DNA Replication PDF

DNA Replication Slides by Reema C.Deshmukh Hypothesis for mode of DNA replication Three models for DNA replication were proposed. If the DNA molecule was untwisted and the two strands separated, each strand could act as a template for the synthesis of a new, complementary strand of DN...

DNA Replication Slides by Reema C.Deshmukh Hypothesis for mode of DNA replication Three models for DNA replication were proposed. If the DNA molecule was untwisted and the two strands separated, each strand could act as a template for the synthesis of a new, complementary strand of DNA that could then be bound to the parental strand. This DNA replication model is known as the semiconservative model, because each progeny molecule retains (“conserves”) one of the parental strands In the conservative model, the two parental strands of DNA remain together or pair again after replication and, as a whole, serve as a template for the synthesis of new progeny DNA double helices. In this model, one of the two progeny DNA molecules is the parental double-stranded DNA molecule, and the other consists entirely of new material. In the dispersive model, the parental double helix is cleaved into double-stranded DNA segments that act as templates for the synthesis of new double-stranded DNA segments. Then the segments reassemble into complete DNA double helices, with parental and progeny DNA segments interspersed. Although the two progeny DNAs are identical with respect to their base-pair sequence, double stranded parental DNA has become dispersed throughout both progeny molecules. It is hard to imagine how the DNA sequences of chromosomes could be kept the same without some sophisticated regulatory mechanisms. Slides by Reema C.Deshmukh  If the DNA molecule was untwisted and the two strands separated, each strand could act as a template for the synthesis of a new, complementary strand of DNA that could then be bound to the parental strand. This DNA replication model is known as the semiconservative model, because each progeny molecule retains (“conserves”) one of the parental strands Slides by Reema C.Deshmukh  In the conservative model, the two parental strands of DNA remain together or pair again after replication and, as a whole, serve as a template for the synthesis of new progeny DNA double helices. In this model, one of the two progeny DNA molecules is the parental double-stranded DNA molecule, and the other consists entirely of new material. Slides by Reema C.Deshmukh  In the dispersive model, the parental double helix is cleaved into double-stranded DNA segments that act as templates for the synthesis of new double-stranded DNA segments. Then the segments reassemble into complete DNA double helices, with parental and progeny DNA segments interspersed. Although the two progeny DNAs are identical with respect to their base-pair sequence, double stranded parental DNA has become dispersed throughout both progeny molecules. Slides by Reema C.Deshmukh Slides by Reema C.Deshmukh Matthew Meselson and Frank Stahl’s experiment In 1958, Matthew Meselson and Frank Stahl obtained experimental evidence that the semiconservative replication model is correct. Meselson and Stahl grew E. coli in a medium in which the only nitrogen source was 15NH4Cl ammonium chloride In this compound, the normal isotope of nitrogen 14N , is replaced with the heavy isotope 15N 15 (Note: Density is weight divided by volume, so N with one extra neutron in its nucleus, is denser 14N) As a result, all the bacteria’s nitrogen-containing compounds, including DNA, contained 15N instead of 14N Slides by Reema C.Deshmukh  Next, the 15N labeled bacteria were transferred to a medium containing nitrogen in the normal form 14N, and the bacteria were allowed to reproduce for several generations. All new DNA synthesized after the transfer was labeled, then, with14N. As the bacteria reproduced in the medium, samples of E. coli were taken at various times (after every 20 mins), and the DNA was extracted and analyzed to determine its density. Slides by Reema C.Deshmukh  In this technique, high-speed centrifugation of a solution of cesium chloride (CsCl) produces a gradient of that salt, with the least dense solution at the top of the tube and the most dense solution at the bottom. DNA that is present in the solution during centrifugation forms a band at a position where its buoyant density matches that of the surrounding cesium chloride. Slides by Reema C.Deshmukh  15 N-labeled DNA ( 15N–15N DNA) and 14 N labeled DNA ( 14N– 14N DNA) form bands at distinct positions in a CsCl gradient After one replication cycle (one generation) in the 14N medium, all of the DNA had a density that was exactly intermediate between that of 15 N–15N DNA and that of 14N– 14N DNA After two replication cycles, half the DNA was of that intermediate density and half was of the density of 14N– 15N DNA. These observations, and those obtained from subsequent replication cycles were exactly what the semiconservative model predicted. Slides by Reema C.Deshmukh  If the conservative model for DNA replication had been correct, after one replication cycle there would have been a band of 15N– 15N DNA (parental) and a band of 14N-14N DNA (newly synthesized). The heavy parental DNA band would have been seen at each subsequent replication cycle, in the amount found at the start of the experiment. 14 All new DNA molecules would then have been N–14N DNA. Therefore, the relative amount of DNA in the14N –14N DNA position would have increased with each replication cycle. For the conservative model of DNA replication, then, the most significant prediction was that at no time would any DNA of intermediate density be seen. The fact that intermediate density DNA was seen ruled out the conservative model. Slides by Reema C.Deshmukh  If the dispersive model for DNA replication had been correct, then all DNA present in the medium 14N after one replication cycle would have been of intermediate ( 15N–14N band) density and this was seen in the Meselson–Stahl experiment. However, the dispersive model predicted that, after a second replication cycle in the same 14N medium, DNA segments from the first replication cycle would be dispersed throughout the progeny DNA double helices produced. Thus, the 15N-15N DNA segments dispersed among new 14N– 14 N DNA after one replication cycle would then be distributed among twice as many DNA molecules after two replication cycles. As a result, the DNA molecules would be found in one band located halfway between the 15N– 14N DNA and 14N-14N DNA positions in the gradient. With subsequent replication cycles, there would continue to be one band, and it would become lighter in density with each replication cycle. The results of the Meselson–Stahl experiment did not bear out this prediction, so the dispersive model was ruled out. Slides by Reema C.Deshmukh DNA Polymerase In 1955, Arthur Kornberg and his colleagues were the first to identify the enzymes necessary for DNA replication. Their work focused on bacteria, because the bacterial replication machinery was assumed to be less complex than that of eukaryotes. Kornberg shared the 1959 Nobel Prize in Physiology or Medicine for his “discovery of the mechanisms in the biological synthesis of deoxyribonucleic acid.” Kornberg’s approach was a biochemical one. He set out to identify all the ingredients needed to synthesize of E. coli DNA in vitro. The first successful DNA synthesis was accomplished in a reaction mixture containing 1.DNA fragments, 2. a mixture of four deoxyribonucleoside triphosphate precursors (dATP, dGTP, dTTP, and dCTP, collectively abbreviated dNTP for deoxyribonucleoside triphosphate), and 3. an E. coli extract (cells of the bacteria, broken open to release their contents). Kornberg used radioactively labeled dNTPs to measure the minute quantities of DNA synthesized in the reaction. Slides by Reema C.Deshmukh Primer Template Junction and dNTPs Slides by Reema C.Deshmukh Mechanism of DNA elongation All DNA polymerases from prokaryotes and eukaryotes catalyze the polymerization of nucleotide precursors (dNTPs) into a DNA chain The reaction has three main features: 1. At the growing end of the DNA chain, DNA polymerase catalyzes the formation of a phosphodiester bond between the 3’-OH group of the deoxyribose on the last nucleotide and the 5’-phosphate of the dNTP precursor. The energy for the formation of the phosphodiester bond comes from the release of two of three phosphates from the dNTP. The lengthening DNA chain acts as a primer in the reaction - a pre existing polynucleotide chain to which a new nucleotide can be added at the free 3’-OH. 2. At each step in lengthening the new DNA chain, DNA polymerase finds the correct precursor dNTP that can form a complementary base pair with the nucleotide on the template strand of DNA. Nucleotides are added rapidly—850 per second in E. coli and 60–90 per second in human tissue culture cells. The process does not occur with 100% accuracy, but the error frequency is extremely low. 3. The direction of synthesis of the new DNA chain is only from to 5’ to 3’. Slides by Reema C.Deshmukh  One of the best understood systems of DNA replication is that of E. coli. For several years after the discovery of DNA polymerase I scientists believed that it was the only DNA replication enzyme in E. coli. However, genetic studies disproved that hypothesis. Scientists have now identified a total of five DNA polymerases, DNA Pol I–V. Functionally, DNA Pol I and DNA Pol III are polymerases necessary for replication DNA Pol I, DNA Pol II, DNA Pol IV, and DNA Pol V are polymerases involved in DNA repair. The DNA polymerases used for replication are different structurally. DNA polymerase I is encoded by a single gene (polA) and consists of one polypeptide. DNA polymerase III have more genes and more no of polypeptide chains Slides by Reema C.Deshmukh  DNA polymerases bound to primer template junction have structure resembling a right hand. They contain three main subdomains: the “palm”, “fingers”, and “thumb”. DNA binding occurs in the cavity formed by these subdomains. The catalytic center is based on conserved amino acid residues of the palm subdomain( composed of beta sheets). This catalytic center bind two divalent metal ions (Mg2+ or Zn2+) Palm domain also monitors the hydrogen bond formation (base pairing) recently added nucleotide. Finger and thumb are composed of alpha helix and involved in attaching the right nucleotide and bringing in close contact with catalytic domain Slides by Reema C.Deshmukh  The core DNA polymerase III contains the catalytic functions of the enzyme and consists of three polypeptides: (alpha, encoded by the dnaE gene), (epsilon, encoded by the dnaQ gene), and (theta, encoded by the holE gene). The complete DNA Pol III enzyme, called the DNA Pol III holoenzyme, contains an additional six different polypeptides. Slides by Reema C.Deshmukh  Both DNA Pol I and DNA Pol III replicate DNA in the 5’ to 3’ direction. Both enzymes also have 3’ to 5’ exonuclease activity, meaning that they can remove nucleotides from the 3’end of a DNA chain. This enzyme activity is used in error correction in a proofreading mechanism. That is, if an incorrect base is inserted by DNA polymerase (an event that occurs at a frequency of about for both DNA polymerase I and DNA polymerase III, meaning that one base in a million is incorrect), in many cases the error is recognized immediately by the enzyme. By a process resembling using a backspace or delete key on a computer keyboard, the enzyme's 3’to5’ exonuclease activity excises the erroneous nucleotide from the new strand. Then, the DNA polymerase resumes forward movement and inserts the correct nucleotide. DNA Pol I also has 5’ to 3 ’exonuclease activity and can remove either DNA or RNA nucleotides from the 5’ end of a nucleic acid strand. This activity is important in DNA replication. Slides by Reema C.Deshmukh  Replication bubble The initiation of replication is directed by a DNA sequence called the replicator. The replicator usually includes the origin of replication, the specific region where the DNA double helix denatures into single strands and within which replication commences. The locally denatured segment of DNA is called a replication bubble. The segments of single strands in the replication bubble on which the new strands are made (with complementary base-pairing rules) are called the template strands. When DNA untwists to expose the two single stranded template strands for DNA replication, a Y-shaped structure called a replication fork forms. A replication fork moves in the direction of untwisting the DNA. When DNA untwists starting within a DNA molecules in a circular chromosome or replication starting within a linear chromosome, there are two replication forks: two Ys joined together at their tops to form a replication bubble. In many cases, each replication fork moves, so that bidirectional replication occurs. Slides by Reema C.Deshmukh DNA replication in E.coli Slides by Reema C.Deshmukh Initiation of replication The E. coli replicator is oriC, which spans 245 bp of DNA and contains a cluster of three copies of a 13-bp AT-rich sequence and four copies of a 9-bp sequence. An initiator protein bind to the replicator and denature the AT-rich region. The E. coli initiator protein is DnaA (dnaA gene), which binds to the 9-bp regions in multiple copies, leading to the denaturing of the region with the 13-bp sequences. DNA helicases (DnaB; encoded by the dnaB gene) are recruited and are loaded onto the DNA by DNA helicase loader proteins (DnaC; encoded by the dnaC gene). The helicases untwist the DNA in both directions from the origin of replication by breaking the hydrogen bonds between the bases. Each DNA helicase recruits the enzyme DNA primase (dnaG gene), forming a complex called the primosome. DNA primase is important in DNA replication because DNA polymerases cannot initiate the synthesis of a DNA strand; they can add nucleotides only to a preexisting strand. The DNA primase (which is a modified RNA polymerase) synthesizes a short RNA primer (about 5–10 nucleotides) to which new nucleotides are added by DNA polymerase. At this point, the bidirectional replication of DNA starts (replication events are identical at both the forks) Slides by Reema C.Deshmukh Elongation (Semi discontinuous replication) DNA helicase -untwist The process of separation of double-stranded DNA to two single strands is called DNA denaturation or DNA melting. Single-strand DNA-binding (SSB) proteins bind to each single-stranded DNA, stabilizing them and preventing them from reforming double-stranded DNA by complementary base pairing (a process called reannealing). The RNA primer made by DNA primase is at the end of the new strand being synthesized on the bottom template strand and on the top template DNA strand Each RNA primer is extended by the addition of DNA nucleotides by DNA polymerase III. The polymerases displace bound SSB proteins as they move along the template strands. The new DNAs synthesized are complementary to the template strands. Slides by Reema C.Deshmukh  DNA polymerases can synthesize DNA only in the 5’to3’ direction To maintain the 5’to 3’ polarity of DNA synthesis on each template, and to maintain one overall direction of replication fork movement, DNA is made in opposite directions on the two template strands The new strand being made in the same direction as the movement of the replication fork is the leading strand (its template strand the bottom strand is the leading-strand template) The new strand being made in the direction opposite that of the movement of the replication fork is the lagging strand (its template strand the top strandis the lagging-strand template). The leading strand needs a single RNA primer for its synthesis, whereas the lagging strand needs a series of primers. Slides by Reema C.Deshmukh  DNA gyrase relaxes the tension produced in the DNA ahead of the replication fork. On the leading strand template (the bottom strand), DNA polymerase III synthesizes the leading strand continuously toward the replication fork. Because of the 5’to3’ direction of DNA synthesis, synthesis of the lagging strand has gone as far as it can.For DNA replication to continue on the lagging-strand template, a new initiation of DNA synthesis occurs: an RNA primer is synthesized by the DNA primase at the replication fork DNA polymerase III adds DNA to the RNA primer to make another DNA fragment. Because the leading strand is synthesized continuously, whereas the lagging strand is synthesized in pieces or discontinuously. DNA replication occurs in a semi discontinuous manner. The fragments of lagging-strand DNA made in semi discontinuous replication are called Okazaki fragments Slides by Reema C.Deshmukh Eventually, the Okazaki fragments are joined into a continuous DNA strand. Joining them requires the activities of two enzymes, DNA polymerase I and DNA ligase. Consider two adjacent Okazaki fragments: The 3’end of the newer fragment is adjacent to the 5’end of primer at the end of the previously made fragment. DNA polymerase III leaves the newer DNA fragment, and DNA polymerase I binds. Slides by Reema C.Deshmukh The DNA polymerase I simultaneously digests the RNA primer strand ahead of it and extends the DNA strand behind it Digesting the RNA strand ahead of it involves using the enzyme’s 5’to3’ exonuclease activity to remove nucleotides from the primer’s 5’end, which also exposes template nucleotides. Extending the DNA strand behind it involves the enzyme’s 5’to3’ polymerase activity to add nucleotides to the DNA strand’s 3’ end, whose sequence is directed by the newly exposed template nucleotides. Slides by Reema C.Deshmukh  When DNA polymerase I has replaced all the RNA primer nucleotides with DNA nucleotides, a single stranded nick (a point at which the sugar–phosphate backbone between two adjacent nucleotides is unconnected) is left between the two DNA fragments. DNA ligase joins the two fragments, producing a longer DNA strand The DNA ligase catalyzes the formation of phosphodiester bond between the 3’ OH and the 5’ phosphate groups on either side of the nick sealing the nick. The steps are repeated until all the DNA is replicated. Slides by Reema C.Deshmukh Slides by Reema C.Deshmukh 1. Termination means the end of replication process. 2. It occurs in the region called Ter region and it is about 20bp. 3. The Tus (terminas utilization substances) proteins bind with the Ter region and form Ter-Tus complex. 4. The Ter-Tus complex works as a trap in unidirectional and halts one of the replication fork. 5. The other replication fork gets halted on colliding with the previously halted fork. 6. The newly formed circular DNA remains interlinked to each other and it is called as catenanes. 7. These covalently closed circular chromosomes are separated by Topoisomerase IV and passed on to daughter cell. Slides by Reema C.Deshmukh Model for replisome The key replication proteins are closely associated to form a replication machine called a replisome which is bound to the replicating DNA where it is being unwound into single strands. The lagging- strand DNA, looped so that its DNA polymerase III is complexed with the DNA polymerase III on the leading strand. These are two copies of the core enzyme held together by the six other polypeptides to form the DNA Pol III holoenzyme. Slides by Reema C.Deshmukh  Only the core enzymes are shown in the figure, for simplicity. The looping of the lagging-strand template brings the 3’end of each completed Okazaki fragment near the site where the next Okazaki fragment will start. The primase stays near the replication fork, synthesizing new RNA primers intermittently on the lagging strand template. Because the lagging-strand polymerase is complexed with the other replication proteins at the fork, that polymerase can be reused over and over at the same replication fork, synthesizing a string of Okazaki fragments as it moves with the rest of the replisome. That is the complex of replication proteins that forms at the replication fork moves as a unit along the DNA and synthesizes new DNA simultaneously on both the leading- strand and lagging-strand templates. Slides by Reema C.Deshmukh  Rolling Circle Replication Virus chromosomes, such as that of bacteriophage , a circular, double-stranded DNA replicates to produce linear DNA; the process is called rolling circle replication The first step in rolling circle replication is the generation of a specific nick in one of the two strands at the origin of replication The end of the nicked strand is then displaced from the circular molecule to create a replication fork The free end of the nicked strand acts a primer for DNA polymerase to synthesize new DNA, using the single-stranded segment of the circular DNA as a template The displaced single strand of DNA rolls out as a free “tongue” of increasing length as replication proceeds. Slides by Reema C.Deshmukh  New DNA is synthesized by DNA polymerase on the displaced DNA in the 5’-to-3’ direction, meaning from the circle out toward the 5’ end of the displaced DNA. With further displacement, new DNA is synthesized again, beginning at the circle and moving outward along the displaced DNA strand Thus, synthesis on this strand is discontinuous because the displaced strand is the lagging-strand template As the single-stranded DNA tongue rolls out, new DNA synthesis proceeds continuously on the circular DNA template. Because the parental DNA circle can continue to “roll,” a linear double-stranded DNA molecule can be produced that is longer than the circumference of the circle. Slides by Reema C.Deshmukh DNA Replication in Eukaryotes In eukaryotes DNA is distributed among many chromosomes rather than just one. Each chromosome is linear DNA double helix. A haploid human genome (24 chromosomes) consist of 3 billion bp of DNA. Each chromosome is 25 times longer than e.coli chromosome. Eukaryotic chromosomes have multiple origins of replication. This helps in efficient and relatively quicker DNA replication. At each origin of replication DNA unwinds and replication proceeds bidirectionally. Eventually each replication replication fork runs into adjacent replication forks, initiated at adjacent origin of replication E.coli chromosome replication (4.6 MB) ○ Rate of nt addition- 1000bp per sec ○ Time for replication - 42 mins Human chromosome replication ○ 10000 to 100000 replicons for replication of 30-300 kb ○ Rate of nt addition -100 bp per sec ○ Whole genome replication time -8 hrs Slides by Reema C.Deshmukh Initiation of replication For correct duplication of the chromosome each origin of replication must be use only once in the cell cycle The events of eukaryotic replication initiate at distinct and specified stage in the cell cycle This regulated by level of different proteins in distinct phases of cell cycle (CDK) This ensures that each chromosome is replicated only once during each cell cycle Initiation 1.Formation of pre replication complex (helicase loading) 2. Activation of the replication complex (helicase activation) Slides by Reema C.Deshmukh Formation of pre replication complex (helicase loading) G1 Phase 1. Recognition of the replicator by ORC (Origin recognition complex ) 2. As the cell enters G1 phase ORC bound to replicator recruits Two helicase loading proteins Cdc6 - cell division cycle 6 Cdt1- Chromatin licensing and DNA replication factor 1 The replication factors Cdt1 and Cdc6 are essential for origin licensing, a prerequisite for DNA replication initiation. Mechanisms to ensure that replication initiate once per cell cycle include degradation of Cdt1 during S phase and inhibition of Cdt1 by the geminin protein. Two copies of Mcm 2-7 Minichromosome maintenance complex (heterohexamer) It shows helicase activity Slides by Reema C.Deshmukh Activation of the complex (helicase activation) Pre replication complex to replication initiation complex (CMG complex) S Phase 1.DDK- DBF4-dependent kinase phosphorylate mcm2-7 2. CDK - Cyclin dependent kinases phosphorylate Sld2, Sld3, Dpb11 3. On phosphorylation these bind to Mcm2-7 and recruits Cdc45, GINS, Pol ε 4. After binding of Cdc45, GINS, Pol ε; Sld2, Sld3, Dpb11 is released to form CMG complex 5. Mcm2-7 starts unwinding of the dsDNA and polɑ/primase and polδ are added to CMG complex and the process of replication is initiated Slides by Reema C.Deshmukh Elongation PCNA-Proliferating cell nuclear antigen (sliding clamp) RPA- Replication protein A (Single -stranded DNA binding protein) Pol ɑ /primase- synthesis of RNA/DNA primer Pol ε- leading strand DNA synthesis Pol δ -lagging strand DNA synthesis Slides by Reema C.Deshmukh Okazaki fragment processing In eukaryotic replication, the lagging strand is synthesized in a discontinuous fashion by the production of Okazaki fragments, and synthesis each of them is initiated by an RNA/DNA primer. When DNA polymerase δ synthesizing an Okazaki fragment faces the primer of preceding fragment, it displaces it forming a flap structure RNase H -Removes rNTPs FEN1- cleaves 1rNTP and following dNTPs However, when the flap is too long, it is partly covered by RPA proteins blocking FEN1 DNA2- enabling FEN1 to cut it off flap by removing RPA. Slides by Reema C.Deshmukh Termination End replication problem in eukaryotes Because DNA polymerases can synthesize new DNA only by extending a primer, there are special problems in replicating the ends (the telomeres) of eukaryotic chromosomes Replication of a parental chromosome produces two new DNA molecules, each of which has an RNA primer at the 5’ end of the newly synthesized strand in the telomere region By contrast, the numerous RNA primers in each lagging strand have been replaced by DNA during the normal DNA replication steps The Okazaki fragment 5’ to the RNA primer is extended in 5’ to 3’ direction to replace the RNA primer. Since there is no Okazaki fragment 5’ to the primers at the 5’ ends, the same mechanism would not work at the 5’ ends of each new strand. Removal of the RNA primers at the 5’ ends of the new DNA strands leaves a single-stranded stretch of parental DNA—an overhang—extending beyond the 5’ end of each new strand. DNA polymerase cannot fill in the overhang. If nothing were done about these overhangs, the chromosomes would get shorter and shorter with each replication cycle. Slides by Reema C.Deshmukh Synthesis of telomeric DNA by telomerase Most eukaryotic chromosomes have species specific tandem repeats in the telomeric region. End replication problem is solved by the enzyme telomerase Telomerase maintain chromosome length by adding repeats to one strand i.e with 3’end. Telomerase is an enzyme made up of protein and RNA RNA- 451 nts 11 bases are template RNA sequence Slides by Reema C.Deshmukh  After 2 rounds of telomerase activity the template DNA strand is extended (multiple round of extensions may occur) On this extended strand new primers are synthesized to provide 3’OH for polymerase to replicate the DNA. Aagin the primer will be removed to still produce the overhang But net shortening of chromosome is reduced by the action of telomerase. Telomere DNA then loops back to form t-loop (single stranded end) And D-loop (double stranded loop) Slides by Reema C.Deshmukh