In this three steps of DNA replication in order post we have briefly explained about DNA replication occurs by three steps, DNA replication in prokaryotes, DNA replication in eukaryotes, steps in eukaryotes DNA replication, enzymes involved in DNA replication.
- DNA Replication is the process by which DNA is essentially doubled, also known as semi-conservative replication. It is a critical process that occurs within the dividing cell. In this article, we’ll take a quick look at the structure of DNA, the precise steps involved in DNA replication (initiation, elongation, and termination).
- Millions of nucleotides make up DNA. These are molecules made up of a deoxyribose sugar, a phosphate, and a nucleobase. To form a ‘sugar-phosphate backbone,’ these nucleotides are linked together in strands by phosphodiester bonds. The bond is formed between the third carbon atom of one nucleotide’s deoxyribose sugar (henceforth known as the 3′) and the fifth carbon atom of another sugar on the next nucleotide (known as the 5’).
- There are two strands that run in opposite or antiparallel directions. These are linked together along the length of the strand by the bases on each nucleotide. Cytosine, Guanine, Adenine, and Thymine are the four bases associated with DNA. Cytosine binds to Guanine in normal DNA strands, while Adenine binds to Thymine. Together, the two strands form a double helix.
Three Steps of DNA Replication in Order
DNA Replication in prokaryotes
- The parent DNA strands are complementary to one another. Both strands replicate at the same time, resulting in two daughter molecules. One-half of the parental DNA (one strand from the original) and one-half of newly synthesised DNA are found in each freshly generated DNA strand. This type of replication is known as semiconservative since half of the original DNA is conserved in the daughter DNA.
- The origin of replication is the location where DNA synthesis begins. In prokaryotes, there is only one origin site, however in eukaryotes, there are several origin sites. The majority of these sites are made up of a short A-T base pair sequence. For replication, a specialised protein termed dna A (20-50 monomers) attaches to the place of origin. The double-stranded DNA separates as a result of this.
- At the replication site, the two complementary strands of DNA split and create a bubble. In eukaryotic DNA molecules, several replication bubbles occur, which is necessary for a fast replication process.
- The creation of a replication fork occurs when the two strands of parent DNA are separated. This is where active DNA synthesis takes place. As the daughter DNA molecules are synthesised, the replication fork advances along the parent DNA.
- DNA helicases: At the replication fork, these enzymes attach to both DNA strands. Helicases separate the strands of DNA by moving along the helix. Their purpose is similar to that of a zip opener. Helicases are ATP-dependent energy producers.
- SSB proteins (single-stranded DNA binding proteins): DNA helix-destabilizing proteins are another name for these proteins. They don’t have any enzyme activity. SSB proteins only attach to single-stranded DNA (separated by helicases), keeping the two strands apart and serving as a template for fresh DNA synthesis. SSB proteins are thought to protect single-stranded DNA against nuclease degradation.
Leading strand synthesis
- Leading strand synthesis is a relatively simple process that begins with the Primase-mediated synthesis of RNA primer at the replication origin. The nucleotides are subsequently added at the 3’end by DNA polymerase III. The leading strand synthesis continues until it reaches the termination sequences, keeping up with the unwinding of the replication fork.
Lagging strand synthesis
- The lagging strand synthesized in short fragments called Okazaki fragments. At first RNA primer is synthesized by primase and as in leading strand DNA polymerase III binds to RNA primer and adds dNTPS. On this level the synthesis of each okazaki fragments seems straight forward but the reality is quite complex.
- The complexicity lies in the co-ordination of leading and lagging strand synthesis. Both the strand are synthesized by a single DNA polymerase III dimer which accomplished the looping of template DNA of lagging strand synthesizing Okazaki fragments.
- Helicase (dnaB) and primase (dnaG) constitute a functional unit within replication complex called primosome. DNA pol III use one set of core sub unit (Core polymerase) to synthesize leading strand and other set of core sub unit to synthesize lagging strand.
- In elongation steps, helicasein front of primaseand pol III, unwind the DNA at the replication fork and travel along lagging strand template along 5’-3’ direction. DnaG primase occasionally associated with dnaB helicase synthesizes short RNA primer. A new B-sliding clamp is then positioned at the primer by B-clamp loading complex of DNA pol III.
- When the Okazaki fragments synthesis is completed, the replication halted and the core sub unit dissociates from their sliding clamps and associates with new clamp. This initiates the synthesis of new Okazaki fragments.
- Both leading and lagging strand are synthesized co-ordinately and simultaneously by a complex protein moving in 5’-3’ direction. In this way both leading and lagging strand can be replicated at same time by a complex protein that move in same direction. Every so often the lagging strands must dissociates from the replicosome and reposition itself so that replication can continue.
- Lagging strand synthesis is not completes until the RNA primer has been removed and the gap between adjacent Okazaki fragments are sealed. The RNA primer are removed by exonuclease activity (5’-3’) of DNA pol-I and replaced by DNA. The gap is then sealed by DNA ligase using NAD as co-factor.
- Eventually the two replication fork of circular E. coli chromosome meet at termination recognizing sequences (ter). The Ter sequence of 23 bp is arranged on the chromosome to create trap that the replication fork can enter but cannot leave. Ter sequences function as binding site for TUS protein.
- Ter-TUS complex can arrest the replication fork from only one direction. Ter-TUS complex encounter first with either of the replication fork and halt it. The other opposing replication fork halted when it collide with the first one. This seems the Ter-TUS sequences are not essential for termination but it may prevent over replication by one fork if other is delayed or halted by damage or some obstacle.
- When either of the fork encounter Ter-TUS complex, replication halted. Final few hundred bases of DNA between these large protein complexes are replicated by not yet known mechanism forming two interlinked chromosome.
- In E. coli DNA topoisomerase IV (type II) cut the two strands of one circular DNA and segrate each of the circular DNA and finally join the strand. The DNA finally transfers to two daughter cell.
DNA replication in Eukaryotes
- DNA replication in eukaryotes occurs only in S-phase of cell cycle. However pre-initiation occur in G1 phase. Due to sheer size of chromosome in eukaryotes, chromosome contains multiple origin of replication. ARS (autonomously replicating sequence) in case of yeast is origin for replication.
- The first steps are the formation of pre-initiation replication complex (pre-RC). It occurs in two stage. 1st stage requires, there are no CDK activities. It occurs in early G1 phase. It is known as licensing but licensed pre-RC cannot initiate replication at G1 phase. 2nd stage is binding of ORC (origin recognition complex).
- The replication begins with binding of ORC to the origin. ORC is a hexamer of related protein and remains bounded even after DNA replication occurs. Furthermore ORC is analogue of prokaryotic dnaA protein.
- After binding of ORC to origin, cdc6/cdc18 and cdtl coordinate the loading of MEM (mini chromosome maintainance) to origin. MEM complex is thought to be major eukaryotic helicase.
- After binding of MEM complex to pre-RC, cdtl get displaced. Then DdK phosphorylates MEM, which activates its helicase activity. Again DdK and CdK recruit another protein called cdc45 which then recruit all the DNA replicating protein such that the origin get fired and replication begins.
- DNA polymerase δ synthesizes and adds dNTPs at 3’ end of RNA primer. The leading and lagging strands are synthesized in the similar fashion as in prokaryotic DNA replication.
- At the end of DNA replication the RNA primer are replaced by DNA by 5’-3’exonuclease and polymerase activity of DNA polymerase ε. Exonuclease activity of DNA polymerase removes the RNA primer and polymerase activity adds dNTPs at 3’-OH end preceding the primer.
- In case of bacteria, with circular genome, the replacement of RNA primer with DNA is not a problem because there is always a preceding 3’-OH in a circular DNA.
- But in eukaryotic organism with linear DNA, there is a problem. When RNA primer at 5’ end of daughter strand is removed, there is not a preceding 3’-OH such that the DNA polymerase can use it to replace by DNA. So, at 5’ end of each daughter strand there is a gap (missing DNA). This missing DNA cause loss of information contain in that region. This gap must be filled before next round of replication.
- For solving this end replication problem; studies have found that linear end of DNA called telomere has G: C rich repeats. This sequence is known as telomere sequence. These repeats of telomere sequence are different among different organisms. Telomere in human cell consists of repeats of TTAGGG/AATCCC. Each species has its own species specific telomere repeats. These telomere sequence do not codes anything but it is essential to fill the gap in daughter strand and maintain the integrity of DNA.
- DNA replication is a highly enzyme-dependent process. There are many enzymes involved in the DNA replication which includes the enzymes DNA-dependent DNA polymerase, helicase, ligase, etc. Among them, DNA-dependent DNA polymerase is the main enzyme.
- DNA polymerase: DNA polymerases are enzymes used for the synthesis of DNA by adding nucleotide one by one to the growing DNA chain. The enzyme incorporates complementary amino acids to the template strand. DNA polymerase is found in both prokaryotic and eukaryotic cells. They both contain several different DNA polymerases responsible for different functions in DNA replication and DNA repair mechanisms.
- DNA Helicase enzyme: This is the enzyme that is involved in unwinding the double-helical structure of DNA allowing DNA replication to commence. It uses energy that is released during ATP hydrolysis, to break the hydrogen bond between the DNA bases and separate the strands. This forms two replication forks on each separated strand opening up in opposite directions. At each replication fork, the parental DNA strand must unwind exposing new sections of single-stranded templates. The helicase enzyme accurately unwinds the strands while maintaining the topography on the DNA molecule.
- DNA primase enzyme: This is a type of RNA polymerase enzyme that is used to synthesize or generate RNA primers, which are short RNA molecules that act as templates for the initiation of DNA replication.
- DNA ligase enzyme: This is the enzyme that joins DNA fragments together by forming phosphodiester bonds between nucleotides.
- Exonuclease: Exonucleases can act as proof-readers during DNA polymerization in DNA replication. They work by scanning along the newly synthesized strand directly behind the DNA polymerase. If the last nucleotide added is mismatched, it will be removed by the exonuclease.
- Topoisomerase: This is the enzyme that solves the problem of the topological stress caused during unwinding. They cut one or both strands of the DNA allowing the strand to move around each other to release tension before it re-joins the ends. And therefore, the enzyme catalysts the reversible breakage it causes by joining the broken strands. Topoisomerase is also known as DNA gyrase in E. coli.
- Telomerase: This is an enzyme found in eukaryotic cells that adds a specific sequence of DNA to the telomeres of chromosomes after they divide, stabilizing the chromosomes over time.