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Steps
in
Prokaryotic
Replication -
When the two strands of DNA double helix are separated, each can serve as a template for the replication of a new complementary strand, producing two daughter molecules each of which contains two DNA strands with an antiparallel orientation. The enzymes involved in DNA replication process are template-directed polymerases that can synthesize the complementary sequence of each strand with extraordinary fidelity.
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This complex leads to the local denaturation and unwinding of an adjacent A + T rich region of DNA. The interaction of proteins with ori defines the start site of replication and provides a short region of ssDNA essential or initiation of synthesis of the nascent DNA strand. Then helicase binds and allows processive unwinding of double stranded DNA into single stranded DNA. As helicase unwinds the DNA, DNA binding (SSB) proteins bind and stabilize the single stranded DNA.
As the two strands unwind and separate the DNA, they form a 'V' shaped structure or region called replication fork. A replication fork consists of four components that form in the following sequence:
1. DNA helicase unwinds a short segment of the parental duplex DNA.
2. A Primase initiates synthesis of an RNA molecule that initiates DNA synthesis by acting as primer.
3. The DNA polymerase initiates daughter strand synthesis.
4. SSBs bind to ssDNA and prevent premature re annealing. It moves along the DNA as synthesis occurs. Some people define replication fork as the site of active DNA synthesis.
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The polymerase III holoenzyme binds to template DNA as a part of a multi protein complex that consists of several polymerase accessory factors. DNA polymerase synthesizes DNA only in the 5 ' to 3 ' direction and only one of the several different types of polymerases is involved at the replication fork. As the DNA strands are anti parallel, the DNA polymerase functions asymmetrically.
On the leading (forward) strand, the DNA is synthesized continuously. On the lagging strand (retro strand) the DNA is synthesized in short (1-5 kb) fragments. These DNA fragments are called as okazaki fragments.
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DNA polymerase present in the replication fork shares three important properties
(i) chain elongation
(ii) Processivity and
(iii) Proofreading.
Chain elongation accounts for the rate at which polymerization occurs. Processivity is expression of the number of nucleotides added to the nascent chain before the polymerase disengages from the template.
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The proof function identifies copying errors and corrects them. Pol III is an enzyme with high processivity and catalysing capacity than others. The initiation of DNA synthesis requires priming by a short length of RNA about 10-200 nucleotides long. This priming process involves the nucleophilic attack by the 3' -OH group of the RNA primer on the aphosphate of the deoxyribonucleotide triphosphate that enters first, with the splitting off of pyrophosphate.
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The 3' -OH group of the recently attacked deoxyribonucleotide monophosphate is then free to carry out a nucleophilic attack on the deoxyribonucleoside triphosphate that enters I?-ext. The selection of the proper deoxyribonucleoside whose terminal 3' -OH is to be attacked is dependent upon proper base pairing with the other strand of the DNA molecule, according to the rules proposed originally by Watson and Crick.
By this stepwise process, the template dictates which deoxyribonucleoside triphosphate is complementary, and by hydrogen bonding holds it in place while the 3' -OH group of the growing strand attacks and incorporates the new nucleotide into the polymer.
DNA pol III not only incorporates a nucleoside. It makes sure that the added nucleoside is correctly matched to its complementary base on the template and edits its mistakes. If by mistake it incorporates a wrong nucleotide, then DNA pol III hydrolytically removes the misplaced nucleotide and replaces it with the correct nucleotide. To ensure that replication occurs without any problem, the helicase acts on the lagging strand to unwind dsDNA in a 5 ' to 3 ' direction.
The helicase associates with the Primase to afford the latter, proper access to the template. This allows the RNA primer to be made and the polymerase to begin replicating the DNA. This is an important reaction sequence since DNA polymerase cannot initiate DNA synthesis de novo. The mobile complex between helicase and primase is called as primosome. This process proceeds as long as the DNA polymerase does not come in contact with RNA.
Once the polymerase encounters the RNA, the lagging strand DNA is released from the polymerase. But the polymerase remains attached to the replication fork complex proteins. The exact mechanism of this process is not clear yet. But research suggests, that the B subunit of DNA pol II carries out the function of releasing and re clamping single-stranded DNA in an energy dependent fashion.
After the entire replication completes, the RNA primers are removed by DNA pol II and fresh base pairs of deoxyribose sugars are added in its position. Then ligase joins the DNA molecules and the process of DNA replication proceeds towards termination.
The termination of the replication of a circular chromosome, E. coli chromosome is circular, presents no major topological problems. At the end of the theta structure both the 'V' structure junctions have preceded around the molecule. The region of termination on the E. coli chromosome, the terminus region, is 180˚ from ori site.
There are six termination sites, the protein product of the tus gene arrests the replication of both the strands of DNA. One interesting aspect of the termination of E. coli DNA replication is that the cells are viable even if the whole terminator gene is deleted. One may question as to why RNA is used to prime DNA synthesis, where DNA can be used directly to avoid the exonuclease and resynthesis activity.
This strategy is followed to have low error rate. Priming is an inherently error prone process since nucleotides are initially added without a stable primer configuration. To prevent long term errors in the DNA, an RNA primer is put in and it can later be recognized and removed thus making very few errors.
The other question that comes to the mind is why DNA synthesis cannot take place in the 3 ' -5' direction. The answer has to do with proofreading and the exonuclease removal of mismatched nucleotides. When an incorrect nucleotide is found and removed, the next nucleotide brought in, in the 5'-3' direction has a triphosphate available to provide the energy for its own incorporation.
Let us consider a polymerase with the ability of adding nucleotides in the opposite direction. The energy for the phosphodiester bond would be coming from the triphosphate already attached in the growing 3 '-5 ' strand.
Then if an error in complementarity were detected and if the polymerase removed the most recently added nucleotides from the 3 ' -5 ' strand, the last nucleotide in the double helix would no longer have a triphosphate available to provide energy for the diester bond with the next nucleotide.
Continued polymerization would thus require additional enzymatic steps to provide the energy needed for the process to continue. This could stop or slow down the process considerably. As it is, the process incorporates about four hundred nucleotides per second with an error rate of about one incorrect pairing per 10 bases.
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