Slide 1 : DNA REPLICATION - PART - I
27-08-2011 6.30 PM
DNA REPLICATION - PART - II
05-09-2011 6.30 PM
By
Dr. Ichha PuraK
http://dripurak.com
http://drichhapurak.webnode.com 8/26/2011 1 DNA REPLICATION PART -I
Slide 2 : DNA REPLICATION PART-I
INTRODUCTION
THE REPLICATION FACTORY
IMPORTANCE OF REPLICATION
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 8/26/2011 2 DNA REPLICATION PART -I
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
PROOF READING BY DP III
SPEED OF REPLICATION
REGULATION OF REPLICATION 8/26/2011 3 DNA REPLICATION PART -I
Slide 4 : DNA replication is a fundamental process by which the parent DNA duplex copies itself and become duplicated and produce two daughter DNA duplexes .
Both strands of DNA ladder separate and act as template, onto which new daughter strands are assembled, for which complementary deoxyribonucleotides are taken from pool of deoxyribonucleotides.
The two DNA duplexes that emerge from this process receive only one side of original DNA and other side newly synthesized. 4 8/26/2011 DNA REPLICATION PART -I
Slide 5 : 5 PARENTAL DNA Semi-conservative Model:
Watson and Crick predicted : the two strands of the parental molecule separate, and each functions as a template for synthesis of a new complementary strand. 8/26/2011 DNA REPLICATION PART -I
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Slide 8 : THE REPLICATION FACTORY
Replication proteins are clustered together in particular locations in the cell and may therefore be regarded as Replication Factory that manufactures DNA copies.
The DNA to be copied is fed through the factory, much as a reel of film is fed through a movie projector. The incoming DNA double helix is split into two single strands and each original strand becomes half of a new double helix. Because each resulting DNA double helix retains one strand of the original DNA. DNA replication is said to be semi-conservative 8 8/26/2011 DNA REPLICATION PART -I
Slide 9 : DNA REPLICATION PART -I 9 8/26/2011
Slide 10 : IMPORTANCE OF REPLICATION
It is one of the most vital processes. It provides means by which genetic instructions can be transmitted from one (parent ) cell to its two daughter cells or from one individual to its offsprings because during Replication Parent DNA duplex is able to make its two identical copies or Replica and It is now well known that DNA is the Genetic Material,able to transmit information over generations. 10 8/26/2011 DNA REPLICATION PART -I
Slide 11 : MODE OF REPLICATION :
Three possibilities have been suggested as mode for replication :
Conservative Of the two duplexes produced after replication one is entirely parental and other entirely new
Semi-conservative Both the two duplexes formed after replication contain one parental strand and one newly synthesized strand. Parent DNA double helix is not conserved as entity, But one parental strand is conserved in both daughter double helices in each generation and it continues for many generations.
Dispersive Both strands of parental molecule break at random, after replication the parental and daughter pieces are joined randomly. 11 8/26/2011 DNA REPLICATION PART -I
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Slide 14 : Of the various ideas proposed regarding mode of replication viz. conservative, semi-conservative and dispersive, the semi-conservative mode is highly accepted , which indicates that one half of the parent duplex is transmitted to each daughter duplex. It has been theoretically predicted by Watson and Crick ( 1953) on the basis of double helical nature and complementary sequence of bases on two strands of DNA. 14 Semi conservative mode of replication has been experimentally proved by Maselson and Stahl (1958) using 15N in the nutrient medium for culturing E.coli and monitoring the density of DNA after each replication 8/26/2011 DNA REPLICATION PART -I
Slide 15 : EXPERIMENTAL EVIDENCE
Steps in the Experiment
1.E. coli cells were grown in culture medium having NH4Cl (with15N) as Nitrogen source for 14 generations, so that all DNA bases become labeled. This heavier ( High density) DNA settles differently after centrifugation than the lighter (Low Density ) DNA having 14 N. 2. E.coli cells were then removed ,washed and transferred to culture medium having NH4Cl ( with 14 N) as Nitrogen source.
3. Some cells of E. coli were removed after every 30 minutes and DNA was extracted and examined for density by centrifugation with Cesium Chloride solution followed by sedimentation. ( E coli takes 30 minutes for one division) 15 8/26/2011 DNA REPLICATION PART -I
Slide 16 : The results were observed :
4. After first generation only one band was observed by ultraviolet absorption(260 nm) and it showed density intermediate between heavier ( DNA 15 N) and lighter (DNA 14 N ) DNA s.
5. After two generations two bands were seen, one comparable to Intermediate (Hybrid) DNA density and other of Normal ( DNA 14 N ) density.
6. In all subsequent generations two bands appeared, but intensity of Hybrid density band gradually decreased and band of lighter DNA gradually increased as expected by Semiconservative mode of replication. 16 8/26/2011 DNA REPLICATION PART -I
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Slide 20 : 20 15 N 14 N 15 N+14 N
Hybrid 8/26/2011 DNA REPLICATION PART -I
Slide 21 : 21 15 N Hybrid 14 N 8/26/2011 DNA REPLICATION PART -I
Slide 22 : PHASE OF REPLICATION
Takes place during ‘S’ (Synthetic) phase of the preparatory stage of cell cycle which commences after G 1 and is followed by G 2. The preparatory stage is also known as Resting stage as cell is supposed to be not active in division. It is also known as Interphase as it is the period in between two cell divisions. During this period cell is metabolically active. 22 8/26/2011 DNA REPLICATION PART -I
Slide 23 : Eukaryotic Replication having many origins PLACE OF REPLICATION
It starts at origins of replication (Ori), appears as bubble under electron microscope, ultimately extend in the form of ‘Y’ shaped replication fork. 8/26/2011 23 DNA REPLICATION PART -I
Slide 24 : CIRCULAR BACTERIAL CHROMOSOME UNDERGOING BIDIRECTIONAL SEMICONSERVATIVE REPLICATION 8/26/2011 24 DNA REPLICATION PART -I
Slide 25 : 25 PROKARYOTIC DNA REPLICATION Parent Duplex Daughter Duplexes 8/26/2011 DNA REPLICATION PART -I
Slide 26 : Direction of Replication
Replication takes place in bi-directional way, proceeds on both sides of origin of replication ( Ori), both in pro as well as eukaryotes. In Bacteria, as DNA is circular and there is single (Ori), bi-directional replication gives a shape. 26 Thetalike configuration assumed by replicating,circular,no-end double helical DNA molecule of Escherichia coli. Arrows indicate the two replication forks. These have progressed bidirectionally from a single replication bubble 8/26/2011 DNA REPLICATION PART -I
Slide 27 : Pattern of replication of long DNA molecule with more than one origin of replication. Replication proceeds out bidirectionally from each origin 8/26/2011 27 DNA REPLICATION PART -I
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Slide 29 : Origin of replication in E.coli
Replication initiates at a Unique site on the E.coli chromosome ,designated as ori. First event is the binding of an initiator protein to ori DNA , which leads to partial unwinding of the DNA double helix giving two templates DNA continues to unwind by the action of Helicase and single stranded DNA binding proteins and
RNA primers synthesized by Primase The two replication forks formed at the origin then move in opposite directions along the circular DNA molecule 8/26/2011 29 DNA REPLICATION PART -I
Slide 30 : Initiation of Replication : Factors Involved
Prokaryotes : Eubacterial cell : E.coli
Initiation begins at A -T rich sequences at Ori C in E.coli. The main components of the replication complex formation are ORC
(Origin Recognition Complex), factors Dna A, Dna B, Dna C, HU
and enzyme helicase. Dna A binding site consists of 9 bp repeats of
5’TGTGAATAA 3’, it binds to the Ori C , sets the platform and promotes double helical opening, it also loads Dna B . 30 8/26/2011 DNA REPLICATION PART -I
Slide 31 : Binding of Dna A protein appears to be the key event ,it appears as ellipsoidal mass (Dna A-Ori C ) under electron microscope. Dna B has 5’ 3’ helicase activity and is also activator of primase Dna C complex. HU is another double stranded binding protein.
As ORC binds DNA with the help of different protein factors, the double helical structure opens in the form of small bubble by the enzyme helicase. Once double helical structure is opened, it is stabilized by single stranded DNA binding proteins. 8/26/2011 31 DNA REPLICATION PART -I
Slide 32 : Eukaryotic Initiation of Replication : Factors Involved
Eukaryotic DNA is complexed with proteins, is assembled as chromosomes. Eukaryotic DNA is many times larger than prokaryotic DNA and therefore have multiple origin of replication (ori) sites. The average human chromosome contains 150x106 nucleotide pairs, which are copied at about 50 bp per sec speed. But due to presence of multiple origin of replication, whole genome is replicated in 1 hour. In order to initiate replication at these multiple sites a pre replicative complex is formed having the following components : 32 8/26/2011 DNA REPLICATION PART -I
Slide 33 : ORC ( Origin Recognition Complex ) ,these remain bound to DNA through out the Initiation process and is a six subunit complex. It has affinity for single stranded DNA. Other factors help ORC in identifying the ori sites. Its binding is coupled with ATP hydrolysis.
CdC6 protein associates with ORC and help MCM proteins to associate with chromatin.
Cdt 1 protein is identified as key factor in Pre-Rc assembly, so mutations in Cdt 1 (In vitro studies ) results in a block to DNA replication. 33 8/26/2011 DNA REPLICATION PART -I
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Slide 36 : MCM (Mini chromosome Maintenance ) proteins - DNA is coated with these proteins with the help of CdC6 and Cdt1. Once replication begins in S phase,Cdt1 and CdC6 leave the ORCs and MCM proteins remain in front of Replication fork on both sides and all these help in stabilizing replication fork and its extension having single stranded DNA.
DNA synthesis begins with the activity of DNA helicase which causes melting of hydrogen bonds between base pairs ( A- T and G-C) . As a result two single stranded structures with their exposed nucleotides are produced. 36 8/26/2011 DNA REPLICATION PART -I
Slide 37 : These single strands have a tendency to wound again (make base pairs) and to avoid this single stranded DNA binding proteins play important role. They also protect the single stranded structures from nucleases. After this DNA polymerase comes into action. It selects the complementary nucleotide from a mixture of dNTPs ( dATP,dCTP, dGTP,dTTP) and add to the template strand and also establish phosphodi-ester bonds between successive nucleotides. 8/26/2011 37 DNA REPLICATION PART -I
Slide 38 : PROTEINS AND ENZYMES INVOLVED IN REPLICATION Many enzymes and proteins are involved in the process of DNA Replication to unwind double helix,replication fork stabilization and synthesizing new DNA strand reading the template.viz. Helicase, SSB Protein, Primase, The sliding Clamp, DNA Polymerase, Rnase H and DNA Ligase. Each enzymes has a specific role.
DNA replication requires a variety of proteins. Each protein performs a specific function in the production of the new DNA strands.
Helicase, made of six proteins arranged in a ring shape, unwinds the DNA double helix into two individual strands. 38 8/26/2011 DNA REPLICATION PART -I
Slide 39 : Single-strand binding proteins, or SSBs, are tetramers that coat the single-stranded DNA. This prevents the DNA strands from reannealing to form double-stranded DNA. Primase is an RNA polymerase that synthesizes the short RNA primers needed to start the strand replication process. DNA polymerase is a hand-shaped enzyme that strings nucleotides together to form a DNA strand. The sliding clamp is an accessory protein that helps hold the DNA polymerase onto the DNA strand during replication. RNAse H removes the RNA primers that previously began the DNA strand synthesis. DNA ligase links short stretches of DNA together to create one long continuous DNA strand. 39 8/26/2011 DNA REPLICATION PART -I
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Slide 41 : DNA Gyrase ( Type II Topoisomerase). Helps unwinding by DNA helicase. Reduces the tension caused by super coil formation during unwinding. For it single nick is created by breaking the phosphodiester bond on one of the strand, which helps to release tension.
DNA helicase Helps in dissolving ‘H’ bonds between base pairs and result in separating the two strands near origin of replication, which gradually extends. Helicase as it requires energy to open the duplex is associated with hydrolysis of ATP.
Singe stranded DNA binding Proteins Maintain the stability of replication fork by binding the separated strands on both sides and keeping them apart to avoid rewinding.These proteins also protect the exposed nucleotides on the separated strands against nucleases. 41 8/26/2011 DNA REPLICATION PART -I
Slide 42 : DNA Polymerase Proceeds along the single stranded templates , recruit complementary dNTPs, form Hydrogen bonds with their appropriate complementary base present on the template and catalyze phosphodiester bond with previous nucleotide of the same strand.In prokaryotes DP III is responsible for synthesis of new DNA strands .DP I is involved in replacing deoxyribonucleotides after removal of RNA primers on the lagging strand.
The sliding clamp is an accessory protein that helps hold the DNA polymerase onto the DNA strand during replication.
RNA Primase Is actually part of aggregates of proteins called primosomes. This enzyme attaches a small RNA primer to the single stranded DNA onto which DP III can add deoxyribonucleotides. 42 8/26/2011 DNA REPLICATION PART -I
Slide 43 : RNase This enzyme dismantles the RNA primers present at the 5’ end of each Okazaki fragment on lagging strand and also single RNA primer present on the leading strand at the 5’ end.
DNA ligase Can catalyse the formation of phosphodiester bond between 3’OH and 5’ phosphate groups of two DNA fragments on lagging strand after removal of RNA primers. They seal the gaps between two such DNA pieces. 43 8/26/2011 DNA REPLICATION PART -I
Slide 44 : REPLICATION FORK
The point where the DNA is separated into single strands, and where new DNA will be synthesized, is known as the replication fork.
For replication to take place the two strands of DNA double helix at weak spots ( Where more A------- T base pairing is present ) separate by dissolving Hydrogen bond. The separated strands appear as bubble under Electron Microscope. The two halves of this bubble look as Y shaped Replication Fork with Neck and Mouth. In prokaryotes there is single origin (Ori) , where as in Eukaryotes several Ori are present. 44 8/26/2011 DNA REPLICATION PART -I
Slide 45 : 45 NECK MOUTH REPLICATION FORK 8/26/2011 DNA REPLICATION PART -I
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Slide 47 : SUMMARY OF THE CLASS DNA REPLICATION PART –I
DNA Replication is a process by which the parent DNA duplex copies itself to produce two identical Daughter DNA duplexes.
DNA Replication takes place during S (synthetic) phase of Interphase so that the two daughter cells after cell division get equal amount of DNA as that of parent cell.
Various models have been proposed to explain DNA Replication of which Semi-conservative mode in which one half of the parent duplex is transmitted to each daughter duplex is highly accepted. 8/26/2011 47 DNA REPLICATION PART -I
Slide 48 : It has been theoretically predicted by Watson and Crick
( 1953) on the basis of double helical nature and complementary sequence of bases on two strands of DNA.
Semi conservative mode of replication has been experimentally proved by Maselson and Stahl (1958) using 15N in the nutrient medium for culturing E.coli and monitoring the density of DNA after each replication
Replication begins at Origin of Replication (Ori )
Replication takes place bidirectionally from origin of replication. 8/26/2011 48 DNA REPLICATION PART -I
Slide 49 : There is a single Ori in prokaryotic circular DNA whereas eukaryotic DNA has many origins of Replication
For semi-conservative replication two strands of DNA separate by breaking Hydrogen bonds between bases
After separation each strand acts as a template onto which new or daughter strand is assembled following strict base pairing rules.
Each origin of replication ( Bubble ) consists of two Y shaped Relication ForKs having Neck and Mouth 8/26/2011 49 DNA REPLICATION PART -I
Slide 50 : For opening of double helix at Ori, stabilization of single strands and synthesis of daughter DNA strands complementary to the template in sequence of bases , many enzymes and protein factors are involved such as Helicase, Topoisomerase, DNA gyrase, ssDNA Binding Proteins, DNA Polymerase, Primase,Rnase and DNA ligase are required
Main enzymes in prokaryotic DNA Replication is DNA Polymerase III which adds complemetary nucleotides on 3’OH end of RNA primer in 5’---- 3’ direction 8/26/2011 50 DNA REPLICATION PART -I
Slide 51 : The Mechanism or steps involved in synthesis of daughter DNA will be discussed in next class on DNA Replication Part - II 8/26/2011 51 DNA REPLICATION PART -I
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