Unit – III B
Semiconservative Replication of DNA
Activation of Nucleotides:
- All the four types of DNA nucleotides are present in nucleoplasm in the form of their monophosphates. They are activated into triphosphates like dATP, dGTP, dTTP and dCTP using ATP in presence of enzyme called phosphorylase. This process is known as activation of nucleotides.
dAMP + ATP → dATP + AMP (Enzyme – Phosphorylase)
Origin or initiation Point:
- DNA replication starts at certain specific sites situated on the molecule. Such sites are called origin points or initiation points.
- In eukaryotes, there are several origin points.
- During replication, DNA molecules split (unzip) by activity of initiator proteins at the origin by forming an incision also called nick. Here the breaking of the hydrogen bond between two strands of DNA starts.
Unwinding of DNA Strand:
- Enzyme DNA Helicase also called rep protein untwists the helix at locations called replication origins.
- The replication origin forms a Y-shape and is called a replication fork. The replication fork moves down the DNA strand, usually from an internal location to the strand’s end. Thus every replication fork has a twin replication fork, moving in the opposite direction from that same internal location to the strand’s opposite end.
- Single-stranded binding proteins (SSB) also called helix destabilizing protein work with helicase to keep the parental DNA helix unwound. It works by coating the unwound strands with rigid subunits of SSB that keep the strands from snapping back together in a helix. The SSB subunits coat the single-strands of DNA in a way as not to cover the bases, allowing the DNA to remain available for base-pairing with the newly synthesized daughter strands.
Synthesis of New Strands:
- Each separated strand acts as a template or mould for the synthesis of the complementary new strand. It takes place with help of RNA molecule called RNA primer. Synthesis of RNA primer is controlled by an enzyme called RNA primase. RNA primer attracts complementary nucleotides from the surrounding nucleoplasm.
- When the two parent strands of DNA are separated to begin replication, one strand is oriented in the 5′ to 3′ direction while the other strand is oriented in the 3′ to 5′ direction.
- But DNA replication, is inflexible: the enzyme that carries out the replication, DNA polymerase, only functions in the 5′ to 3′ direction. This characteristic of DNA polymerase means that the daughter strands synthesize through two different methods, one adding nucleotides one by one (continuous) in the direction of the replication fork, while the other adds nucleotides only in chunks (non-continuous). The first strand, which replicates nucleotides one by one is called the leading strand; the other strand, which replicates in chunks, is called the lagging strand.
- The strand which opens from 3′ to 5′ is called leading template and its complementary strand is called leading strand. It is a continuous and fast process. Triggered by RNA primase, which adds the first nucleotide to the nascent chain, the DNA polymerase simply sits near the replication fork, moving as the fork does, adding nucleotides one after the other, preserving the proper anti-parallel orientation.
- The strand which opens from 5′ to 3′ is called lagging template and its complementary strand is called lagging strand. DNA polymerase on the leading strand can simply follow the replication fork, because DNA polymerase must move in the 5′ to 3′ direction, on the lagging strand the enzyme must move away from the fork. But if the enzyme moves away from the fork, and the fork is uncovering new DNA that needs to be replicated. This was explained by Okazaki.
- He proposed the formation of lagging strand is discontinuous and takes place at a slower rate. the fragments of lagging strand formed during formation of complementary strands are called Okazaki fragments. These fragments are then joined by enzyme DNA ligase. Each Okazaki fragment requires RNA primer. Later RNA primer is removed by the enzyme RNase.
Formation of Daughter DNA Molecules:
- For each old strand, a new complementary strand is formed. Simultaneously the two strands undergo coiling and two identical DNA molecules are formed at the end of the process.
- As new molecules formed in the replication process retain 50% from parent DNA molecule while remaining 50% is newly formed. Hence the replication is called semi-conservative replication.
Errors of DNA replication and its Repair:
- There is a high degree of accuracy in DNA replication. Still, it may have an error of 1 in 1 billion base pairs formed. The initial rate of base pair error is high it is 1 error in 0.1 million base pairs.
- But it is immediately corrected by repair enzymes (nucleases) within the DNA polymerase complex. This enzyme proofreads each base pair formed and then remove and replace the mistake in the base pair in coordination with enzyme ligase.
In Prokaryotes circular DNA is present. There is only one origin and the replication is called theta replication.