Mechanism of DNA Replication
Replication is a vital process for life because, after every cell division, the two new daughter cells that are formed must contain the same genetic information, or DNA, as the parent cell. The human genome contains approximately 6.6 billion base pairs of DNA, carrying information for making all of the cell’s proteins. So the process of replication has to be very accurately done each time the cell undergoes cell-division.
During the period of replication, both the strand of the DNA molecule separate, and each strand is used as a template for the synthesis of a new strand. Thus results in the formation of two identical DNA double helices. This is called semiconservative replication. This term semiconservative defines that in each round of DNA replication, it produces two hybrid DNA molecules each of which contains one old strand and one newly synthesized strand.
The polymerase required for DNA replication:
The molecules that are required for the replication of the DNA are enzymes, called as DNA polymerase. The DNA polymerase – DNA polymerase are the enzymes that are responsible for creating new DNA molecule, by joining the nucleotide molecules, called as the building blocks of DNA molecule. DNA polymerase was first identified in E. coli by Arthur Kornberg in 1956. In prokaryotes, five different types of polymerases are known:
DNA pol I accounts for repairing activity, containing both 5’ to 3’ and 3’ to 5’ activity.
DNA pol II is also invoved in the repairing activity, but only 3’ to 5’ exonuclease activity. The enzyme is involved in repairing DNA damage caused during polymerase activity.
DNA pol III is a holoenzyme and the main polymerase for replication, the core – consisting of three subunits (α, ɛ and θ), a clamp loading complex and two beta subunit forming sliding clamp, allowing the polymerase activity on both the leading and lagging DNA strands.
DNA Pol IV and DNA Pol V, both the enzymes belongs to the Y family of DNA polymerases, among which DNA Pol IV is an frequent error causing polymerase that has no 3’ to 5’ proofreading activity and also involved in activity that can alter the DNA, thus giving rise to a mutation. The DNA Pol V, which allows DNA damage to happen in order for replication to continue without any proofreading activity.
Processivity of a DNA polymerase – The processivity of a DNA polymerase during DNA synthesis can define as the number of nucleotides that a polymerase can add to the synthesizing DNA nucleotide chain during any single template‐binding event, before dissociating from a DNA template.
Fidelity of a DNA polymerase – The fidelity of a DNA polymerase can be explained as the ability of a polymerase to accurately replicate a template without any error.
Process of DNA replication:
1) Initiation: There is a specialized 245 base pairs long sequence also called as OriC or Origin of Replication, rich in AT sequences, making the DNA strands easier to separate because less stronger bond compared to GC. This specific sequence of AT is recognized by an initiator protein called DnaA, having the helicase activity, it unwind the DNA to form two Y-shaped structures called replication forks. Single-strand binding proteins bind with the newly separated strands of the DNA near the replication fork, thus stabilizing the strands and preventing from forming double helix.
Chain initiation starts, when a specialized enzyme called, primase, synthesizes RNA primer that is about five to ten nucleotides long and complementary to the DNA, the primer provides a 3′ end for DNA polymerase for elongation.
2) Elongation: DNA polymerase III uses the primer synthesized by primase as a precursor to elongate the strand but here arises a problem, the DNA polymerases can synthesize DNA only in the 5′ to 3′ direction, i.e.; the direction in which the replication fork is moving. One strand, which is complementary to the 3’ to 5’ direction, is continuously synthesized by DNA polymerase III, the same direction in which the replication fork is moving. This strand is called as leading strand. The strand which is complementary to the 5’ to 3’ direction is discontinuous, called as lagging strand. This strand is synthesized by joining the small fragments, also called as Okazaki fragments. This lagging strand requires new primer for synthesizing each small Okazaki fragments.
It is the DNA gyrase, also called as topoisomerase II, ahead of the replication fork introduces ATP dependent negative coiling by unwinding the double helix from getting entangle during replication.
The lagging strand is looped around the polymerase in such a manner that the synthesis of the strand continues along with the leading strand. DnaB helicase and DnaG primase are two enzymes which together forms a complex called primosome, the complex unwind the DNA and synthesize primers. The primer is then extended by the core subunit of DNA polymerase III complex. The replication is stopped after the completion of Okazaki fragment synthesis. The core subunit get dissociate from the completed Okazaki fragment and get associated with a new beta- sliding clamp, loaded by the clamp loading complex of pol III on the primers to be extended. The primers are removed and filled with deoxyribonucleotides by the DNA polymerase I, whereas the nick that remains after the DNA polymerase I activity is sealed by an enzyme called DNA ligase.
3) Termination: Bacterial replication terminates at a position diagonally opposite to the OriC. The Tus protein makes complex with the DnaB, terminating the DnaB helicase activity at a region containing multiple copies of 20bp sequence called as Ter sequence, or Terminus sequence.
Mechanism of DNA Replication
- What is semiconservative DNA replication?
- DNA polymerase was first identified by __________
a) Marshall W. Nirenberg
b) Arthur Kornberg
c) Jerard Hurwitz
d) Har Gobind Khorana
- What is processivity and fidelity of a DNA polymerase?
- Explain the role of DNA gyrase in replication.
- The primer that provides the 3′ end for DNA polymerase for elongation during chain initiation is –