-32PdNTP) into probes, nucleic acid molecular hybridization experiments, is an important technology of modernmolecular biology.

many experiments have confirmed that DNA polI. is not the main enzyme in DNA replication, and that its role is mainly related to post-DNA repair.

2.DNA polymerase II. (DNA pol II.)

This enzyme molecular weight is 120KD, about 1 per cell 00 enzyme molecules, but only 5% of the activity of DNa polI. , it has a polymerization activity of 5' →3' and 3' →5' exoches activity, but not 5' →3' exoches activity, its role may be related to DNA damage repair.

3.DNA polymerase III. (DNA pol III.)

this is the polymerase that plays a major role in DNA replication, it is composed of a multi-sub-baseprotein molecule, with a molecular weight of >600kDa the entire enzyme molecule forms an asymmetric secondary, with only 10?0 enzyme molecules per E. coli cell, but the rate at which catalytic dNTP is involved in the DNA chain is the fastest, at about 9000 nucleotides per minute/per enzyme molecule.

also proves that DNa pol III. is the enzyme that mainly play a role in DNA replication. When THE E. coli chromosome DNA is copied, the DNA polymerase III. Whole enzyme does not act alone, but forms a replicator (replisome) with the trigger, mesolycerase, etc. Because of the replica, the pilot chain and the entourage chain can be copied at the same time.

DNa pol III. is an asymmetric djure made up of polyaquits, which may be responsible for both pilot and entourage chain replication, and in φ×174 replication it was observed that the trigger was always accompanied by the presence of a DNA loop.

it can be seen that because the template DNA of the entourage chain rotates 180 degrees around the DNA polymerase III. whole enzyme to form a slug, the synthesis direction of the Gangsaki fragment can be consistent with the synthesis direction of the pilot chain and the direction of the replicator movement.

as DNA pol III. moves forward, the synthesis of the pilot chain gradually extends, as does the Gangsaki fragment, which is also expanding. When the Gangsaki fragment is synthesized to the 5' end of the previous fragment, the slug is released, and as the copy fork moves forward, the template of the other part of the entourage chain can be replaced, the new quotation is synthesized by the trigger, and then a small slug is formed to synthesize the new Gangzaki fragment.

from this model, it is not difficult to see that the synthesis of the entourage chain needs to be triggered periodically, so its synthesis progress is always one gangsaki fragment from the leading chain. Upon completion of the Gangsaki fragment, the RNA quotation at the 5' end is removed by DNa pol I.'s 5' →3' excision enzyme activity, and the resulting void is filled by DNA polI.'s 5' →3' polymerization activity catalytic dNTP.

so DNA replication is done in conjunction with DNa pol III. and DNA polI.

list below shows the properties of the three E. coli DNA polymerases

Table 16-1 E. coli DNA polymerase characteristics