In DNA replication, one “parental” double-stranded DNA molecule is converted to two identical “daughter” molecules and makes a copy of itself during cell division. Each daughter molecule will be identical to the parent in composition, but neither one is completely new which is the main feature of DNA replication. The original parental DNA strand serves as a template strand. Thus, preservation of the parent molecule is termed semiconservative replication which helps to explain the reliability and fidelity of replication.
A simple difference between prokaryotic DNA replication and Eukaryotic DNA replication in tabular form is given below
|Characteristics||Prokaryotic DNA Replication||Eukaryotic DNA Replication|
|Initiation||Initiation is carried by DNA a and DNA b||Initiation is carried by complex protein and ORC|
|Type of process||Continues process||Occurs in the S-phase of cell cycle.|
|Location||DNA replicates in the cytoplasm||DNA replicates in the nucleus|
|Size of DNA||Small amount of DNA||The DNA is 50 times more than prokaryotic DNA|
|Origin of replication||Single origin of replication||Multiple origins of replication|
|Types of DNA polymerase||5||14|
|DNA polymerase||DNA polymerase I and III are involved||DNA polymerase ɑ, δ and ε are involved|
|Presence of histone||Absent||Present|
|DNA gyrase||DNA gyrase is required||DNA gyrase is not required|
|Synthesis of telomeres||Absent||Present|
|Okazaki fragments||1000-2000 nucleotides large||100-200 nucleotides in length|
|Speed of replication process||Rapid process 500 nucleotide per second||Slow process 50 nucleotide per second|
|RNA primer removal||DNA Polymerase I||RNase H|
|Number of Replication fork||One replication fork||Multiple replication fork|
|Sliding clamp||Sliding clamp||PCNA|
What is Prokaryotic DNA
DNA of prokaryotes is circular packed in a single chromosome and is present inside the cytoplasm. It is not enclosed in a nuclear membrane. In addition to chromosomal DNA, small, single-stranded circular plasmid DNA is also present in prokaryotes which contain additional information and certain genes like antibiotic resistance genes which are essential for bacterial survival. Plasmid DNA is an essential component in genetic engineering techniques.
Depending upon the type of species, the DNA of prokaryotes ranges from few thousand to million base pairs (about 160,000-12.2000000 bp).
There is only one origin of replication in the chromosome of prokaryotes hence during the process of replication single replication folk and bubble will be formed. As the prokaryotic genome is less complex with less repetitive DNA sequences, therefore, the replication process is more efficient with a rapid speed of 2000 nucleotides/second.
Prokaryotic DNA replication
DNA replication requires a great deal of energy which is supplied from the nucleotides, which are actually nucleoside triphosphates. DNA replication by some prokaryotic organisms like bacteria, such as E. coli, goes bi-directionally around the chromosome. Two replication forks (Y- shaped) move in opposite directions away from the origin of replication as the replication proceed.
As we know the bacterial chromosome is a closed-loop, the replication forks even meet when replication is completed. The two loops must be separated by a topoisomerase which relaxes the supercoiling of the DNA strand.
DNA replication require a number of enzymes which are following
Helicase: Unwinds double-stranded DNA
Primase: Synthesizing an RNA primer
DNA polymerase III: helps in Addition of bases to the new DNA chain; proofreading the chain for mistakes
DNA polymerase I: DNA polymerase I helps for removing primer, closing gaps, repairing mismatches
Ligase: Helps in final binding of nicks in DNA during synthesis and repair
Methylase: this enzyme helps to add methyl group to selected bases in newly made DNA
RNA primase: help in the synthesis of RNA primer
Topoisomerase: relax supercoiling ahead of the replication fork
DNA replication starts at a fixed region on the chromosome called the replication origin (oriC), which is a sequence of about 250 base pairs rich in adenine and thymine. Initiator proteins along with other enzymes bind at the origin of replication and form two “replication factories” in which DNA synthesis will occur.
Before the replication helicases (unzipping enzymes) untwist the helix and break the hydrogen bonds holding the two strands together, resulting in two strands that act as the template.
The other proteins (enzymes) needed for replication will then add to this so-called “replication factory.” The process of synthesizing a new daughter strand of DNA using the parental strand as a template is carried out by the enzyme DNA polymerase III and must have this short strand of RNA to serve as a starting point for adding nucleotides.
As the DNA Polymerase moves towards the replication fork one new DNA strand called the leading Strand is continuously synthesized by the free nucleotides present in the cytoplasm. The DNA is always synthesized in the 5’ to 3’ direction. The nucleotides are matched up to exposed bases of the single-stranded parental DNA.
DNA polymerase can only add a new nucleotide to the 3’ end, thus RNA primer made by the primase start synthesis and required for the initiation of Okazaki fragments as intermediate in the synthesis of lagging strand. Okazaki fragments are short pieces of about 1000 nucleotides. The lagging strand is formed as the DNA polymerase moves away from the replication fork.
DNA polymerase remove the RNA primer and newly made DNA fragments are joined by DNA ligase which seal the nick left between the fragments creating the continuous DNA strand.
What is Eukaryotic DNA
DNA of prokaryotes is in the linear form present inside the nucleus enclosed in the nuclear membrane. Some membrane-bound organelles like mitochondria and chloroplast also have some quantities of DNA inside them which is not part of genomic DNA. Bundles of chromosomes have DNA packed inside them.
Linear DNA is coiled around an alkaline protein known as histone which gives a more complex structure to DNA. Histones are the proteins that assist in the packing chromosomes in the nucleus and are responsible for the structural organization of DNA in eukaryotic chromosomes. Eukaryotic DNA contains telomeres at both ends which are repetitive sequences of nucleotides and provide protection to chromosomes from deterioration.
Eukaryotic genes contain non-coding sequences known as introns. In the human genome, about 97% region consists of non-coding DNA sequences which is not involved in protein-encoding.
DNA replication in Eukaryotes:
DNA replication is a highly regulated process in eukaryotes than prokaryotes and requires external signals.
As we know that the eukaryotic DNA is bound to histones proteins to form structures called nucleosomes. DNA replication initiate when the origin recognition complex (ORC) binds to the origins of replication during the G1 phase of the cell cycle. This binding of proteins allows other necessary proteins for replication to binds in the same region.
The enzyme helicase helps the DNA to unwind and separate the DNA helix into single-stranded DNA and DNA open into Y Y-shaped structure. Replication forks are formed at each replication origin as the DNA unwinds and allow replication to occur simultaneously in hundreds to thousands of locations along each chromosome.
For the elongation three polymerase enzymes α, δ and ε is required to add DNA nucleotides to the 3′ end of the newly synthesized polynucleotide strand. DNA polymerase cannot add nucleotides in new strand, for this purpose it requires RNA primer (short stretch of RNA nucleotides) synthesized at the template DNA which is synthesized by the RNA Primase. Then DNA polymerase extends the new strand with nucleotides complementary to the template DNA.
DNA pol α: Add short (20 to 30 nucleotides) DNA fragment to the RNA primer
DNA pol δ: synthesis the leading strand and displace the primer from the DNA template.
DNA pol ε: synthesize the lagging strand
RNase H removes the displaced primer RNA and replaced it with DNA nucleotides. template strands at each replication fork are antiparallel
The newly synthesizes strand grows in opposite direction due to the antiparallel template strand at each replication fork. As helicase unwinds the template double-stranded DNA the “leading strand” is synthesized continuously toward the replication fork while the “lagging strand” is synthesized in the direction away from the replication fork and synthesized in pieces called Okazaki fragments. The synthesis of Okazaki fragments begins with its own RNA primer.
There is bubble of duplicated DNA on either side of the origin of replication which are formed by each origin of replication. When the leading strand of one replication bubble reaches the lagging strand of another bubble, and the discontinuous strands will reach the 5′ end of the previous Okazaki fragment in the same bubble both the continuous and discontinuous strands are formed.
Then exonuclease including proteins FEN1 (flap endonuclease 1) and RNase H, removes all RNA primers from the original strands. Proofreading by the polymerase enzyme replaced RNA primer with appropriate bases. Ligase enzyme joins the sugar-phosphate backbones at each nick site of Okazaki fragments, forming a single unified strand. Telomerase aids in their replication and prevents chromosome degradation by catalyzes the synthesis of telomere sequences at the ends of the DNA.