The enzymes which take part in replication are able to copy DNA molecules which may contain millions of bases. They perform this function with utmost accuracy and at high speed, even though DNA molecule is highly compact and is bound with proteins. Maintenance of integrity of genetic information is the main feature of replication.
In E. coli, two main enzymes that take part in polymerization are DNA polymerase I and DNA polymerase III. Of these DNA polymerase III is the main enzyme involved in replication. Polymerization involves addition of new nucleotides to a growing strand.
In addition, there is an enzyme DNA polymerase II which takes part in DNA repair. DNA polymerase IV and DNA polymerase V have also been discovered. DNA polymerases are capable of adding 1000 nucleotides per second. The speed of DNA synthesis is known as processivity.
DNA replication would not occur without enzymes that catalyze various steps in the process. Enzymes that participate in the eukaryotic DNA replication process include:
● DNA helicase - unwinds and separates double stranded DNA as it moves along the DNA. It forms the replication fork by breaking hydrogen bonds between nucleotide pairs in DNA.
● DNA primase - a type of RNA polymerase that generates RNA primers. Primers are short RNA molecules that act as templates for the starting point of DNA replication.
● DNA polymerases - synthesize new DNA molecules by adding nucleotides to leading and lagging DNA strands.
● Topoisomerase or DNA Gyrase - unwinds and rewinds DNA strands to prevent the DNA from becoming tangled or supercoiled.
● Exonucleases - group of enzymes that remove nucleotide bases from the end of a DNA chain.
● DNA ligase - joins DNA fragments together by forming phosphodiester bonds between nucleotides.
Polymerization
Nucleotide monophosphates, which are building blocks of DNA, cannot be added to the growing strand of DNA. The DNA polymerase can act only on deoxyribose triphosphate nucleotides. They are dATP, d GTP, d CTP and dTTP. Triphosphate nucleotides possess high-energy phosphate bonds.
DNA polymerase I and DNA polymerase III can add new nucleotides at the 3′ OH end of the growing strand. Inorganic pyrophosphate is released. Hydrolysis of pyrophosphate is the driving force for DNA synthesis. Phosphodiester bond is formed between 3′-end of growing strand and 5′-end of first phosphate of incoming nucleotide.
Template
The two DNA strands separate and each acts as a template for the formation of new strand. Polymerization-reaction is dictated by a template strand according to the base pairing rules where if adenine is present in the template, thymine is added to the new strand and guanine pairs with cytosine.
Primer
DNA polymerase needs primer to synthesize new strand on it. Primer is a small strand segment which is complementary to the template. It has a free 3′-OH end to which a new nucleotide can be added. In this way part of the new strand is already in place. Primer is hydrogen bonded to the template to form primer: template junction. Primer is short nucleotide strand (oligonucleotide). The primer for both the new strands is RNA primer. The RNA primer is synthesized by an enzyme, primase.
DNA polymerase moves along the template adding nucleotides. Thus DNA is synthesized by extending the 3′-end of the primer. Afterwards, primer is removed by DNA polymerase I and RNAse H.
Leading strand requires only a single RNA primer. On the other hand, discontinuous synthesis of lagging strand requires primer for each Okazaki fragments. For synthesis of lagging strand hundreds of Okazaki fragments with their associated RNA primers are required.
In this way two conditions are necessary for DNA synthesis. They are a template and a primer with 3′-OH end.
DNA Synthesis takes Place in 5. → 3′ Direction Only
A new strand of DNA is always synthesized in 5′ → 3′ direction. The free 3′-end enables it to be elongated. Because the two strands are antiparallel, orientation of new growing strand is opposite to the template strand.
DNA Replication is Discontinuous in One Strand
If the synthesis is to proceed in 5′ → 3′ direction, only one strand can be synthesized in correct 5′ → 3′ direction. As the strands are antiparallel, the other strand will have to be synthesized in 3′ 5′ direction.
This constraint is overcome in an ingenious way. As the two strands unwind and the replication fork grows, one strand is synthesized in 5′ 3′ direction correctly in a continuous manner. This is called leading strand. It grows in the same direction as the replication fork.
Mr. Reiji Okazeki discovered that the other strand is synthesized discontinuously and a little after the leading strand. Therefore this is called lagging strand. The lagging strand also grows in 5′ 3′ direction which is opposite to the direction of replication fork. In lagging strand, the DNA synthesis does not occur continuously but in small fragments, which are called Okazaki fragments. Later these fragments are joined and sealed by the action of DNA ligase enzyme to form a continuous strand.
Okazaki fragments are about 1000-2000 nucleotides long in E. coli and about 100 200 nucleotides long in eukaryotes. Both the strands begin the synthesis starting on a primer segment. Leading strand synthesis starting on a primer proceeds continuously keeping pace with the unwinding of DNA at the replication fork.
Each Okazaki fragment is synthesized on a short RNA primer. DNA polymerase III binds to RNA primer and adds deoxyribonucelotides. Lagging strand proceeds in opposite direction from the fork movement.
The primer ribonucleotides are removed and replaced by deoxyribornucleotides and then joined. The removal of RNA primer is done by exonuclease activity of DNA polymerase I.
