DNA Replication With All Enzymes Carrer780

 

DNA  REPLICATION:-

• When a cell divides to form two daughter cells, the cellular content gets divided almost equally 
• into two daughter cells. As, every cell has only one nucleus, so how is it possible that one  nucleus be given to two daughter cells. Hence, in cell cycle, before a cell divides into two, it  replicates its DNA in S phase of cell cycle which gets separated into two nuclei in a process called karyokinesis (which has four phases known a prophase, metaphase, anaphase and telophase) and thenthe cell divides into two by a process of cytokinesis.

• DNA replication is a process in which original DNA molecule (present in nucleus of eukaryotes and cytoplasm of prokaryotes) called as parent DNA is replicated to form two new DNA molecules called as daughter DNA molecules. Parental strands act as template (guide) strand for the synthesis of daughter strands.

 

Three different hypotheses had been proposed for the mode of DNA replication

1. Conservative Mode-

• In this model, the two strands of DNA unwind from each other, and each acts as a template for synthesis of a new, complementary strand. This results in two DNA molecules with one original 
• strand and one new strand. An entirely new molecule is synthesized from a DNA template and  no change occurs in template (parent) DNA i.e. it remains conserved.

2. Semi-Conservative Model-

• In this model, DNA replication results in one molecule that consists of both original DNA 
• strands (identical to the original DNA molecule) and another molecule that consists of two new 
• strands (with exactly the same sequences as the original molecule). It means after replication 
• each DNA molecule has one parental strand and one newly synthesized strand.

3. Dispersive Model-

• In the dispersive model, DNA replication results in two DNA molecules that are mixtures, or 
• ―hybrids,‖ of parental and daughter DNA. In this model, each individual strand is a patchwork of 
• original and new DNA. New molecules are made up of segments of new and old DNA.

Different Model Of DNA Replication

Meselson and Stahl Experiment: 

• DNA Replication is Semi-Moderate Watson and Cramp model recommended that DNA replication is semi-moderate. That's what it infers a big part of the DNA is preserved. Just a single new strand is blended, the other strand is the first DNA strand (layout) that is held. Each parental DNA strand fills in as a layout for one new corresponding strand. Every one of these strands of the twofold helix contains one unique parental strand and one recently framed strand.

• Semi-conservative replication of DNA was proved by the work of Mathew Meselson and Franklin Stahl (1958). In their experiment, Escherichia coli was grown for several  generations on a medium containing heavy isotope of nitrogen 15N, till the bacterial DNA  became completely labelled with heavy isotope. The labelled bacteria were then cultured onto  fresh medium with normal nitrogen 14N. DNA was isolated from each generation of bacteria and  tested for the presence of heavy isotope of nitrogen through density gradient centrifugation using  cesium chloride (CsCl). When centrifuged at high speed the salt forms a density gradient with  heaviest region at the bottom and less concentrated lighter one towards the surface.

• Meselson and Stahl found that DNA of the first generation was hybrid or intermediate ( 15N and 14N). It settled in CsCl solution at a level higher than the fully labelled DNA of parent  bacteria (15N15N). Because N-15 has one extra neutron, it's slightly heavier than N-14 and  therefore makes the DNA molecule denser. The second generation of bacteria after 40 minutes  contained two types of DNA, 50% light (14NI4N) and 50% intermediate (15N I4N). The third  generation of bacteria after 60 minutes contained two types of DNA, 25% intermediate (15N 14N) and 75% light (14N 14N) in 1 : 3 ratio. 

• This perception is conceivable provided that the two strands of DNA duplex separate at the hour of replication and go about as a layout for the union of new corresponding strands of DNA having typical or 14N. This will deliver two DNA duplexes with one old strand (15N) and one new strand (14N). In every one of the girl DNA atom one strand is parental and another strand is recently incorporated DNA strand. Consequently the course of DNA replication is semi-conservative.

The complete process of DNA replication can be divided into three steps:

1. Initiation-

• The first step occurs when DNA helicase unwinds the double helix by breaking the hydrogen bonds between the parent strands of DNA at locations called replication origins. This opens up the DNA molecule to form a Y-shape structure, which is called as replication fork. 

• Singlestranded binding proteins (SSB) work with helicase to keep the parental DNA helix unwound.

• Before the synthesis of daughter DNA strands, an RNA primer is made by the enzyme called RNA Primase. RNA Primase is the enzyme that builds an RNA primer on the parent strand to  initiate DNA replication. Once the RNA primer is built, then the next enzyme, DNA polymerase, begins synthesis of DNA strands. 

• During the course of replication DNA polymerase is situated behind the RNA groundwork. The ―Topoisomerase‖ proteins encompass the unfastening strands and loosen up the winding that could harm the loosening up DNA.

2. Elongation-

• After separation of helix separated and primer synthesis DNA polymerase starts adding 
• complementary nucleotides and synthesis of daughter strand begin. Addition of complementary  nucleotides means that if A is present on parental strand then nucleotide with base T will be  added on daughter strand, if G is present on daughter strand then nucleotide with base C will be added on daughter strand and vice versa. Now at this stage where two parent strands of DNA are unwound or separated, one strand is oriented in the 5' to 3' direction and the other strand has  opposite orientation in the 3' to 5' direction. 

• Single DNA polymerase will catalyze replication of both the strands. DNA polymerase works simply in 5' to 3' bearing. This element makes combination of girl strands through various strategies, one adding nucleotides individually toward the replication fork, the other ready to add nucleotides just in pieces. The primary strand, which duplicates nucleotides one by one, is known as the main strand; the other strand, which reproduces in pieces, is known as the slacking strand.

(a) The Leading Strand-

• Because DNA polymerase moves along the parent strand in the 5' to  3' direction, replication can occur very easily on the leading strand. Addition of nucleotides  occurs in the 5' to 3' direction. 

• Set off by RNA Primase, which adds the underlying nucleotides (invtype of preliminary) to the new chain, the DNA polymerase moves along the fork and keeps on adding integral nucleotides in a steady progression as per the grouping of nucleotides present on parental strand. Union of driving strand is a persistent interaction.

(b) The Lagging Strand-

• Whereas the DNA polymerase III on the leading strand can simply  follow the replication fork, because DNA polymerase III must move in the 5' to 3' direction, on  the lagging strand the enzyme must move away from the fork. The lagging strand replicates in  small segments, called Okazaki fragments. These fragments are stretches of 100 to 200  nucleotides in humans (1000 to 2000 in bacteria) that are synthesized in the 5' to 3' direction  away from the replication fork.

• For combination of each Okazaki piece another groundwork is made before the section is duplicated. After replication of over these preliminaries are corrupted. Presently in the empty spaces DNA is combined by DNA polymerase I. These sections are then sewed together by DNA ligase, making a constant strand. The amalgamation of slacking strand is called intermittent.

3. Termination-

• After elongation is complete, two new double helices have been made from the original parental  DNA molecule. The process of DNA replication is summarized in Replication Enzymes DNA replication would not occur without enzymes that catalyze various steps in the process.  Enzymes that participate in the eukaryotic DNA replication process include:

1. DNA helicase-

• Unwinds and separates double stranded DNA as it moves along the DNA. It  forms the replication fork by breaking hydrogen bonds between nucleotide pairs in DNA.

2. DNA primase-

• A type of RNA polymerase that generates RNA primers. Primers are short  RNA molecules that act as templates for the starting point of DNA replication.

3. DNA polymerases: 

• Synthesize new DNA molecules by adding nucleotides to leading and lagging DNA strands.

4. Topoisomerase or DNA Gyrase-

• unwinds and rewinds DNA strands to prevent the DNA from becoming tangled or supercoiled.

5. Exonucleases-

• group of enzymes that remove nucleotide bases from the end of a DNA chain.

6. DNA ligase-

• joins DNA fragments together by forming phosphodiester bonds between nucleotides.

summary of DNA replication-

(A) Parent DNA helix, (B) Helicase loosen up DNA, (C) SSB ties to isolated strands, Primase combines groundwork, (D) DNA Polymerase-III beginnings combination of driving and slacking strand, (E) Complete preliminaries are taken out after replication (F) DNA Polymerase-I combine DNA in the holes left after evacuation of preliminary and Ligase joins different DNA sections. (G) Two little girl DNA particles are created.