Most organisms, including mammals, use bi-directional replication. There are four basic components required to initiate and propagate DNA synthesis. They are substrates, templates, primer and enzymes.
Four deoxyribonucleotide triphosphates (dNTPs) are required for DNA synthesis (note the only difference between deoxyribonucleotides and ribonucleotides is the absence of an OH group at position 2′ on the ribose ring) as they are building blocks of DNA. As a result, the substrates for DNA synthesis are called 2′ deoxyribonucleotides. These are dATP, dGTP, dTTP and dCTP. The high energy phosphate bond between these nucleotides and phosphates is cleaved and the deoxynucleotide monophosphate is incorporated into the new DNA strand.
Ribonucleoside triphosphates (NTP’s) are also required to initiate and sustain DNA synthesis. NTP’s are used in the synthesis of RNA primers and ATP is used as an energy source for some of the enzymes needed to initiate and sustain DNA synthesis at the replication fork.
The nucleotide that is to be incorporated into the growing DNA chain is selected by base pairing with the template strand of the DNA. The template is the DNA strand that is copied into a complementary strand of DNA.
The enzyme that synthesizes DNA, DNA polymerase, can only add nucleotides to an already existing strand or primer of DNA or RNA that is base paired with the template.
An enzyme, DNA polymerase, is required for the covalent joining of the incoming nucleotide to the primer. To actually initiate and sustain DNA replication requires many other proteins and enzymes which assemble into a large complex called a replisome. It is considered that the DNA is spooled through the replisome and replicated as it passes through. Some enzymes used in DNA synthesis are;
There are many types of DNA polymerases which can excise, fill gaps, proofread, repair and replicate.
These proteins bind and partially denatures the origin of DNA while binding to another enzyme called helicase.
It unwinds double stranded DNA.
Single-stranded DNA Binding Protein (SSB)
They enhance the activity of the helicase and prevent the unwound DNA from renaturing.
It synthesizes the RNA primers required for initiating leading and lagging strand synthesis.
It recognizes the RNA primers and extends them in the 5′ to 3′ direction.
It helps load the polymerase onto the primer-template while anchoring the polymerase to the DNA.
It removes the positive supercoils that form as the fork is unwound by the helicase.
It removes RNA portions from Okazaki fragments.
It seals the nicks after filling in the gaps left by DNA polymerase.
The enzymes and other protein factors that carry out DNA replication in E. coli and in eukaryotic cells are similar, suggesting that the biochemical mechanism of DNA replication is similar in all cells.