Slide 1 : DNA REPLICATION
04-09-2011 5.30 PM
DNA REPLICATION - PART - II
05-09-2011 6.30 PM
By
Dr. Ichha PuraK
http://dripurak.com
http://drichhapurak.webnode.com DNA REPLICATION PART-II 1 9/4/2011
Slide 2 : DNA REPLICATION PART-I
INTRODUCTION
IMPORTANCE OF REPLICATION
THE REPLICATION FACTORY
MODE OF REPLICATION : SEMI CONSERVATIVE
EXPERIMENTAL EVIDENCE
PHASE OF REPLICATION
PLACE OF REPLICATION
DIRECTION OF REPLICATION
INITIATION OF REPLICATION: FACTORS INVOLVED –PROKARYOTES
EUKARYOTIC INITIATION OF REPLICATION : FACTORS INVOVED
PROTEINS AND ENZYMES INVOLVED IN REPLICATION
REPLICATION FORK DNA REPLICATION PART-II 2 9/4/2011
Slide 3 : DNA REPLICATION PART-II
REPLICATION FORK
MECHANISM OF REPLICATION : STEPS
LEADING AND LAGGING STRANDS
OKAZAKI FRAGMENTS
DNA POLYMERASES : PROKARYOTES
EUKARYOTIC DNA POLYMERASE
DIFFERENCE IN PROKARYOTIC AND EUKARYOTIC REPLICATION
TELOMERASE ACTIVITY
PROOF READING BY DP III
THE BIOCHEMICAL REACTION
SPEED OF REPLICATION
REGULATION OF REPLICATION DNA REPLICATION PART-II 3 9/4/2011
Slide 4 : DNA REPLICATION PART-II 4 NECK MOUTH REPLICATION FORK 9/4/2011
Slide 5 : DNA REPLICATION PART-II 5 MECHANISM OF REPLICATION
Mechanism of replication was studied for the first time for the bacterium Escherichia coli. DNA replication in higher organisms is less well understood, but involves almost same type of mechanism with some differences.
For Initiation of DNA Replication ,recognition of Origin of Replication is required. It is recognised by Pre Priming Complex. PPC consists of Dna A protein, Single stranded DNA binding proteins and enzyme Helicase. PPC help in separation of two parental strands locally and stabilize Replication Fork . 9/4/2011
Slide 6 : 9/4/2011 DNA REPLICATION PART-II 6 For semi conservative mode of DNA replication the two strands must get separated by dissolving the hydrogen bonds between base pairs gradually in a Zipper like manner and then two separated strands act as templates for synthesis of new ( daughter ) strands Steps of DNA Replication in Prokaryotes
SEPARATION OF TWO PARENTAL STRANDS
The two strands separate after formation of Initiation complex by the enzyme helicase at A-T rich sequences (Ori). A single ori is present in prokaryotic circular DNA. Many Ori are present in Eukaryotic linear DNA. Ori are the consensus sequences composed almost exclusively of AT base pairs.
Slide 7 : SOLVING PROBLEM OF SUPERCOIL FORMATION
2. The opening is facilitated by topoisomerase, which causes a nick or cut in one of the two strands.Nick is formed by breaking one phospho di-ester bond near replication fork.The nick helps to solve the problem of supercoil formation tendency of DNA double helix on separation of strands. DNA REPLICATION PART-II 7 9/4/2011
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Slide 9 : 3. This localized opening appears as bubble under electron microscope, consisting of two ‘Y’ shaped replication forks.
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Slide 11 : 9/4/2011 DNA REPLICATION PART-II 11 4.The single stranded DNA structure of replication fork is stabilized by SSDNA binding proteins which act in co-operative way . Binding of one SSBp help other SSBPs to bind on both sides of single strands
5. DP III starts synthesis by adding complementary deoxyribonucleotides onto 3’ OH end of RNA primer 6.A short sequence of Ribonucleotides (RNA Primer) is synthesized prior to DNA synthesis by primase ROLE OF RNA PRIMERS IN DNA REPLICATION
DNA Polymerase can not initiate DNA synthesis simply on single stranded DNA template. They require an RNA Primer ,short oligo ribonucleotide that makes base pair with few of exposed bases on DNA template having a free 3’OH group , which acts as first acceptor of incoming deoxyribonucleotide introduced by DNA polymerase.
Slide 12 : DNA REPLICATION PART-II 12 PRIMASE (A Specific RNA Polymerase ) synthesizes RNA primer of about 10 nucleotides long that are complementary and antiparallel to DNA template. In the resulting hybrid duplex U of RNA pairs with A of DNA. Only one RNA primer is synthesized in one origin on Leading strand but many RNA primers are synthesized on Lagging strand
PRIMOSOME
Pre Priming Compex of several proteins is assembled and binds to single stranded DNA displacing some of SSBPs .This protein complex along with enzyme Primase is called as Primosome. It initiates Okazaki Fragment formation by moving along the template for Lagging strand in the 5’→3’ direction for synthesis of RNA Primers 9/4/2011
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Slide 14 : DIRECTION OF REPLICATION
7. AS DNA polymerase can only add deoxyribonucleotides in 5’ 3’ direction and as two strands of DNA double helix run antiparallel, so synthesis on two strands differ DNA REPLICATION PART-II 14 9/4/2011 LEADING STRAND REPLICATION
8.On one of the strand of replication fork running in 3’ 5’ direction towards the fork, DNA is synthesized continuously in 5’3’ direction using only one RNA primer with its free 3’ OH at the beginning by DP III movement along the template. It is called as leading strand.
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Slide 16 : DNA REPLICATION PART-II 16 LAGGING STRAND REPLICATION
9.On other strand of the replication fork running in 5’ 3’ direction away from the replication fork , DNA is synthesized in a discontinuous manner in 5’ 3’ direction as small stretches . Each time it requires RNA primer with its free 3’OH for adding dNTPs by movement of DP III along the template. As a result many RNA-DNA pieces are formed which are termed as Okazaki Fragments after their discoverer Okazaki (Japanese Molecular Biologist). This strand is termed as lagging strand as it has to wait for replication fork to open gradually. 9/4/2011
Slide 17 : DNA REPLICATION PART-II 17 CHAIN ELONGATION
10.Although both strands replicate simultaneously but DNA synthesis on lagging strand require help of another two enzymes. RNase dismantles first the primers and then DP I replaces deoxyribonuleotides to fill the gaps.
11.After that DNA ligase catalyses phophodiester bond formation between two successive DNA pieces as a result of which continuous DNA strand is synthesized.
12.As both strands synthesize DNA simultaneously it is presumed that two molecules of DP III move in opposite directions along their respective template strands. This is accomplished by having the lagging strand template looped back on itself. (Fig Next slide) 9/4/2011
Slide 18 : DNA REPLICATION PART-II 18 Replication of the leading and lagging strands in E coli is accomplished by two DNA Polymerases III working together as part of a single complex 9/4/2011
Slide 19 : DNA REPLICATION PART-II 19 Replication of the leading and lagging strands in E coli is accomplished by two DNA Polymerases III working together as part of a single complex
The two DNA Polymerase III molecules travel together, even though they are moving towards the opposite ends of their respective templates. This is accomplished by causing the lagging strand template to form a loop. The polymerase releases the lagging strand template when it encounters the previously synthesized Okazaki fragment. The polymerase that was involved in the assembly of the previous Okazaki fragment has now rebound the lagging strand template farther along its length and is synthesizing DNA onto the end of RNA Primer that has just been constructed by the primase 9/4/2011
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Slide 21 : DNA REPLICATION PART-II 21 LEADING AND LAGGING STRANDS
To catalyse the polymerization reaction, DP enzyme requires all the four d NTPs, a template strand to copy, a primer containing free 3’ OH to which nucleotides can be added. The primer is required because DP is unable to initiate the formation of a DNA strand de novo. Rather, it is capable of adding nucleotides to a 3’OH terminus of the existing strand. DP is only capable of polymerizing a strand in 5’ 3’ direction. The two new strands are synthesized in opposite direction and mode of synthesis is quite different.One of newly synthesized strands ( leading strand) grows towards the replication fork and is synthesized continuously.The other newly synthesized strand ( lagging strand ) grows away from the fork and is synthesized discontinuously 9/4/2011
Slide 22 : DNA REPLICATION PART-II 22 . In bacterial cells, the lagging strand is synthesized as okazaki fragments approximately 1000 to 2000 nucleotide long. In eukaryotes also DNA synthesis on lagging strand is discontinuous but the okazaki fragments are considerably smaller
Okazaki fragments have RNA primers and DNA stretches, For ligation, first RNA primers are removed by RNases and the gaps are filled by deoxyribonucleotides by DPI. DNA ligase establishes phospho - diester bond between two DNA pieces as a result continuous stretch of new DNA is synthesized on lagging strand away from the replication fork. Two molecules of DP II are thought to move together as a complex in opposite directions along their respective template strands. 9/4/2011
Slide 23 : DNA REPLICATION PART-II 23 OKAZAKI FRAGMENTS 9/4/2011
Slide 24 : DNA REPLICATION PART-II 24 DNA Polymerase enzyme in prokaryotes
In bacterial cell,3 different types of DP are present, although all of them have the same basic catalytic activity,which is to add deoxyribonucleotides onto the 3’OH end of a single stranded primer (short RNA sequences). However they differ in their various roles within the cell and their molecular structure.
The enzyme that acts in DNA strand formation during replication in E.coli is DP III and is often called as replicase. DP III holoenzyme ( core consisting of 3 subunits ,, ) is much larger than other two polymerases, consisting of a single catalytic subunit and at least nine different subunits having various functions. 9/4/2011
Slide 25 : One of subunit , appears responsible for keeping DP III associated with DNA template. DP III has two contrasting properties. At one end it remains associated with the template over long stretches to synthesize a continuous complementary strand. At the same time it has to move from one nucleotide to other, which is probably helped by its subunits. The function of DP II is yet unknown. DP I is thought to be involved primarily in DNA repair , it also removes RNA primers at the 5’ end of each Okazaki fragment and replaces them with deoxyribonuleotides. DNA REPLICATION PART-II 25 9/4/2011
Slide 26 : DNA REPLICATION PART-II 26 9/4/2011
Slide 27 : The DNA polymerase III holoenzyme is a multisubunit complex, which consists of 17 polypeptides. It contains four sub assemblies. First, the core polymerase consists of three subunits: α (the polymerase); ε (the 3'–5' exonuclease); and θ (the stimulator of the 3'–5' exonuclease). Second, the π subunit is responsible for dimerization of the core DNA polymerase. Third, the sliding clamp comprises two homo dimers of the β subunit, which provides the ring structure that is needed for processivity. Fourth, five subunits have β clamp-loader functions . DNA REPLICATION PART-II 27 9/4/2011
Slide 28 : DNA REPLICATION PART-II 28 Eukaryotic DNA polymerase
To date 5 different DNA polymerases have been isolated from eukaryotic cells and are designated as , , , and . Polymerase is tightly associated with the primase, which initiates synthesis of primers at the 5’ end of each okazaki fragment as the polymerase – primase complex moves along the lagging strand template.
All the DP require some divalent cat ions as prokaryotic DP
It appears that the leading strand and most of the fragments of the lagging strand are assembled by DP ( delta). 9/4/2011
Slide 29 : DP replicates mitochondrial DNA and DP functions in DNA repair. DP (epsilon) binds to template of lagging strand and synthesize discontinuous okazaki fragments. Like prokaryotic polymerases, all the eukaryotic enzymes elongate DNA strands in the 5’ 3’ direction by addition of nucleotides to a 3’ OH group and non of them is able to initiate the synthesis of DNA chain without a primer. Eukaryotic polymerases posses a 3’ 5’ exonuclease activity ensuring that replication occurs with very high accuracy. DNA REPLICATION PART-II 29 9/4/2011
Slide 30 : DNA REPLICATION PART-II 30 Table : ACTIVITIES OF EUKARYOTIC
DNA POLYMERASES (Pols) 9/4/2011
Slide 31 : DNA REPLICATION PART-II 31 DIFFERENCE IN PROKARYOTIC AND EUKARYOTIC REPLICATION
As eukaryotic DNA is many times larger than prokaryotic DNA and is linear, there are multiple origins of replication ( Fig.3).
Both in prokaryotes and Eukaryotes replication proceeds bi-directionally from the origin of replication.
In eukaryotes all the origins of replication start DNA synthesis simultaneously and ultimately meet each other to complete replication from one end to other.
The Okazaki fragments formed in prokaryotes are much larger (1000-2000 nucleotides long) than that formed in eukaryotes averaging about 250 nucleotides only in length. All rest of the steps in eukaryotic replication are same as prokaryotic 9/4/2011
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Slide 33 : DNA REPLICATION PART-II 33 Eukaryotic DNA Replication with many origins of replication.
Replication proceeds bi-directionally from each origin 9/4/2011
Slide 34 : DNA REPLICATION PART-II 34 TELOMERASE ACTIVITY
As eukaryotic DNA is linear , it faces problem in replicating the ends. Folllowing removal of RNA primer from the extreme 5’ end on lagging strand , there is no way to fill the gap by deoxyribonucleotides.
To solve this problem and to protect the ends of DNA (chromosome ) against nucleases , the terminities of DNA has generally Non coding sequence which are highly repetitive having T/G . The ends are known as telomeres. The TG strand is longer than its complement ,leaving a region of single stranded DNA at the 3’ end of the double helix that is a few hundred nucleotides long. 9/4/2011
Slide 35 : DNA REPLICATION PART-II 35 RNA of telomerase base pairs with terminal nucleotides at the single stranded 3’ end of DNA . The RNA then serves as a template for extending the DNA strand , once the next repeat sequence is complete, telomerase RNA is translocated to newly synthesized end of DNA ,where it again hydrogen bonds and the proccess is completed Telomerase is a special kind of Reverse Transcriptase that carries its own RNA molecule of about 150 nucleotide long having copies of A/C sequence that is complementary to the T/G repeat sequence 9/4/2011
Slide 36 : DNA REPLICATION PART-II 36 Mechanism of action of telomerase Reproduced –from lipincott’s Illustrated Reviews:Biochemistry 9/4/2011
Slide 37 : DNA REPLICATION PART-II 37 PROOF READING BY DP III
Exact Replication of DNA is partly due to strict base pairing provision and partly due to proof reading property of DNA Polymerase I & III in 3’ 5’ direction by hydrolyzing the wrong nucleotides.
To ensure Replication fidelity(accuracy), DNA Polymerase III has, in addition to its 5’3’ polymerase activity , a proof reading activity in 3’5’direction,acts as exonuclease. As each nucleotide is added to the chain, DP III checks to make sure the added nucleotide is in fact, correctly matched to its complementary base on the template and edits its mistakes. For example if the template base adenine and the enzyme by mistake has introduced cytosine instead of Thymine to the new chain, DP III hydrolytically removes the mismatched nucleotide and replaces it with correct nucleotide (T). 9/4/2011
Slide 38 : DNA REPLICATION PART-II 38 1
DPIII inserts correct nucleotide to its complementary base on DNA template and is added to growing chain 2
Proof reading function of DP III (3’-------→5’) . If DP III mispairs a nucleotide on the template , it utilizes its 3’-------→5 exonuclease activity to excise the wrong nucleotide 9/4/2011
Slide 39 : DNA REPLICATION PART-II 39 THE BIOCHEMICAL REACTIONS
DNA replication begins with the "unzipping" of the parent molecule as the hydrogen bonds between the base pairs are broken.
Once exposed, the sequence of bases on each of the separated strands serves as a template to guide the insertion of a complementary set of bases on the strand being synthesized.
The new strands are assembled from deoxyribonucleoside triphosphates.
Each incoming nucleotide is covalently linked to the "free" 3' carbon atom on the pentose as the second and third phosphates are removed together as a molecule of pyrophosphate (p-p) .
By release of Pyrophosphate energy is liberated which helps in formation of Phoshpho di ester bond with subsequent nucleotide. 9/4/2011
Slide 40 : The nucleotides are assembled in the order that complements the order of bases on the strand serving as the template.
Thus each C on the template guides the insertion of a G on the new strand, each G a C, and so on.
When the process is complete, two DNA molecules have been formed identical to each other and to the parent molecule. DNA REPLICATION PART-II 40 9/4/2011
Slide 41 : DNA REPLICATION PART-II 41 DNA SYNTHESIS : PHOSPHO-DIESTER BOND FORMATION 9/4/2011
Slide 42 : DNA REPLICATION PART-II 42 USE OF RNA PRIMER TO INITIATE DNA SYNTHESIS 9/4/2011
Slide 43 : DNA REPLICATION PART-II 43 9/4/2011
Slide 44 : DNA REPLICATION PART-II 44 Speed of Replication
Prokaryotes : Genome size of E.coli is 4.7 x 106 nucleotide pairs. Replication proceeds from single origin of replication at the speed of 1000 nucleotides per sec completing replication of whole genome in 40 minutes.
Eukaryotes : Speed is very slow in comparision to prokaryotes. The average human chromosome contains 150x 106 nucleotide pairs which are copied at about only 50 bp per sec. But because there are multiple origins of replication present along the linear DNA, it is completed in a hour. 9/4/2011
Slide 45 : DNA REPLICATION PART-II 45 Control or Regulation of Replication
As there are multiple origins of replication in Eukaryotes, which origin has replicated and which awaits replication , becomes problematic for DNA polymerase. Two control mechanisms have been identified- one positive and one negative.
.Licensing positive control of replication
and Geminin negative control of replication
In order to be replicated, each origin of replication must be bound by ORC(Origin Recognition Complex) of proteins which remains on DNA throughout the process. Accessory proteins called Licensing factors accumulate in the nucleus during G 1 of the cell cycle. They include CdC6 and Cdt1 which bind to ORC and are essential for coating of DNA with MCM proteins because only after becoming 9/4/2011
Slide 46 : coated with MCM proteins DNA can be replicated. Once replication begins in ‘S’ phase, Cdt1 and CdC6 leave the ORC whereas MCM proteins remain ahead of replication fork. So DP III can easily know which Ori has to be replicated
Geminin protein controls replication through G 2 phase.
G2 nuclei contain protein called geminin that prevents assembly of MCM proteins on freshly synthesized DNA. As the cell completes mitosis, geminin is degraded so the DNA of the two daughter cells will be able to respond to licensing factor and be able to replicate their DNA at the next S phase DNA REPLICATION PART-II 46 9/4/2011
Slide 47 : DNA REPLICATION PART-II 47 SUMMARY OF THE CLASS DNA REPLICATION PART –II
The overall pattern of replication is semiconservative,that is each new molecule produced is made up of one intact strand of the old parental molecule and one newly synthesized strand
Replication begins at specific points called origin. There is only one origin in prokaryotic circular DNA and many origins in linear eukaryotic DNA.
DNA replication takes place simultaneously at each fork.
The process begins when the helicase enzyme unwinds the double helix to expose two single DNA strands .
Single-strand binding proteins, or SSBs, coat the single DNA strands to prevent them from snapping back together. SSBs are easily displaced by DNA polymerase. 9/4/2011
Slide 48 : Initiation requires a short piece of RNA onto which the nucleotides are added.
Replication proceeds bidirectionally from origin
Nucleotides are added in 5’→3’ direction
DNA polymerase begins to synthesize a new DNA strand by extending an RNA primer in the 5' to 3' direction.
Each parental DNA strand is copied by one DNA polymerase. Both template strands move through the replication factory in the same direction, and DNA polymerase can only synthesize DNA from the 5’ end to the 3’ end. Due to these two factors, one of the DNA strands must be made discontinuously in short pieces which are later joined together. DNA REPLICATION PART-II 48 9/4/2011
Slide 49 : 9/4/2011 DNA REPLICATION PART-II 49 As replication proceeds, RNAse H recognizes RNA primers bound to the DNA template and removes the primers by hydrolyzing the RNA.
DNA polymerase I can then fill in the gap left by RNase H.
The DNA replication process is completed when the ligase enzyme joins the short DNA pieces together into one continuous strand
The Replication process is semidiscontinous , occurring fairly continuously on leading strand and discontinuouly on the other strand
The short fragments produced are joined together to complete synthesis
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