Ritu's Biology Hub

What is DNA replication?

It is a process by which DNA in living organisms can multiply and make new copies of itself so that it can be passed on to new daughter cells and perpetuate itself for generations. This is how DNA has been transferred from one cell to another in living beings for years. Cells have this unique capability to multiply in numbers by cell division. When the cells divide, in order to survive and live, they also need a DNA molecule. So how can that one DNA in the mother cell be passed to the two new daughter cells that are created when a cell divides? This is accomplished by the mechanism of DNA replication. The two strands of the parent DNA separate and new strands are replicated using the strands of the old DNA. When replication is done, two new DNA molecules are made and each of them is transferred to a new daughter cell.

This is just an overview of the basic process. To learn more about this, continue to read below –

Requirements for DNA polymerase to catalyse DNA synthesis –

• Template strand that can be read in 5’ to 3’ direction
• Primer to provide 3’-OH end to which new nucleotides can be added
• dNTPs
• Mg²⁺ ions

Process of DNA replication (Theta model) –

• Initiation of replication occurs at a specific region called origin of replication where the ds-DNA denatures to form ss-DNA and within which replication commences
• The locally denatured segment of DNA is called the replication bubble and the 2 strands in this region using which new complimentary strands are synthesized are called the template strands
• As the DNA unwinds, a y-shaped structure is formed at either ends of the replication bubble. It is known as the replication fork. In such cases, bidirectional replication occurs
• The fork is generated by a complex of 7 proteins called primasome that includes – Dna G primase, Dna B helicase, Dna C helicase assistant, Dna T, Primase A, B and C
• In coli, the OriC region spans 245 bp and contains clusters of 3 copies of 13-mer and 4 copies of 9-mer sequences
• To initiate replication, an initiator protein called Dna A ATP (encoded by dna A gene) binds to 9-mer sequences and denatures the region connecting it to 13-mer sequences by breaking A-T bonds which are weaker as they are held by 2 hydrogen bonds. This requires energy from ATP. This forms the initial complex
• DNA helicase (Dna B) is loaded onto the DNA strands by a helicase loader (Dna C). DNA helicase untwists the DNA. This forms the pre-priming complex
• SSBP (Single-stranded Binding Protein) binds the open strands of DNA to prevent rewinding. Gyrase, a type of topoisomerase, releases the tension generated by rapid unwinding of the DNA strands at 3000 rpm
• The DNA helicase recruits primase enzyme that synthesizes a short segment of 5-10 nucleotides called primer which allows elongation of DNA. This happens because DNA polymerase III can only add nucleotides but cannot initiate synthesis of a new strand.
• Elongation is carried out by replisome which is made up of DNA polymerase III and the primasome complex. The DNA pol III enzyme tethers itself to the ss-DNA via its core enzyme. The core enzyme catalyzes the DNA synthesis by adding complementary nucleotides
• DNA synthesis takes place in 5’ to 3’ direction towards the replication fork. The strand that is being synthesized in this direction continuously is called the leading strand while the strand that is synthesized in the opposite direction is called lagging strand
• The leading strand requires just 1 primer whereas the lagging strand requires many such primers. Since, leading strand is synthesized continuously and simultaneously along with the discontinuous synthesis of lagging strand, the entire process of DNA synthesis is a semi-discontinuous process
• Fragments of lagging strand are known as okazaki fragments. After the strands have been completely synthesized, these okazaki fragments are joined together.
• DNA pol III is removed. DNA pol I removes the primers by its 5’ to 3’ exonuclease activity exposing the template nucleotides. It then adds complementary nucleotides by its 5’ to 3’ polymerase activity to the 3’-OH end of the previous okazaki fragment, thereby replacing the primers
• The nicks that remain behind are joined by ligase by creating a phosphodiester bond. This is known as nick translation
• Any errors in base-pairing is removed by DNA polymerase III by its 3’ to 5’ exonuclease activity immediately before proceeding onto the next nucleotide. This is called proof-reading
• Termination of this process occurs when the replication forks reach the ter sites. Tus proteins (Terminus Utilization Substance) bind to ter sites and halt progression of forks. In coli, there are 10 replication termini (Ter sites) each spanning 23 bp
• Ter B and C terminate the clockwise fork while ter A, D and E terminate anti-clockwise fork
• In circular chromosomes, the daughter chromosomes remain interlocked and are called catenanes. Topoisomerase II resolves this problem by breaking some bonds in DNA molecules so as to separate the strands – Decatenation

DNA Replication by Rolling Circle Model –

• This occurs when a circular ds-DNA genome needs to be made in multiple copies such as in lambda phage
• A nick is made at the origin of replication on the outer strand, also called the (+) strand, making 2 ends of the (+) strand – 5’ and 3’ end
• The 3’-OH end is extended by replication enzymes which is the leading strand using the inner or (-) strand as the template. As the 3’ end is being lengthened, the 5’ end gets displaced and forms an ever lengthening tail
• The 5’ end of the (+) strand acts as a template for the complementary lagging strand making it double stranded
• The leading strand and the template of the lagging strand remain covalently attached to each other. Concatamers are formed. They are then cut apart to give separate genomes

Continued to Part 2