Enzymes involved in DNA replication
DNA topoisomerases/gyrase: It helps to relieve helical winding and DNA tension problems.
DNA clamp: is a protein which prevents elongating DNA polymerases from dissociating from the DNA parent strand.
DNA-dependent DNA polymerase: It catalyzes the polymerization of nucleotide triphosphate and also the overall process of DNA replication with the support of other enzymes in 5’ to 3’ direction. DNA polymerase is of three types:
DNA Polymerase I: It is a DNA repair enzyme. It is involved in three activities:
- 5′-3′ polymerase activity
- 5′-3′ exonuclease activity
- 3′-5′ proof reading exonuclease activity
- It fills the gap between the lagging strand fragments
DNA Polymerase II: It is responsible for primer extension and proofreading.
DNA Polymerase III: It is responsible for in vivo DNA replication. It has two subunits, one each for a strand. The alpha subunit is the actual polymerase.
Helicase: Helicase is the enzyme which unzips the DNA strands by breaking the hydrogen bonds between them. Thus, it helps in the formation of the replication fork.
Ligase: It is the enzyme which joins the discontinuous DNA strands by forming phosphodiester bonds between 3’OH and 5’ Phosphate group in 5’ to 3’ direction.
Primase: This enzyme helps in the synthesis of short RNA primers complementary to the DNA template strand in 5’ to 3’ direction.
Single strand Binding Proteins: It binds to single-stranded DNA and protects it from forming secondary structures and thus keeping the strand unwound.
Steps involved in DNA Replication
- The two strands of DNA unwind at the origin of replication.
- Helicase enzyme then opens the DNA by cutting the hydrogen bond and replication fork is formed at the point of separation of strands.
- The DNA is coated by the single-strand binding proteins around the replication fork to prevent rewinding of DNA.
- Topoisomerase prevents the supercoiling of DNA and release the tension.
- RNA primers are synthesised by primase complementary to the DNA strand.
- Initiation in prokaryotes is at a single site OriC.
- There are multiple origin sites in eukaryotic DNA
- In E.coli. the primary initiator protein is DnaA
- In Yeast, this is the Origin Recognition Complex (ORC)
- Sequences used by initiator proteins tend to be “AT-rich” (rich in adenine and thymine bases), because A-T base pairs have two hydrogen bonds (rather than the three formed in C-G pair) and thus are easier to strand-separate.
- DNA polymerase III starts adding nucleotides at the end of the primers.
- The leading and lagging strands continue to elongate with the formation of Okazaki fragments in the lagging strand.
- The primers are removed and the gaps are filled with DNA Polymerase I and sealed by ligase.
- Termination occurs when a termination site sequence in the DNA is reached, and a protein binds to this sequence to physically stop DNA replication, the DNA replication terminus site-binding protein, Ter protein, an inhibitor of DnaB helicase which works along with a Tus factor (forming a complex).
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