DNA replication

[SUMAN-SAJAN]

DNA Replication

Of particular importance for cell survival is the accurate, efficient and rapid duplication of the cellular genome. This process is termed DNA replication



Types of DNA Replication
• Coservative mode of replication: The cellular DNA is duplicated and one cell receives the pre-existing(original) DNA while the other cell receives the newly synthesized DNA.
• “in other words, the integrity of pre-existing DNA is conserved”.




Semiconservative mode of DNA Replication
Two DNA molecules — identical to each other and identical to the original — have been produced. Each strand of the original molecule has
• remained intact as it served as the template for the synthesis of a complementary strand.
This mode of replication is described as semi-conservative: one-half of each new molecule of DNA is old; one-half new.



E. coli are grown in heavy nitrogen (15N) for many generations.
• This caused the nitrogen in the DNA molecule of each cell to contain 15N, a heavier than typical isotope.
• The E. coli were then grown for one or two cell divisions in 14N, the lighter and typical isotope.
• DNA was spun in a cesium chloride gradient. Meselson and Stahl actually invented this technique, called density centrifugation, which now has many other applications, just for the purposes of this experiment.





• The cesium chloride gradient and centrifugation separates molecules based on their density.
• The DNA molecules with 15N are more dense than those with 14N, and band below DNA with 14N.
• If two bands were observed after one division in 14N, there would have been wholly old strands and wholly new strands. This would have been consistent with and meant the replication was conservative.
• If there was just one band after one division, replication could be either dispersive or semiconservative.
• The result was just one band after one division.





• If one or a long smear was observed after two divisions in 14N containing medium, dispersive replication would have been the mode.
• If intermediate weight and light weight molecules were found, semiconcervative would be the mode.
• This is what was found; the replication was semiconservative.

Basic requirement for DNA Synthesis
• Substrates
• Template
• Primer
• Enzymes


Substrates

The four deoxynucleoside triphosphates(Dntps)- deoxyadenosine triphosphate(dATP), deoxyguanosine triphosphate (dGTP), deoxycytidine triphosphate (dCTP), and deoxythymine triphosphate (dTTP) are needed as substrates for DNA synthesis.



Primer

A short sequence (often RNA) that is paired with one strand of DNA and provides a free 3’-OH end at which a DNA Polymerase start the synthesis of deoxyribonucleotide chain.



Template

A molecular “mold” that dictates the structure of another molecule; most commonly, one starnd of DNA that direct synthesis of a complementry DNA strand during DNA replication or of an RNA during transcription.




Enzymes


• Helicase: The enzymes, also referred as unwinding protein, is responsible for the melting of DNA and formation of SS DNA at the beginning of the replication fork.
• It requires ATP for its action.
• The active form of helicase is the homo-hexamer of a 330 KD protein.








DNA Gyrase or Topoisomerases
Introduces negative supercoils to compensate for the strain that results from positive supercoiling at the replication fork in prokaryotes. It is a Type II topoisomerase.





DNA ligase
• It can join two of DNA bye the formation of a phodiester bond between these molecules.
• The should have a free 3’OH and a 5’PO4 groups, repectively.
• It should be noted that the DNA ligase can only close a nick but canot incoprate any new nucleotide to fill a gap.





Primase

• It is sepecific RNA polymerase which is responsible for the synthesis of a sequence specific RNA molecule which serve as the primer to the DNA synthesis.





DNA polymerase I

• DNA polymerase was originally isolated from E.coli by Arther Kornburg and is also referred as kornberg’s enzyme.
• It is single polywith a molecular weight 103 KD.
• It has three distinct properties
• 5’--------3’ polymerase activity
• 5’--------3’ exonuclease activity
• 3’--------5’ exonuclease activity (proof reading)
• DNA poly I is found in the highest concentration of all DNA polymerases; it is involved in DNA repair and assists with primary DNA replication




Klenow fragment
• The DNA polymerase I enzyme is 103 kD.
• Of this a 68 kD portion is called the Klenow fragment.
• Klenow has the 3’ to 5’ exonuclease and the polymerase activity in different regions of the fragment.
Another 35 kD fragment has a 5’ to 3’ exonuclease activity





DNA polymerase II
• Molecular weight 120 KD
• It is very specialised enzyme involve in DNA repair.





DNA polymerase III
• DNA polymerase III is the main polymerase for E. coli chromosome replication.
• It is made up of many subunits (hetro multimeric ).
• It has several different activities.
• It is found in different forms ( subunit associations).
• E. coli's has 3’ to 5’ exonuclease proofreading.
• The gene dna E codes for the 130 kD fragment which has the polymerase activity.
• The alpha subunit works to increase error proofreading ability





genes and their role in E. coli DNA replication.

Gene
• dnaA,I,P
• dnaB,C
• dnaE,N,Q,X,Z
• dnaG
• gyrA,B
• lig
• oriC
• polA
• polB
• rep
• ssb



Function
• Initiation
• Helicase at oriC
• Subunits of DNA polymerase III
• Primase
• Subunits of gyrase
• Ligase
• Origin of Replication
• DNA polymerase I
• DNA polymerase II
• Helicase
• Single-stranded DNA binding proteins





Protein involve in DNA replication
• Dna A: recognizes and binds at the origin of replication.
Dna A is monomer of 82 KD protein.
• Dna T: Dna T binding with helicase which cause melting of small stretch.
• SSB protein: the association of these protein prevent the reformation of ds DNA.
SSB is tetramer of 74 KD protein.
• Dna B : activation of primase which synthesis the RNA primer, hexamerthe activity of Dna B is facilitated by binding of another protein
One monomer of Dna C bind to each of the subunit of Dna B hexamer
One primer initiate the DNA synthesi, DNA pol take new nucleotide.





Process of DNA replication
Initiation: In the initiation step, several key factors are recruited to an origin of replication. This origin of replication is unwound, and the partially unwound strands form a "replication bubble", with one replication fork on either end.
The factors involved are collectively called the pre-replication complex It consists of the following:
• A helicase, which unwinds and splits the DNA ahead of the fork. Thereafter, single-strand binding proteins(SSB) swiftly bind to the separated DNA, thus preventing the strands from reuniting.
• A primase, which generates an RNA primer to be used in DNA replication.
A DNA holoenzyme, which in reality is a complex of enzymes that together perform the actual replication









Elongation
• After the helicase unwinds the DNA, single-strand binding protein is used to hold the DNA strands apart. RNA primase is then bound to the starting DNA site.
• At the beginning of replication, an enzyme called DNA polymerase binds to the RNA primase, which indicates the starting point for the replication. DNA polymerase can only synthesize new DNA from the 5 to 3’(of the new DNA). Because of this, the DNA polymerase can only travel on one side of the original strand without any interruption.





• This original strand, which goes from 3’ to 5’, is called the leading strand. The complement of the leading strand, from 5’ to 3’, is the lagging strand.
• Each time the helicase unwinds additional DNA, new DNA polymerase needs to be added to ensure there remains enough. As a result, the DNA of the lagging strand is replicated in a piecemeal fashion. Another enzyme, DNA ligase, is used to connect the so-called Okazaki fragments.
• In prokaryotes, coupled leading strand and lagging strand synthesis is achieved by the action of the DNA polymerase III holoenzyme.











Termination
• Termination occurs when DNA replication forks meet one another or run to the end of a linear DNA molecule.
• Termination may occur when a replication fork is deliberately stopped by a special protein, called a replication terminator protein, that binds to specific sites on a DNA molecule .





Some antimicrobials inhibit normal nucleic acid replication in bacteria
• Fluoroquinolones (norfloxacin, lomefloxacin, fleroxacin, ciprofloxacin, enoxacin, trovafloxacin, etc.) work by inhibiting one or more of a group of enzymes called topoisomerase , enzymes needed for bacterial nucleic acid synthesis.
• Sulfonamides and Trimethoprim (co-trimoxazole - a combination of sulfamethoxazole and trimethoprim, sulfanilamide, etc.) block enzymes in the bacteria pathway required for the synthesis of tetrahydrofolic acid, a cofactor needed for bacteria to make the nucleotide bases thymine, guanine, uracil, and adenine .
• Metronidazole is a drug that is activated by the microbial proteins flavodoxin and feredoxin found in
microaerophilc and anaerobic bacteria and certain protozoans. Once activated, the metronidazole puts
nicks in the microbial DNA strands.





Theta mode of replication
Many bacteria such as e.coli the bubble is formed at thje origin of replication and DNA synthesis start both direction from this point.thus the shape of DNA looks similar to the latin latter theta at the beginning of replication.this region of replication continues to grow until entire DNA is duplicated. This mode of replication of circular DNA is known as Theta mode of replication.








DNA Replication in Eukaryotes
As in prokaryotes, the linear chromosomes of eukaryotes replicate by strand separation and complementary base pairing (def) of free deoxyribonucleotides (def) with those on each parent DNA strand . As with prokaryotes, DNA replication in eukaryotic cells is bidirectional. However, unlike the circular DNA in prokaryotic cells that usually has a single origin of replication the linear DNA of a eukaryotic cell contains multiple origins of replication