Explain the process of replication in Eukaryotes in detail
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The process of replication in eukaryotes involves the following steps:
1. Initiation:
– The origin recognition complex (ORC) binds to the origin of replication.
– The Mcm complex is recruited, and the double helix is unwound by helicases.
2. Unwinding:
– The double helix is unwound by helicases, creating a replication fork.
– Topoisomerase relaxes the tension in the DNA.
3. Synthesis:
– DNA polymerase alpha (Pol α) begins synthesizing the leading strand.
– DNA polymerase delta (Pol δ) synthesizes the lagging strand in short, discontinuous segments (Okazaki fragments).
4. Elongation:
– The leading strand is continuously synthesized.
– The lagging strand is synthesized in short segments, which are later joined.
5. Ligation:
– DNA ligase seals the gaps between the Okazaki fragments, forming a continuous strand.
6. Proofreading and editing:
– DNA polymerase and other enzymes correct errors and ensure the new DNA is error-free.
7. Completion:
– The replication fork closes, and the new DNA molecule is complete.
8. Separation:
– The replicated chromosomes separate, each containing a complete copy of the genetic material.
This process is regulated by various proteins and enzymes, ensuring accurate and efficient replication of eukaryotic DNA.
DNA replication in eukaryotes occurs in three stages: initiation, elongation, and termination.
Initiation
Eukaryotic DNA is bound to proteins known as histones to form structures called nucleosomes. During initiation, the DNA is made accessible to the proteins and enzymes involved in the replication process. There are specific chromosomal locations called origins of replication where replication begins. Certain proteins recognize and bind to the origin of replication and then allow the other proteins necessary for DNA replication to bind the same region.
Two copies of an enzyme called helicase are among the proteins recruited to the origin. Each helicase unwinds and separates the DNA helix into single-stranded DNA. As the DNA opens up, Y-shaped structures called replication forks are formed. Because two helicases bind, two replication forks are formed at the origin of replication; these are extended in both directions as replication proceeds creating a replication bubble. There are multiple origins of replication on the eukaryotic chromosome which allow replication to occur simultaneously in hundreds to thousands of locations along each chromosome.
Elongation
During elongation, an enzyme called DNA polymerase adds DNA nucleotides to the 3′ end of the newly synthesized polynucleotide strand. The template strand specifies which of the four DNA nucleotides (A, T, C, or G) is added at each position along the new chain. Only the nucleotide complementary to the template nucleotide at that position is added to the new strand.
DNA polymerase cannot initiate new strand synthesis; it only adds new nucleotides at the 3′ end of an existing strand. All newly synthesized polynucleotide strands must be initiated by a specialized RNA polymerase called primase.
Primase initiates polynucleotide synthesis and by creating a short RNA polynucleotide strand complementary to template DNA strand. This short stretch of RNA nucleotides is called the primer. Once RNA primer has been synthesized at the template DNA, primase exits, and DNA polymerase extends the new strand with nucleotides complementary to the template DNA.
The Leading and Lagging Strands
DNA polymerase can only synthesize new strands in the 5′ to 3′ direction. Therefore, the two newly-synthesized strands grow in opposite directions because the template strands at each replication fork are antiparallel. The “leading strand” is synthesized continuously toward the replication fork as helicase unwinds the template double-stranded DNA.
The “lagging strand” is synthesized in the direction away from the replication fork and away from the DNA helicase unwinds. This lagging strand is synthesized in pieces because the DNA polymerase can only synthesize in the 5′ to 3′ direction, and so it constantly encounters the previously-synthesized new strand. The pieces are called Okazaki fragments, and each fragment begins with its own RNA primer.
Termination
Once all the template nucleotides have been replicated, the replication process is not yet over. RNA primers need to be replaced with DNA, and nicks in the sugar-phosphate backbone need to be connected.
The group of cellular enzymes that remove RNA primers include the proteins FEN1 (flap endonulcease 1) and RNase H. The enzymes FEN1 and RNase H remove RNA primers at the start of each leading strand and at the start of each Okazaki fragment, leaving gaps of unreplicated template DNA. Once the primers are removed, a free-floating DNA polymerase lands at the 3′ end of the preceding DNA fragment and extends the DNA over the gap. However, this creates new nicks (unconnected sugar-phosphate backbone).
In the final stage of DNA replication, the enyzme ligase joins the sugar-phosphate backbones at each nick site. After ligase has connected all nicks, the new strand is one long continuous DNA strand, and the daughter DNA molecule is complete.
The process of replication in eukaryotes involves the following steps:
1. Initiation:
– The origin recognition complex (ORC) binds to the origin of replication.
– The Mcm complex is recruited, and the double helix is unwound by helicases.
2. Unwinding:
– The double helix is unwound by helicases, creating a replication fork.
– Topoisomerase relaxes the tension in the DNA.
3. Synthesis:
– DNA polymerase alpha (Pol α) begins synthesizing the leading strand.
– DNA polymerase delta (Pol δ) synthesizes the lagging strand in short, discontinuous segments (Okazaki fragments).
4. Elongation:
– The leading strand is continuously synthesized.
– The lagging strand is synthesized in short segments, which are later joined.
5. Ligation:
– DNA ligase seals the gaps between the Okazaki fragments, forming a continuous strand.
6. Proofreading and editing:
– DNA polymerase and other enzymes correct errors and ensure the new DNA is error-free.
7. Completion:
– The replication fork closes, and the new DNA molecule is complete.
8. Separation:
– The replicated chromosomes separate, each containing a complete copy of the genetic material.
This process is regulated by various proteins and enzymes, ensuring accurate and efficient replication of eukaryotic DNA.
DNA replication in eukaryotes occurs in three stages: initiation, elongation, and termination.
Initiation
Eukaryotic DNA is bound to proteins known as histones to form structures called nucleosomes. During initiation, the DNA is made accessible to the proteins and enzymes involved in the replication process. There are specific chromosomal locations called origins of replication where replication begins. Certain proteins recognize and bind to the origin of replication and then allow the other proteins necessary for DNA replication to bind the same region.
Two copies of an enzyme called helicase are among the proteins recruited to the origin. Each helicase unwinds and separates the DNA helix into single-stranded DNA. As the DNA opens up, Y-shaped structures called replication forks are formed. Because two helicases bind, two replication forks are formed at the origin of replication; these are extended in both directions as replication proceeds creating a replication bubble. There are multiple origins of replication on the eukaryotic chromosome which allow replication to occur simultaneously in hundreds to thousands of locations along each chromosome.
Elongation
During elongation, an enzyme called DNA polymerase adds DNA nucleotides to the 3′ end of the newly synthesized polynucleotide strand. The template strand specifies which of the four DNA nucleotides (A, T, C, or G) is added at each position along the new chain. Only the nucleotide complementary to the template nucleotide at that position is added to the new strand.
DNA polymerase cannot initiate new strand synthesis; it only adds new nucleotides at the 3′ end of an existing strand. All newly synthesized polynucleotide strands must be initiated by a specialized RNA polymerase called primase.
Primase initiates polynucleotide synthesis and by creating a short RNA polynucleotide strand complementary to template DNA strand. This short stretch of RNA nucleotides is called the primer. Once RNA primer has been synthesized at the template DNA, primase exits, and DNA polymerase extends the new strand with nucleotides complementary to the template DNA.
The Leading and Lagging Strands
DNA polymerase can only synthesize new strands in the 5′ to 3′ direction. Therefore, the two newly-synthesized strands grow in opposite directions because the template strands at each replication fork are antiparallel. The “leading strand” is synthesized continuously toward the replication fork as helicase unwinds the template double-stranded DNA.
The “lagging strand” is synthesized in the direction away from the replication fork and away from the DNA helicase unwinds. This lagging strand is synthesized in pieces because the DNA polymerase can only synthesize in the 5′ to 3′ direction, and so it constantly encounters the previously-synthesized new strand. The pieces are called Okazaki fragments, and each fragment begins with its own RNA primer.
Termination
Once all the template nucleotides have been replicated, the replication process is not yet over. RNA primers need to be replaced with DNA, and nicks in the sugar-phosphate backbone need to be connected.
The group of cellular enzymes that remove RNA primers include the proteins FEN1 (flap endonulcease 1) and RNase H. The enzymes FEN1 and RNase H remove RNA primers at the start of each leading strand and at the start of each Okazaki fragment, leaving gaps of unreplicated template DNA. Once the primers are removed, a free-floating DNA polymerase lands at the 3′ end of the preceding DNA fragment and extends the DNA over the gap. However, this creates new nicks (unconnected sugar-phosphate backbone).
In the final stage of DNA replication, the enyzme ligase joins the sugar-phosphate backbones at each nick site. After ligase has connected all nicks, the new strand is one long continuous DNA strand, and the daughter DNA molecule is complete.