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I.Satish Kumar
Lecturer in Biochemistry http://biochemistryden.blogspot.com www.biochemistryden.com
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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)
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.
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.
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.
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 30 to 40 per minute (1 mm=100).
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.
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.
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.
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 5OH group would remain at the ends of the molecule.
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 - and , appear to function at the eukaryotic growing fork. Polymerase (pol. ) is largely responsible for leading synthesis; polymerase (pol. ), 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. , whose activity is stimulated by replication factor.C. Binding of PCNA (Proliferating Cell Nuclear Antigen) at the primer template terminus then displaces Pol., 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 -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.
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
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)
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 II and II, 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.
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.
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.
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 5diphosphates, 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--phosphorous of the incoming deoxynucleoside 5triphosphate. 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
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.
Sub units
Core enzyme
The core enzyme, which contains the essential enzyme activities. The assembly of the holoenzyme in vivo occurs as follows: the -sub unit functions as a dimer and forms a ring (or) clamp, which can slide along single-stranded DNA. The -sub unit is located onto template-primer by the -complex, an ATP-dependent process, to form the Pre initiation complex. The loading of the -sub unit allows the core enzyme to bind, and addition of the -sub unit facilitates dimerization. The holoenzyme is Symmetrical except for the -complex, which is associated with only one of the monomers. The -complex is required for both loading and unloading the -sub unit from DNA. The presence of the -complex allows the -sub unit to dissociate from the template primer when the polymerization encounters the 5end of a previously synthesized okazakii fragments on the retrograde template.
Sub units of the E.Coli DNA polymerase III holoenzyme and their proposed functions
Sub unit
Core Sub units
Gene
dna.E (pol.C)
dna .Q (mut.D)
Unassigned
dna.Xa
for
dna.Xa
Associates with four peptides to form a DNA dependent ATPase known as the -complex required for initiation, facilitates -sub unit binding. Associate with to form the -complex.
,x,
Unassigned
dna.N
Sliding clamp, which increases processivity of the holoenzyme. binds to DNA to form a precipitation complex, a process which requires the ATP-dependant activity of the -complex dna.N is induced by the SOS response
DNA polymerase :
The role is unclear. It is structurally very similar to DNA pol but does not associate with PCNA. It may be involved in DNA repair, like DNA polymerase .
DNA polymerase :
Responsible for the replication of mitochondrial DNA and a similar enzyme has been isolated from plant chloroplast.
I
1 Polymerization 5 3 Yes
II
Yes
III
Yes
Exonuclease 3 5
Yes
Yes
Yes
Exonuclease 5 3
Yes
Yes
Yes
Yes
No
No
Yes
No
No
Molecular weight
400
Not known
10-20
Genes
Pol A
Pol B
Upto 1000
Upto 50
Upto 15,000
10
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 Principles of Biochemistry, 3/e, Lehninger, Nelson & Cox Genetics, P.K.Guptha, Rastoji publications Fundamentals of Biochemistry, Voet & Voet