Elementary plant Biochemistry and Biotechnology (2+1)

Mechanism of DNA replication

Initiation

• Most organisms contain one or more enzymes called topoisomerases, which can produce variety of topological changes in DNA. The brief outline of replication mechanism in E. coli is described here. An enzyme called as helicase binds with single strand binding (SSB) protein along with DnaB protein and unwinds the helix. The unpaired bases are coated with SSB. DNA gyrase (Eco topoisomerase II) has the ability to produce negative superhelicity generated during replication. That is, positive superhelicity is removed by gyrase introducing negative twists by binding ahead of the advancing replication fork.

Elongation

• The leading strand advances along one parental strand by nucleotide addition catalyzed by the pol III holoenzyme (DNA polymerase III). The term holoenzyme refers to an enzyme that contains several different subunits and retains some activity even when one or more subunits are missing. In E. coli, two types of DNA polymerases exist viz., pol I and pol III. They are able to synthesize DNA from four precursor molecules, four-deoxynucleoside 5’-triphosphates (dNTPs viz., dATP, dGTP, dTTP and dCTP), as long as a DNA molecule to be copied (template DNA) is provided. Neither 5’monophosphates nor 5’ diphosphates, nor 3’-mono, di, tri-phosphates can be polymerased; only the 5’triphosphates are substrate for the polymerization reaction. In addition they require nucleic acid fragment to initiate the polymerization. The overall chemical reaction catalyzed by the DNA polymerase is:
Poly (nucleotide)n-3’-OH + dNTP Poly (nucleotide)n+1-3’-OH + PPi

• The polymerase also catalyzes depolymerization. In order to drive the reaction to the right, pyrophosphate must be removed, and this is done by a potent pyrophosphatase, a widely distributed enzyme that breaks down pyrophosphate to inorganic phosphate.


• In addition, polymerization occurs only is the presence of primer- that is an oligonucleotide hydrogen bonded to the template strand and whose terminal 3’-OH group is available for reaction. Because polymerization consists of a reaction between a 3’OH group at the end of the growing strand and an incoming nucleoside-5’ triphosphate. When the nucleotide is added it supplies another free 3’-OH group.


• The primer for both leading and lagging strand synthesis is a short RNA oligonucleotide that consists of 1 to 60 bases; the exact number depends on the particular organism. This RNA primer is synthesized by copying a particular base sequence from one DNA strand and differs from a typical RNA molecule, in that after its synthesis the primer remains hydrogen bonded to the DNA template.


• In bacteria two different enzymes are known that synthesize primer RNA molecules – RNA polymerase and Primase. The DnaB protein complex moves along the other parental strand, prepriming it so that primase will synthesis a primer RNA. Pol III holoenzyme adds nucleotides to the primer, thereby synthesizing a precursor fragment. This synthesis continues up to the primer of the preceding precursor fragment.


• Apart from this function, DNA polymerases also has 3’→5’ exonuclease, 5’→3’ exonuclease and endonuclease activity and so they can perform nick translation and strand displacement. By nick translation the RNA is removed and replaced by DNA. Once the RNA is gone, DNA ligase seals the nick, thereby joining the precursor fragment to the lagging strand. Pol II moves back along the DNA (in the direction of advancement of the fork) until it encounters the next primer and the process continue again and again.


• Since each strand has 5’-P terminus and 3’-OH terminus, strand growth is said to proceed in the 5’→3’ direction (Fig). The advance of the replication fork continues until replication is completed. An unsolved question is how the rates of growth of the leading and lagging strands are coordinated.

Termination

• In a unidirectionally replicating molecule, replication terminates at the origin. In bidirectionally replicating molecule, it may be of two types: 1. there might be definite termination sequence. 2. two growing points collide and termination occurs where ever the collision point happens to be.

Replication in Eukaryotes

• The complete mechanism of initiation, elongation and termination of linear DNA molecule and chromatin replication has not yet been elucidated. However, it is believed that there might be multiple replication forks exist during replication. Similarly different isoforms of DNA polymerases have been identified in eukaryotes with specific functions.

Fidelity of DNA replication

• There is no single molecule whose integrity is as vital to the cell as DNA. Thus, in the course of hundreds of millions of years there have evolved efficient systems for correcting the occasional mistakes that occur during replication. DNA repair/damage can occur as the result of exposure to environmental stimuli such as alkylating chemicals or ultraviolet or radioactive irradiation and free radicals generated spontaneously in the oxidizing environment of the cell. These phenomena can, and do, lead to the introduction of mutations in the coding capacity of the DNA. Mutations in DNA can also, but rarely, arise from the spontaneous tautomerization of the bases (the rare imino form of adenine can form a stable hydrogen bond with cytosine and the enol form of thymine can pair with guanine).


• E. coli cells possess at least five distinct mechanisms for the repair of defects in DNA: 1) light-dependent repair or photoreactivation, 2) excision repair, 3) mismatch repair, 4) post-replication repair and 5) error-prone repair. Mammals seem to possess all of the repair mechanisms found in E. coli except photoreactivation.