Docstoc

DNA replication - Molecular Biology

Document Sample
DNA replication - Molecular Biology Powered By Docstoc
					Life Science Study materials Molecular Biology --------------------------------------------------------------------

(Life Science Study Materials)

The material is prepared by

I.Satish Kumar
Lecturer in Biochemistry http://biochemden.blogspot.com www.biochemistryden.com
Email: Satishkumar7777@gmail.com

My Sweet Name_________________________________________________________

-------------------------------------------------------------------------------------------- 1 Class ______________ Group _________________________Roll No ______________ I.SATISH KUMAR Lecturer in Biochemistry
College Name ___________________________________________________________ Email: Satishkumar7777@gmail.com
http://biochemden.blogspot.com www.biochemistryden.com

Life Science Study materials Molecular Biology --------------------------------------------------------------------

MOLECULAR BIOLOGY

DNA Replication
Genetic information is transferred from parent to progeny organisms by a faithful replication of the parental DNA molecules. Usually the information resides in one or more double-stranded DNA molecules. Replication of double-stranded DNA is a complicated process that is not completely understood. This complexity results in part from the following facts: 1) 2) 3) A supply of energy is required to unwind the helix; The single strands resulting from the unwinding tend to form intra-strand base pairs; A single enzyme can catalyze only a limited number of physiological and chemical reactions and many reactions are needed in replication; Several safe guards have evolved that are designed both to prevent replication errors and to eliminate the rate errors that do occur; and Both circularity and the enormous size of DNA molecules impose genomic constraints on the replicative system, and how these fits into the system have to be understood.

4)

5)

The basic rule for replication of all Nucleic acids
All genetically relevant information contained in any nucleic acid molecule resides in its base sequence, so the prime role any mode of replication is to duplicate the base sequence of the parent molecule. The specificity of base pairing – Adenine with Thymine and Guanine with Cytosine – provide the mechanism used by all replication systems. Further more 1. Nucleotide monomers are added one by one to the end of a growing strand by an enzyme called a DNA polymerase. The sequence of base in each new or daughter strand is complementary to the base sequence in the old or parent strand being copied – that is, if there is an adenine in the parent strand, a thymine will be added to the end of the growing daughter strand when the adenine is being copied.

2.

In the following section we consider how the two strands of a daughter molecules are physically related to the two strands of the parent molecule. Replication can be broadly defined as genome duplication, an essential process for the propagation of cellular genome and those of ‘Molecular parasites’ – Viruses, plasmids and transposable elements. The genome to be duplicated is the parental genome, and the copies are daughter genomes.

Nucleic acids as genetic material:
Chromosomes mainly consist of nucleoproteins, having two components, nucleic acids and proteins. One of these should obviously constitute the genetic material. The following experiments demonstrating that nucleic acids and not the proteins contain the genetic information will be discussed. 1. Transformation experiments
2. 3. Experiments with bacteriophage (T2) infection) Experiments with Tobacco Mosaic Virus (TMV)

-------------------------------------------------------------------------------------------- 2 I.SATISH KUMAR Lecturer in Biochemistry
http://biochemden.blogspot.com www.biochemistryden.com
Email: Satishkumar7777@gmail.com

Life Science Study materials Molecular Biology --------------------------------------------------------------------

1. Transformation experiments:
F.Griffith initially conducted transformation experiments in 1928. He injected a mixture of two strains of Pneumococcus (Diplococcus pneumoniae) into mice. One of these two strains, S III was virulent and the other strain R II was non-virulent (causing no infection). Heat killed virulent strin SIII when injected individually did not cause death, showing that infectivity after heat killed is lost. The mice injected with a mixture of R II (living) and S III (heat killed) died and virulent pneumococci could be isolated from these mice.

2. Experiments with bacteriophage (T2) infection):
The fact that DNA is the genetic material could also be demonstrated with the help of infecting bacteria by bacteriaphage known as T2. This phage consists of proteins and DNA. The head region consists of protein coat and DNA. The tail similarly consists of tail core, tail sheath and tail fibers. In 1952, A.D.Hershey and M.J.Chase reported results of experiments of which demonstrated that only DNA of the phage enters host cells and that this DNA carries all the genetic information necessary for assembly of new phage progeny. In these experiments, phage DNA was made radioactive by growing infected bacteria on a medium containing 32PO4. Since phage proteins do not contain phosphorous, only DNA would be labeled. With the help of 35SO4. Since DNA does not contain sulfur, only protein would be labeled with S35. Hershey and chase then allowed both kinds of labeled bacteria were immediately agitated in a warning blender. After shaking, only radioactive 32P was found associated with bacterial cells and 35 S was found only in surrounding medium and not in bacterial cells. When phage progeny was studied for radioactivity in this experiment, it was found that the phage progeny carried label only with 32 P. The progeny was not labeled with 35S. Conclusion: Hershay and Chase proved that DNA is the sole genetic material in DNA bacteriophages.

3. Experiments with Tobacco Mosaic Virus (TMV):
The experiment conducted by H.Fraenkel - Conrat in California showing that RNA is the genetic material. Techniques were first developed for separating TMV particles into RNA and Proteins. Later by using RNA and proteins separately in tests for infectivity, it could be shown that RNA alone was able to cause infection. Such a property was not found in the protein fraction. It was also possible to synthesize chimeric virus particles using RNA from one strain and protein from another strain. Such chimeras had serological properties of those strains from which protein was derived, but in other properties, it resembled the other strain from which RNA was used.

DNA Replication: General Features:
Both DNA as well as genetic RNA is capable of undergoing self-replication. Theoretically, replication of double stranded DNA, could be a. Conservative b. Dispersive c. Semi-conservative a) Conservative replication model:
The conservative replication would mean that double stranded molecule is conserved as such and a new copy is synthesized from old molecule.

-------------------------------------------------------------------------------------------- 3 I.SATISH KUMAR Lecturer in Biochemistry
http://biochemden.blogspot.com www.biochemistryden.com
Email: Satishkumar7777@gmail.com

Life Science Study materials Molecular Biology -------------------------------------------------------------------b) Dispersive replication model:
In dispersive replication, the old molecule should disintegrate and two new molecules would be synthesized.

c) Semi-conservative replication model:
In the semi-conservative replication the two strands would separate from one another, maintain their integrity and each will synthesize, from the pool of nucleotides, its complementary strand. The result would be, that the newly synthesized molecule would carry (or) conserve one of the two strands from the parent molecule and the other strand would be newly assembled.

Experiments of Semi-conservative model evidence:
There is sufficient evidence to prove that double stranded DNA really replicates by semiconservative method.

a) Meselson and Stahl’s Experiment: (Using N15):
M.Meselson and F.W.Stahl in 1958 reported the results of an experiment, which was designed to test whether double stranded DNA replicates in a semiconservative manner. M.Meselson and F.W.Stahl (1958) allowed Escherichia coli cells to grow on N15 culture medium for about 14 cell generations, so that almost all nitrogen (N14) in DNA is replaced by N15. Then the cells were abruptly transferred to N14 culture medium. Since the time required for onecell generations was determined to be about 30 minutes. It was possible to remove the cells after one or more known number of generations of replication. Their DNA could then be analyzed and the following results were obtained. DNA sample obtained one cell generation after the transfer of N14 culture medium showed only one density band as observed through ultraviolet absorption pattern. This band indicated uniform homogenous density of DNA after one cell generation. Moreover, the band was exactly between the bands formed by N15 DNA and N14 DNA, indicating that all DNA found after first generation had intermediate density. This is what one would expect if DNA replicates in a semiconservative manner.

b) Cairn’s auto-radiography experiment: (Tritiated Thymidine[H3-TdR])
J.Cairns, using the technique autoradiography also demonstrated semi conservative mode of replication of bacterial chromosome. In autoradiography technique, the cells are first supplied with a suitable material like tritiated thymidine (H3-TdR; H3 is heavy isotope of hydrogen and it replaces normal hydrogen in tymidine to give rise to tritiated thymidine). Tritiated thymidine is used since this will selectively label only DNA and will not label RNA, since thymine base is absent in RNA.the tritiated thymine gets incorporated into DNA and replaces ordinary thymidine. The cellular material is then sanctioned or else the cells may be broken down to release the intact bacterial chromosomes on slides. These slides are then covered by photographic emulsion and stored in dark. During this storage the particles emitted by tritiated thymidine will expose the film, which can be developed. This photograph will then show the regions of the presence of tritium and thus indirectly show the presence of labeled DNA. Using the above technique, replication of DNA could be easily followed by J.Carins and the results were reported in 1963. During incorporation of tritiated thymidine, autoradiographs could be prepared at regular known intervals. Autoradiography from this replicating material prepared at regular known intervals demonstrated semi-conservative mode of replication.

-------------------------------------------------------------------------------------------- 4 I.SATISH KUMAR Lecturer in Biochemistry
http://biochemden.blogspot.com www.biochemistryden.com
Email: Satishkumar7777@gmail.com

Life Science Study materials Molecular Biology -------------------------------------------------------------------Lighter density of dots is considered to indicate that only one of the two strands is labeled, while a heavier density of dots will indicate that both strands are labeled, Such a situation was actually observed. The rate at which the replication proceeds could also be worked out by measuring the length of DNA undergoing replication in a known interval of time. Carins worked out the generation time as 30 minutes in E.coli. The length of the chromosome was worked thus would be approximately 30µ to 40µ per minute (1 mm=100µ).

DNA Mechanism of DNA replication in E.Coli:
DNA replication starts at unique chromosomal origins, processed bidirectionally, and is semi-conservative. DNA replication mechanisms depend on prior events: identification of a system capable of sustaining in vitro replication of small plasmids carrying “Ori.C” The synthesis of a DNA molecule can be divided into THREE stages: 1. Initiation 2. Elongation 3. Termination

1. Initiation:
The E.Coli replication origin, called “Ori.C”, consists of 245 base pairs, many of which are highly conserved among bacteria. The key sequences for this discussion are two series of short repeats; there repeats of a 13 base pair sequence and four repeats of a 9 base pair sequence. At least “Eight different enzymes” (or) “Proteins” participate in the initiation phase of replication. They open the DNA helix at the origin and establish a preparing complex that sets the stage for subsequent reactions. The key component in the initiation process is the Dna.A protein. A complex of about twenty Dna.A protein molecule binds to the four Nine bp repeats in the origin. In a reaction that requires ATP and is facilitated by the bacterial histone like protein “HU”, the Dna.A protein recognizes and successfully denatures the DNA in the region of the there 13 base pairs repeats, which are rich in A=T pair. The Dna.B protein then binds to this region in a reaction that requires the Dna.C protein. Dna.B is a “Helicase” that unwinds duplex DNA. The helicases constitute a class of enzymes that can move along a DNA duplex utilizing a class of enzymes that can move along a DNA duplex utilizing the energy of ATP hydrolysis to separate the strands. The separated strands are inhibited from subsequently re-annealing by the E.Coli Single Stranded Binding protein (SSBP), which binds to both separated strands. Multiple molecules of SSB bind cooperatively to single – stranded DNA, stabilizing the separated DNA strands and preventing re-naturation. DNA replication must be occurs only once in each cell cycle. Initiation is the only phase of replication that is regulated, but the mechanism is not yet well understood. The Dna.A protein hydrolyses its tightly bound ATP slowly (about 1 hour) to form an inactive Dna.A – ADP complex. Reactivating this complex is facilitated by an interaction between Dna.A protein and acidic phospholipids in the bacterial plasma membrane. Initiation at in appropriate times is prevented by the presence of the inactive Dna.A-ADP complex by the binding of a protein called “Ici.A Inhibitor of chromosomal initiation) to the 13 base pair repeats, and perhaps by other factors.

-------------------------------------------------------------------------------------------- 5 I.SATISH KUMAR Lecturer in Biochemistry
http://biochemden.blogspot.com www.biochemistryden.com
Email: Satishkumar7777@gmail.com

Life Science Study materials Molecular Biology --------------------------------------------------------------------

-------------------------------------------------------------------------------------------- 6 I.SATISH KUMAR Lecturer in Biochemistry
http://biochemden.blogspot.com www.biochemistryden.com
Email: Satishkumar7777@gmail.com

Life Science Study materials Molecular Biology --------------------------------------------------------------------

2. Elongation:
The elongation phase of replication consists of two seemingly similar operations that are mechanically quite distinct: Leading strand synthesis and Lagging synthesis. Several enzymes at the replication fork are important to the synthesis of both strands. “DNA helicases” unwind the parental DNA. “DNA topoisomerases” relieve the tropological stress included by the “Helicases”, and “SSBP” stabilizes the separated strands. a) Leading strand synthesis: It begins with the synthesis by “Primase” of a short (10 to 60 nucleotide) RNA primer at the replication origin. Deoxyribonucleotides are then added to this primer by DNA polymerase-III once begun, leading stand synthesis proceeds continuously, keeping pace with the replication fork. b) Lagging strand synthesis: It must be accomplished in short fragments (Okaazakki fragments) synthesized in the direction opposite to the fork movement. Each fragment must have its own RNA primer synthesized by “PRIMASE”, and positioning of the primers must be controlled and coordinated with fork movement. The regulatory apparatus for “lagging strand synthesis” is a traveling protein machine called a “ PRIMOSOME”, which consists of several different proteins including the Dna.B protein, Dna.C protein and Primase. The primosome moves along the lagging strand template in the 5’ 3’ direction, keeping pace with the replication fork. As it moves the primosome at intervals compels primase to synthesize a short (10 to 60) residues. RNA primer to which DNA is then added by DNA polymerase-III. The direction of the synthetic reactions of “Primase” and “Polymerase-III” is opposite to the direction of primosome movement. When the new okazakii fragment is complete, the RNA primer is removed by DNA polymerase-I and is replaced with DNA by the same enzyme. The remaining nick is sealed by “DNA ligase”. Dimeric DNA polymerase-III containing two “Catalytic sites” for nucleotide addition. The lagging strand template wraps around one of the catalytic sub units, the inverting the physical direction of the growing lagging strand. Special loop like arangement will be form, which is known as “THROMBONE”.

-------------------------------------------------------------------------------------------- 7 I.SATISH KUMAR Lecturer in Biochemistry
http://biochemden.blogspot.com www.biochemistryden.com
Email: Satishkumar7777@gmail.com

Life Science Study materials Molecular Biology --------------------------------------------------------------------

-------------------------------------------------------------------------------------------- 8 I.SATISH KUMAR Lecturer in Biochemistry
http://biochemden.blogspot.com www.biochemistryden.com
Email: Satishkumar7777@gmail.com

Life Science Study materials Molecular Biology --------------------------------------------------------------------

3) Termination:
a) Termination of Replication of a circle:
There are two possible modes of replication: • There are defined termination sequences, or • Two growing points collide and termination occurs whenever the collision point happens to be. In both cases, termination might occur exactly halfway around the circle. Both termination modes have been observed. Termination has a “Topological problem”. When double – stranded circular DNA replicates semiconservatively, the result is a pair of circles that are linked as in a chain. Such a structure is called “CATENANE”. Catenated molecules have been observed in numerous systems, and evidence is accumlating to indicate that they result from replication. “DNA gyrase” is capable of decatenating two circles, which enzyme is responsible for separation of daughter molecules. E.coli chromosome and several plasmids carry specific sequences, called “ter sites”, where TBP ( ter binding protein) or ‘Tus protein’ binds. In the termination zone of E.coli, there are three ter sites (ter A, ter D and terE) for counter-clockwise fork. These six sites are arranged in overlapping manner, laving no “Replication – free” gap on the chromosome. TBP-ter complexes formed at “ter” sites stalls the replication fork, by inhibiting the DNA helicase or DnaB. When this termination zone is deleted, replication stops, simply by the meeting of opposite replication forks, suggesting that the termination zone is not essential.

-------------------------------------------------------------------------------------------- 9 I.SATISH KUMAR Lecturer in Biochemistry
http://biochemden.blogspot.com www.biochemistryden.com
Email: Satishkumar7777@gmail.com

Life Science Study materials Molecular Biology -------------------------------------------------------------------b) Termination in ‘Linear’ DNA molecules:
E.Coli phage T7 DNA replicates as a linear molecule. The origin is located 17% of the total distance from the left end of the molecule and replication is bidirectional. Initially there is a single replication bubble (molecule-I), and when the leftward fork reaches the terminus, the molecules assumes a “Y”-form (molecule-III). “Ribonucleases” exist that can remove this RNA but once it has been removed, either a 5’OH group would remain at the ends of the molecule.

Interaction of “Tus protein” with termination sites stops DNA replication:
Although much research attention has been focused on replication origins, E.Coli DNA also has been shown to contain termination (TER) sites, which bind a specific protein called Tus. This protein may act to stop replication by preventing “Helicase (DnaB) from unwinding duplex DNA, thereby interrupting the function of the growing fork. The details of the mechanism that arrests the growing fork are not yet clear. Replication of the circular E.coli chromosome produces two interlocking daughter chromosomes, which must be separated.

-------------------------------------------------------------------------------------------- 10 I.SATISH KUMAR Lecturer in Biochemistry
http://biochemden.blogspot.com www.biochemistryden.com
Email: Satishkumar7777@gmail.com

Life Science Study materials Molecular Biology --------------------------------------------------------------------

Eukaryotic DNA Replication:
The primary effort has focused on replication of the SV 40 (Simian Virus 40) chromosome; these studies have progressed so rapidly in recent years that the SV40 chromosome now can be replicated in vitro using only eight purified components from mammalian cells. The specific functions of these proteins are highly reminiscent of proteins required for replication of plasmids carrying “Ori.C”. The DNA molecules in eukaryotic cells are considerably larger than those in bacteria and are organized into complex nucleoprotein structure.

Initiation:
Similar to E.Coli, replication is initiated at a unique location on the SV40 DNA by interactivation of a virus-encoded, site-specific DNA binding protein called “T.antigen”. This multifunctional protein locally unwinds duplex at the SV40 origin also requires ATP and replication factor A (RF-A). A host-cell single strand binding protein with a function similar to that of SSB in E.Coli cells.

Elongation:
As in E.coli, eukaryotic DNA replication occurs “bidirectionally from RNA primers made by a “Primase” synthesis of the leading strand is continuous, while synthesis of lagging strand is discontinuous. Two distinct ‘Polymerases’ - α and δ”, appear to function at the eukaryotic growing fork. Polymerase δ (pol. δ) is largely responsible for leading synthesis; polymerase α (pol. α), which is tightly associated with a Primase”, is thought to synthesize the lagging strand. RNA primers, formed by the action of “Primase”, these are elongated for a short stretch by “Pol. α”, whose activity is stimulated by replication factor.C. Binding of “PCNA (Proliferating Cell Nuclear Antigen)” at the primer template terminus then displaces Pol.α, thus interrupting leading-strand synthesis. PCNA increases the “Processivity of the enzyme”. The function of PCNA thus appears to be highly analogous to that of the β-sub unit of E.Coli polymerase-III.

Termination:
The termination of replication on linear eukaryotic chromosomes involves the synthesis of special structures called “Telomeres” at ends of the cheromosmome. The telomers consists of repetitive “Oligomeric sequences”. The enzyme that prevents this progressive shortening of the lagging strand is a “modified reverse transcriptase” called “Telomerase”, which can elongate the lagging-strand template from its 3’-hydroxyl end. This unusual enzyme contains a catalytic site that polymerizes deoxyribonucleotides directed by a RNA template as well as the RNA molecule that functions as that template.

-------------------------------------------------------------------------------------------- 11 I.SATISH KUMAR Lecturer in Biochemistry
http://biochemden.blogspot.com www.biochemistryden.com
Email: Satishkumar7777@gmail.com

Life Science Study materials Molecular Biology --------------------------------------------------------------------

Enzymes involved in the DNA Replication & its role:
Replication means “Synthesis of daughter nucleic acid molecules identical to the parental nucleic acids”. In the replication nucleic acids will be double by the help of some organic catalysts (or) enzymes. Some enzymes, 1) 2) 3) 4) 5) Single Stranded Binding Protein (SSBP) DNA Helicases Topoisomerases DNA primases DNA polymerases

1) Single Stranded Binding Protein (SSBP):
• • • • • Molecular weight of the SSB protein is 75,600 It contains FOUR identical subunits, which binds single stranded DNA. The main function of the SSB protein is “Prevents reannealing” in Replication process. As Polymerase-III holoenzyme advances, it must displace the SSB protein in order that base pairing of the nucleotide being added can occur. “RFA” is a single stranded DNA binding protein equivalent in function to the E.Coli SSB protein.

2) DNA helicases:
• • • • • • DNA helicase enzyme functions “Unwinds DNA”. They have molecular weight 300,000, which contain SIX identical sub units. “Okazakii fragments” are short stretches of 1000-2000 bases produced during discontinuous replication, they are later joined into a covalently intact strand. The “Dna.B helicase” and “Dna.G Primase” constitute a finctional unit within the replication complex, called the “PRIMOSOME”. The DNA is around by the Dna.B helicase at the replication fork, DNA primase occasionally associates with Dna.B helicase and synthesizes a short RNA primer. ”Helicase” and “Nuclease” activities of the Rec B,C,D enzyme is believed to help initiate homologous genetic recombination in E.Coli. It is also involved in the repair of double – strand breaks at collapsed replication fork.

Function:
A “Helicase” is an enzyme that separates the strands of DNA usually the hydrolysis of ATP to provide the necessary energy.

3) Topoisomerases:
• • “Topoisomerases” is an enzyme that can change the “Linking number”. Every cell has enzymes that increas (or) decrease the extent of DNA unwinding are called “Topoisomerases” the property of DNA that they change is the linking number. Topoisomerses”, these enzymes play an especially important role in processes such as “Replication” and “DNA packaging”. There are two classes of topoisomerases.

• •

What is linking number? The linking number (Lk) is a topological property. Lk can be defined as “ the number of times the second strand pierces the second strand surface”

-------------------------------------------------------------------------------------------- 12 I.SATISH KUMAR Lecturer in Biochemistry
http://biochemden.blogspot.com www.biochemistryden.com
Email: Satishkumar7777@gmail.com

Life Science Study materials Molecular Biology -------------------------------------------------------------------a) Type-I b) Type-II Topoisomerases Topoisomerases

a) Type-I Topoisomerases:
This act by transiently breaking one of the two DNA strands, rotating one of the ends about the unbroken strand, and rejoining the broken ends; they change Lk in increments of 1.

b) Type-II Topoisomerases:
The enzyme breaks both DNA strands and change Lk in increments of 2.

Prokaryotic Topoisomerases:
FOUR different Topoisomerases (I and IV) occur in E.Coli.
1)

Type.I (Topoisomerase I and III), generally relax DNA by removing negative
supercoils (increasing Lk) Type.II (Topoisomerase II and IV), Topoisomerase II are also called “DNA gyrase”, can introduce negative supercoils. (Decrease Lk). • • It uses the energy of ATP and a surprising mechanism to accomplish this. The degree of supercoiling of bacterial DNA is maintained by regulation of the net activity of topoisomerase-I and II.

2)

Eukaryotic Topoisomerases:
Eukaryotic cells also have type-I and type-II topoisomerases.Topoisomerases-I & II are both type-I. The two type-II topoisomerases, topoisomerases IIα and IIβ, can not unwind DNA (introduce negative supercoils). Although both can relax positive and negative supercoils. We consider one probable origin of negative supercoils in eukaryotic cells. • • The DNA gyrase molecualr weight is 400,000, which contain FOUR sub units and functions “Super coiling”. Supercoiled DNA is a higher ordered structure occurring in circular DNA molecules wrapped around a core.

3) DNA Primase:
In replication, before DNA polymerase can begin synthesizing DNA primers must be present on the template generally short segments of RNA synthesized by enzyme called “Primases”. • • • DNA primase have molecular weight 60,000 daltons and contain only single sub unit, which functions synthesize RNA primers. The Dna B helicase and Dna G primase constitute a functional unit within the replication complex, called the “Primosome”. The RNA primer typically is 15-50 bases long. It synthesizes primers starting with the sequence pppAG, opposite the sequence 3’-GTC-5’ in the template.

-------------------------------------------------------------------------------------------- 13 I.SATISH KUMAR Lecturer in Biochemistry
http://biochemden.blogspot.com www.biochemistryden.com
Email: Satishkumar7777@gmail.com

Life Science Study materials Molecular Biology --------------------------------------------------------------------

4) DNA Ligase:
An enzyme that creates a phosphodiester bond between the 3’ end of one DNA segment and the 5’ end of another. Once the RNA primer has been removed and replaced the adjacent Okajakii fragments must be linked together. The 3’-OH end of one fragment is adjacent to the 5’-Phosphate end of the previous fragment. The responsible for sealing this nick lies with the enzyme DNA ligase. Ligases are present in both prokaryotes and eukaryotes.

Mechanism of DNA ligase activity:
The E.Coli and T4 ligases share the property of sealing nicks that have 3'’-OH and 5’- P termini. Both enzymes under take a two step reaction, involving an ‘enzyme-AMP complex’. • • The E.Coli and T4 enzyme use different cofactors. The E.Coli enzymes uses NAD as a cofactor, T4 enzyme uses ATP.

The AMP of the enzyme complex becomes attached to the 5’-Phosphate of the nick; and then a phosphodiester bond is formed with the 3’-OH terminus of the nick, releasing the enzyme and the AMP.

-------------------------------------------------------------------------------------------- 14 I.SATISH KUMAR Lecturer in Biochemistry
http://biochemden.blogspot.com www.biochemistryden.com
Email: Satishkumar7777@gmail.com

Life Science Study materials Molecular Biology --------------------------------------------------------------------

5) DNA Polymerases:
In 1957, “Arthur Korenberg” showed that extracts of E.Coli contain a DNA polymerase (now called Polymerase I or Pol I ). This enzyme able to synthesize DNA from four precursor molecules, namely the four deoxynucleotides 5’-Phosphate (dNTP), dATP, dGTP, dCTP and dTTP, as long as a DNA molecule to be copied (a template DNA) is provided. Neither 5’-monophosphates nor 5’diphosphates, nor 3’-(mono-, di-, or tri-) phosphates can be polymerized only the 5’-triphosphates are substrates for the polymerization reaction. E.Coli has at least Three DNA polymerases: 1) DNA Polymerases-I 2) DNA Polymerases-II 3) DNA Polymerases-III

1) DNA Polymerases-I:
DNA pol-I is far from irrelevant, however. This enzyme serves a host of “Clean-up” function during replication, recombination and repair. These special functions are enhanced by an additional enzymatic activity of DNA polymerase I, a 5’ 3’ exonuclease activity. This activity is distinct from the 3’ 5’ proofreading exonuclease and is located in a distinct structural domain that can be separated from the enzyme by mild protease treatment. When the new okazakii fragment is complete, the RNA primer is removed by DNA polymerase I, and is replaced with DNA by the sea enzyme. When the 5’ 3’ exonuclease domain is removed, the remaining fragment (Mr. 68,000) retains the polymerization and proofreading activities and is called the “Large (or) Klenow fragment”. This klenow fragment lacks the “5’ 3’ exonuclease activity”. The structure of the klenow fragment has been demonstrated, and it is this fragment of DNA polymerase I, the 5’ 3’ exonuclease activity of intact DNA polymerase I permits it to extend DNA strand even if the template is already paired to an exiting strand of nucleic acid.

Klenow fragment:
Also known as Klenow polymerase, which is produced commercially by expressing a truncated pol.A gene.

Using this activity, DNA polymerase I can degrade (or) displace a segment of DNA (or RNA) paired to the template and replace a segment of DNA (or RNA) paired to the template and replace it with newly synthesized DNA. Most other DNA polymerizes including DNA polymerase III, lack a 5’ 3’ exonuclease activity.

Fundamental Reaction:
The fundamental (dNMP)n + dNTP (dNMP) n+1 + PPi reaction is a ‘Nucleophilic attack’ by the 3’-hydroxyl group of the nucleotide at the 3’ end of the growing strand on the 5’-α-phosphorous of the incoming deoxynucleoside 5’triphosphate. Inorganic phosphate is release in the reaction.

2) DNA polymerase II :
DNA polymerase II is a minor component of the cell during normal growth but is inducible by the SOS response. It appears that this enzyme allows nucleotide incorporation opposite AP sites. DNA polymerase II appears to have highly specialized DNA repair function. This enzyme participates in “Base-Excision repair” and “ Nucleotide-Excision repair”

-------------------------------------------------------------------------------------------- 15 I.SATISH KUMAR Lecturer in Biochemistry
http://biochemden.blogspot.com www.biochemistryden.com
Email: Satishkumar7777@gmail.com

Life Science Study materials Molecular Biology -------------------------------------------------------------------Base-Excision repair:
Every cell has a class of enzymes called “DNA glycosylases” that recognize particularly common DNA lesions and remove the affected base by cleaving the N-glycosyl bond. This creates an “Apurinic” (or) “Apyrimidinic site” in the DNA, both commonly referred to as a “basic” (or) “AP sites”. Each DNA glycosylase is generally specific for one type of lesion.

Nucleotide-Excision repair:
DNA lesions that cause large distortions in the helical structure of DNA generally are repaired by the nucleotide-excision system. In E.Coli the key enzyme is made up of THREE sub units, products of the “uvr.A, uvr B, uvr C genes”, and is called the “ABC Excinuclease”. This enzyme recognizes many types of lesions, including “Cyclobutane-pyrimidine Dimers”, “6,4-photoproducts” and several other types of “base adducts”. The term “Excinucleas” is meant to distinguish this activity from that of standard endonucleases.

3) DNA Polymerases-III:
E.Coli Polymerase III is a very complex enzyme. In its most active form it is associated with nine (or) more other proteins to form the “Pol III HOLOENZYME”, occasionally termed Pol III. The term holoenzyme refers to an enzyme that contains several different subunits and retains some activity even when one (or) more subunits is missing. The smallest aggregate having enzymatic activity is called the “CORE ENZYME”. The activity of the core enzyme and the holoenzyme are usually very different. The DNA polymerase III has highly complex protein composed of 10 different polypeptides. Overall, the enzyme has an “Assymmetric dimeric structure”. It contains two copies of most sub units and two catalytic sites for nucleotide addition.

Prokaryotic DNA polymerase III:
DNA polymerase III is principle replicative DNA polymerase of E.Coli. it is a multisubunit complex. The holoenzyme (Apoenzyme [protein part] + Coenzyme = Holoenzyme) functions as a “Heterodimer” of complexes at the replication fork, with each monomer seeing to the synthesis of one daughter strand.

α +

ε

+

θ Sub units

Core enzyme

The core enzyme, which contains the essential enzyme activities. The assembly of the holoenzyme in vivo occurs as follows: the β-sub unit functions as a dimer and forms a ring (or) clamp, which can slide along single-stranded DNA. The β-sub unit is located onto template-primer by the γ-complex, an ATP-dependent process, to form the “Pre initiation complex”. The loading of the β-sub unit allows the core –enzyme to bind, and addition of the τ-sub unit facilitates dimerization. The holoenzyme is “Symmetrical” except for the γ-complex, which is associated with only one of the monomers. The γ-complex is required for both loading and unloading the β-sub unit from DNA. The presence of the γ-complex allows the β-sub unit to dissociate from the template primer when the polymerization encounters the 5’end of a previously synthesized okazakii fragments on the retrograde template.

-------------------------------------------------------------------------------------------- 16 I.SATISH KUMAR Lecturer in Biochemistry
http://biochemden.blogspot.com www.biochemistryden.com
Email: Satishkumar7777@gmail.com

Life Science Study materials Molecular Biology --------------------------------------------------------------------

Sub units of the E.Coli DNA polymerase III holoenzyme and their proposed functions

Sub unit
Core Sub units

Gene
dna.E (pol.C)

Properties and proposed function
5’ 3’ polymerase activity, required for DNA synthesis

α ε
θ

dna .Q (mut.D)

3’ 5’ exonuclease activity required for proof reading.

Unassigned

Function uncertain, may help to assemble other sub units.

Accessory sub units

dna.Xa

τ

DNA dependent ATPase, required initiation. Promotes dimerization

for

γ

dna.Xa

Associates with four peptides to form a DNA dependent ATPase known as the γ-complex required for initiation, facilitates β-sub unit binding. Associate with “ γ “ to form the γ-complex.

δ,δ’x,ψ δ ψ β

Unassigned

dna.N

“Sliding clamp”, which increases processivity of the holoenzyme. β binds to DNA to form a precipitation complex, a process which requires the ATP-dependant activity of the γ-complex dna.N is induced by the “SOS response”

-------------------------------------------------------------------------------------------- 17 I.SATISH KUMAR Lecturer in Biochemistry
http://biochemden.blogspot.com www.biochemistryden.com
Email: Satishkumar7777@gmail.com

Life Science Study materials Molecular Biology --------------------------------------------------------------------

Eukaryotic DNA polymerase:
Eukaryotic cells contain FOUR nuclear DNA polymerases and a fifth, which is responsible for organelle genome replication. The nuclear enzymes are DNA polymerases α, β, δ and ε.

DNA polymerase α and δ:
• • • • • • DNA polymerase α and δ are responsible for chromosomal replication. DNA polymerase δ has a proof reading capability. DNA polymerase δ binds an accessory factor called “Proliferating cell nuclear antigen (PCNA), a cylin analogous to the E.Coli polymerase III β sub unit in that it acts as a sliding ring to increase enzyme processivity. δ Synthesizes both the leading and lagging strands. To the lagging strand synthesis provide the template for an accessory fator, “Replication factor.C (RF.C)”, this role analogous to that of the E.Coli γ-complex. DNA polymerase α associates with “Primase” activity but no 3’ 5’ exonuclease activity, whereas DNA pol δhas a “Proof reading capability”.

DNA polymerase ε:
• • • The role is unclear. It is structurally very similar to DNA pol δ but does not associate with PCNA. It may be involved in DNA repair, like DNA polymerase β.

DNA polymerase γ:
• Responsible for the replication of mitochondrial DNA and a similar enzyme has been isolated from plant chloroplast.

Functions of DNA polymerase sub units:
DNA polymerase α DNA polymerase β DNA polymerase γ DNA polymerase δ DNA polymerase ε Lagging strand priming Repair polymerase Organell polymerase Principle relicative polymerase Unknown

-------------------------------------------------------------------------------------------- 18 I.SATISH KUMAR Lecturer in Biochemistry
http://biochemden.blogspot.com www.biochemistryden.com
Email: Satishkumar7777@gmail.com

Life Science Study materials Molecular Biology --------------------------------------------------------------------

A comparison of properties of DNA polymerases of E.Coli
Property DNA Polymerases

I
1 Polymerization 5’ 3’ Yes

II
Yes

III
Yes

2

Exonuclease 3’ 5’

Yes

Yes

Yes

3

Exonuclease 5’ 3’

Yes

Yes

Yes

4

Use of primed single strands

Yes

No

No

5

Use of nicked duplex

Yes

No

No

6

Molecular weight

109,000 (Single chain)

120,000 (Single Chain)

>250,000 (Hetero multimeric chain)

7

Molecules per cell

400

Not known

10-20

8

Genes

Pol A

Pol B

Pol C (dna E), dnaN, dnaZX, dnaQ, dnaT

9

Nucleotides polymerized at 37 , molecules/ minute

0

Upto 1000

Upto 50

Upto 15,000

10

Affinity for nucleoside triphosphate (TPs) precursors

Low

Low

High

Reference books:
1. 2. 3. 4. 5. 6. Molecular Cell Biology, 3/e Lodish and Baltimore Advanced Molecular Biology, Richard M. Twyman Molecular Biology, David Frefielder Principle’s of Biochemistry, 3/e, Lehninger, Nelson & Cox Genetics, P.K.Guptha, Rastoji publications Fundamentals of Biochemistry, Voet & Voet

-------------------------------------------------------------------------------------------- 19 I.SATISH KUMAR Lecturer in Biochemistry
http://biochemden.blogspot.com www.biochemistryden.com
Email: Satishkumar7777@gmail.com