DNA Replication

The process by which DNA molecule makes its identical copies is known as DNA replication or DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule

1. Dr. Pawan Kumar Kanaujia Assistant Professor Molecular Biology (Theory ) DNA Replication

2. The process by which DNA molecule makes its identical copies is known as DNA replication or DNA replication is the biological process of producing two identical replicas of DNA from one original DNA molecule . It takes place in S-phase of interphase . Replication patterns are somewhat different in procaryotes and eucaryotes . In procaryotes when the circular DNA chromosome of E. coli is copied, replication begins at a single point, the origin. Synthesis occurs at the replication fork, the place at which the DNA helix is unwound and individual strands are replicated. Two replication forks move outward from the origin until they have copied the whole replicon , that portion of the genome that contains an origin and is replicated as a unit. When the replication forks move around the circle, a structure shaped like the Greek letter theta ( θ ) is formed. Finally, since the bacterial chromosome is a single replicon , the forks meet on the other side and two separate chromosomes are released.

3. In eukaryotes Eucaryotic DNA is linear and much longer than procaryotic DNA ; E. coli DNA is about 1,300 µm in length, whereas the 46 chromosomes in the human nucleus have a total length of 1.8 m (almost 1,400 times longer). Clearly many replication forks must copy eucaryotic DNA simultaneously so that the molecule/DNA can be duplicated in a relatively short period, and so many replicons are present that there is an origin about every 10 to 100 µm along the DNA. Replication forks move outward from these sites and eventually meet forks that have been copying the adjacent DNA stretch. In this fashion a large molecule is copied quickly.

4. FIGURE 10.12 Mechanism of action of DNA polymerases: covalent extension of a DNA primer strand in the 5’ → 3’ direction. The existing chain terminates at the 3 end with the nucleotide deoxyguanylate (deoxyguanosine-5-phosphate). The diagram shows the DNA polymerase- catalyzed addition of deoxythymidine monophosphate (from the precursor deoxythymidine triphosphate , dTTP ) to the 3’end of the chain with the release of pyrophosphate (P 2 O 7 ).

5. Initiation ,  elongation  and  termination  are three main steps in DNA replication . Let us now look into more detail of each of them: Step 1: Initiation The point at which the replication begins is known as the Origin of Replication ( oriC ) . Helicase brings about the procedure of strand separation, which leads to the formation of the replication fork. Step 2: Elongation The enzyme DNA Polymerase III makes the new strand by reading the nucleotides on the template strand and specifically adding one nucleotide after the other. If it reads an Adenine (A) on the template, it will only add a Thymine (T). Step 3: Termination When Polymerase III is adding nucleotides to the lagging strand and creating Okazaki fragments , it at times leaves a gap or two between the fragments. These gaps are filled by ligase . It also closes nicks in double-stranded DNA.

6. Process of Replication During replication Each enzyme and protein have their own specific function. Let us look at the process step by step

7. Initiation Helicase  – The point at which the replication begins is known as the Origin of Replication. Helicase brings about the procedure of strand separation, which leads to the formation of the replication fork. It breaks the hydrogen bond between the base pairs to separate the strand. It uses energy obtained from ATP Hydrolysis to perform the function. SSB Protein   – Next step is for the Single-Stranded DNA Binding Protein to bind to the single-stranded DNA. Its job is to stop the strands from binding again. DNA Primase  – Once the strands are separated and ready, replication can be initiated. For this, a primer is required to bind at the Origin. Primers are short sequences of RNA, around 10 nucleotides in length. Primase synthesizes the primers.

8. Elongation DNA Polymerase III  – This enzyme makes the new strand by reading the nucleotides on the template strand and specifically adding one nucleotide after the other. If it reads an Adenine (A) on the template, it will only add a Thymine (T). It can only synthesize new strands in the direction of 5’ to 3’. It also helps in proofreading and repairing the new strand. Now you might think why does Polymerase keep working along the strand and not randomly float away? Its because a ring-shaped protein called as sliding clamp holds the polymerase into position.

9. Now when replication fork moves ahead and the Polymerase III starts to synthesize the new strand a small problem arises. If you remember, I mentioned that the two strands run in the opposite directions. This means that when both strands are being synthesized in 5’ to 3’ direction, one will be moving in the direction of the replication fork while the other will move in the opposite. The strand, which is synthesized in the same direction as the replication fork, is known as the ‘leading’ strand. The template for this strand runs in the direction of 3’ to 5’. The Polymerase has to attach only once and it can continue its work as the replication fork moves forward. However, for the strand being synthesized in the other direction, which is known as the ‘lagging’ strand, the polymerase has to synthesize one fragment of DNA.  Then as the replication fork moves ahead, it has to come and reattach to the new DNA available and then create the next fragment. These fragments are known as Okazaki fragments (named after the scientist Reiji Okazaki who discovered them).

10. Termination DNA Polymerase I  – If you remember, we had added a RNA primer at the Origin to help Polymerase initiate the process. Now as the strand has been made, we need to remove the primer. This is when Polymerase I comes into the picture. It takes the help of RNase H to remove the primer and fill in the gaps. DNA ligase  – When Polymerase III is adding nucleotides to the lagging strand and creating Okazaki fragments, it at times leaves a gap or two between the fragments. These gaps are filled by ligase . It also closes nicks in double-stranded DNA. The Replication process is finally complete once all the primers are removed and Ligase has filled in all the remaining gaps. This process gives us two identical sets of genes, which will then be passed on to two daughter cells. Every cell completes the entire process in just one hour! The reason for taking such short amount of time is multiple Origins. The cell initiates the process from a number of points and then the pieces are joined together to create the entire genome!`

11. Mechanism of DNA Replication: Mechanism of DNA replication is the direct result of DNA double helical Structure proposed by Watson and Crick. It is a complex multistep process involving many enzymes. Initiation: It involves the origin of replication. Before the DNA synthesis begins, both the parental strands must unwind and separate permanently into single stranded state. The synthesis of new daughter strands is initiated at the replication fork. In fact, there are many start sites. Elongation: The next step involves the addition of new complementary strands. The choice of nucleotides to be added in the new strand is dictated by the sequence of bases on the template strand. New nucleotides are added one by one to the end of growing strand by an enzyme called DNA polymerase. There are four nucleotides, deoxyribrnucleotide triphosphates dGTP , dCTP , dATP , dTTP present in the cytoplasm. Termination: All the end termination reactions occur. Duplicated DNA molecules are separated from one another. The purpose of DNA replication is to create two daughter DNA molecules which are identical to the parent molecule.

12. Conservative replication model Dispersive replication model Semiconservative replication Proposed DNA Replication Models

13. Figure 12.1 Three proposed models of replication are conservative replication, dispersive replication, and semiconservative replication.

14. Initially, three models were proposed for DNA replication. In conservative replication (Figure 12.1a), the entire double-stranded DNA molecule serves as a template for a whole new molecule of DNA, and the original DNA molecule is fully conserved during replication. In dispersive replication (Figure 12.1b), both nucleotide strands break down (disperse) into fragments, which serve as templates for the synthesis of new DNA fragments, and then somehow reassemble into two complete DNA molecules. In this model, each resulting DNA molecule is interspersed with fragments of old and new DNA; none of the original molecule is conserved. Semiconservative replication (Figure 12.1c) is intermediate between these two models; the two nucleotide strands unwind and each serves as a template for a new DNA molecule.

15. Two isotopes of nitrogen: 14 N common form; 15 N rare heavy form E. coli were grown in a 15 N media first, then transferred to 14 N media. Cultured E. coli were subjected to equilibrium density gradient centrifugation. Meselson and Stahl’s Experiment

16. Bacterial DNA Replication Initiation: 245 bp in the oriC (single origin replicon ); an initiation protein

17. In prokaryotic chromosome contain a single “ Orgin of replication” called oriC . oriC is 245bp long and contain two different conserved repeat sequences of 13bp (AT-rich)sequence is present as three tandem repeats and another conserved component of 9bp sequence that is repeated four times and interspersed with other sequences. these four sequences are binding sites for a protein that play a key role in the formation of the replication bubble.

18. Unwinding of DNA is performed by Helicase . Gyrase removes supercoiling ahead of the replication fork. Single stranded DNA is prevented from annealing by single stranded binding proteins. Primers: an existing group of RNA nucleotides with a 3 ′ -OH group to which a new nucleotide can be added; usually 10 ~ 12 nucleotides long Primase : RNA polymerase One particular protein called Dna A is responsible for the recognition of Orgin of replication. DnaA protein bind with 9mer, this step facilitates the subsequent binding of helicase ( DnaB & DnaC )

19. Bacterial DNA Replication Elongation: carried out by DNA polymerase III Removing RNA primer: DNA polymerase I DNA ligase : connecting nicks after RNA primers are removed Termination: when a replication fork meets or by termination protein

20. Figure Summary of DNA synthesis at a single replication fork. Various enzymes and proteins essential to the process are shown.

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23. The Taylor–Woods–Hughes experiment, demonstrating the semi-conservative mode of replication of DNA in root tips of Vicia faba .

24. A portion of the plant is shown in the top photograph. (a) An unlabeled chromosome proceeds through the cell cycle in the presence of 3H-thymidine. As it enters mitosis, both sister chromatids of the chromosome are labeled, as shown, by autoradiography.

25. (b), this time in the absence of 3H-thymidine, only one chromatid of each chromosome is expected to be surrounded by grains. Except where a reciprocal exchange has occurred between sister chromatids (c), the expectation was upheld. The micrographs are of the actual autoradiograms obtained in the experiment.

26. Eukaryotic DNA replication 1957, J. Herbert Taylor, Philip Woods, and Walter Hughes presented evidence that Semiconservative Replication also occurs in Eukaryotic organism. They were used root-tip cells of the broad bean, Vicia faba , researchers were able to monitor the process of replication by lebeling DNA with 3 H-thymidine , a radioactive precursor of DNA, and by performing autoradiography. Chromosomal DNA replication occurs only during the S phase of the cell cycle. In eukaryotes, chromosome contain multiple replication origins are called autonomously Replicating sequences (ARSs) ARSs consist 120 bp containing a consensus sequence of AT rich 11bp . Helicase loading required four separate proteins to act at each replicator. Replication origins are recognize by a six-protein complex known as an Origin Recognisition complex (ORC), which tags the orgin as a site of initiation. The first step in helicase loading is the recognition of the replicator by the eukaryotic initiator , ORC, bound to ATP . As cells enter the G1 phase of the cell cycle, ORC bound to the origin recruits two helicase loading proteins (Cdc6 and Cdt1) and two copies of the Mcm2-7 helicase to the origin.

27. 9-30 F I G U R E. Eukaryotic helicase load ing . Loading of the eukaryotic replicative DNA helicase is an ordered process that is ini tiated by the association of the ATP-bound origin recognition complex (ORC) with the replicator. Once bound to the replicator, ORC recruits ATP-bound Cdc6 and two copies of the Mcm2-7 helicase bound to a second helicase loading protein, Cdt1. This assembly of proteins triggers ATP hydrolysis by Cdc6, resulting in the loading of a head-to-head dimer of the Mcm2-7 com plex encircling double-stranded origin DNA and the release of Cdc6 and Cdt1 from the origin. Subsequent ATP hydrolysis by ORC is required to reset the process (illustrated as release from Mcm2-7). Exchange of ATP for ADP allows a new round of helicase loading.

28. ATP hydrolysis by Cdc6 results in the loading of a head-to-head dimer of the Mcm2-7 complex such that they encircle the double-stranded origin DNA . During this event, Cdt1 and Cdc6 are released from the origin. Helicases that are loaded during G1 are only activated to unwind DNA and initiate replication after cells pass from the G1 to the S phase of the cell cycle. Loaded helicases are activated by two protein kinases : CDK ( cyclin dependent kinase ) and DDK (Dbf4-dependent kinase ). DDK targets the loaded helicase , and CDK targets two other replication proteins. Phosphorylation of these proteins results in the Cdc45 and GINS proteins binding to the Mcm2-7 helicase (Fig 9-31) . Importantly, Cdc45 and GINS strongly stimulate the Mcm2-7 ATPase and helicase activities and together form the Cdc45–Mcm2-7–GINS (CMG) complex, which is the active form of the Mcm2-7DNA helicase . Helicase is initially loaded around dsDNA as a head-to-head dimer , at the replication fork it is thought to act as a single Mcm2-7 hexamer encircling ssDNA . Its mean one strand of DNA must be ejected from the central channel of each helicase and start unwinding (the interactions between the two Mcm2-7 complexes must be disrupted) Fig 9-32 .

29. 9-31 F I G U R E. Activation of loaded helicases leads to the assembly of the eukaryotic replisome . As cells enter into the S phase of the cell cycle, two kinases , CDK and DDK, are activated. DDK phosphorylates loaded Mcm2-7 helicase , and CDK phosphorylates Sld2 and Sld3. Phosphorylated Sld2 and Sld3 bind to Dpb11, and together these proteins facilitate binding of the helicase -activating proteins, Cdc45 and GINS, to the helicase . Cdc45 and GINS form a stable complex with the Mcm2-7 helicase (called the Cdc45/ Mcm2-7/GINS, or CMG, complex) and dramatically activate Mcm2-7 helicase activity. The leading-strand DNA polymerase (Ɛ) is recruited to the helicase at this stage (before DNA unwinding). After formation of the CMG complex, Sld2, Sld3, and Dpb11 are released from the origin. DNA Pol α / primase and DNA Pol δ (which primarily act on the lagging strand) are only recruited after DNA unwinding. The protein–protein interactions that hold the DNA polymerase at the replication fork remain poorly understood.

30. F I G U R E 9-32 Helicase activation alters helicase interactions. Before helicase activation, loaded helicases encircle double-stranded DNA and are in the form of a head-to-head double hexamer (mediated by interactions between the Mcm2-7 amino termini). After helicase activation, the Mcm2-7 protein in the CMG complex is proposed to encircle single-stranded DNA, and the interaction between the two Mcm2-7 complexes has been broken.

31. F I G U R E 9-34 Cell cycle regulation of CDK activity controls replication. In S. cerevisiae cells, CDK levels tightly regulate helicase loading and activation. DuringG1,CDK levels are low, allowing helicases to be loaded, but the loaded helicases cannot be activated (because of the requirement of CDK for this event). During S phase, elevated CDK activity inhibits new helicase loading and activates previously loaded helicases . When a loaded helicase is used for the initiation of replication, it is incorporated into the replication fork and leaves the origin. Similarly, passive replication of origin DNA also removes the helicase from the origin DNA (not shown). Because CDK levels remain high until the end of mitosis, no new helicase loading can occur until chromosome segregation is complete and the daughter cells have returned to G1.Without a new round of helicase loading, reinitiation is impossible.

32. Unwound single strands are kept in the extended state by a replication protein A ( Rp -A) ( Lewin's Genes X) Topoisomerase released torsonal strain which is develop on the head of replication fork. The three eukaryotic DNA polymerases α , δ & Ɛ are the major form of enzyme involved in initiation and elongation of DNA synthesis. DNA polymerases α / primase synthesize RNA primer of length of ~10 ribo - nucleotides at both strands then pol α dissociates from the strands abd is replaced by pol δ & Ɛ. The event polymerase switching occurs, pol Ɛ synthesizes DNA on leading strand and pol δ synthesizes DNA on laging strand and these enzymes ( pol Ɛ & pol δ ) exhibits 3` to 5` exonuclease activity , thus having the potential to proofreading. However , they do not have 5`to 3` exonuclease activity . Thus they can not remove RNA primers. pol Ɛ & δ must interact with PCNA (Proliferating Cell Nuclear Antigen) and replication factor ( Rf -C). PCNA is a trimeric protein that forms a closed ring; Rf -C induces a change in the conformation of PCNA that allow it to encircle DNA, providing the essential sliding clamp to prevent the polymerase from falling off the strands.

33. The RNA primer are excised by RNase H or two nucleases ribonuclease H1 (which degrades RNA present in RNA-DNA duplexes) & ribonuclease FEN-1 (F1 nuclease1). pol Ɛ & δ fill the gap at lagging and leading strands respectively, except a nicks or break in the phosphodiester back bone between the 3’-OH & 5’ phosphate. This nick in the new DNA strands repaired by an enzyme called DNA ligase . A number of proteins are involved in the disassembly and assembly of nucleosomes during chromosome replication in eukaryotes. Two of the most important are nucleosome assembly protein-1 (Nap-1) and chromatin assembly factor-1 (CAF-1). Nap-1 transports histones from their site of synthesis in cytoplasm to the nucleus & CAF-1 delivers histones to the site of DNA replication by binding to PNCA clamp that lead (tethers) to DNA polymerase Ɛ & δ to DNA template.

34. FIGURE 10.32

35. FIGURE 10.32 Some of the important components of a replisome in eukaryotes. Each replisome contains three different polymerases, α , δ , and Ɛ. The DNA polymerase α -DNA primase complex synthesizes the RNA primers and adds short segments of DNA. DNA polymerase δ then completes the synthesis of the Okazaki fragments in the lagging strand, and polymerase Ɛ catalyzes the continuous synthesis of the leading strand. PCNA ( proliferating cell nuclear antigen) is equivalent to the β subunit of E. coli DNA polymerase III; it clamps polymerases δ and Ɛ to the DNA molecule facilitating the synthesis of long DNA chains. Ribonucleases H1 and FEN-1 (F1 nuclease 1) remove the RNA primers, polymerase δ fills in the gap,and DNA ligase (not shown) seals the nicks, just as in E. coli

36. FIGURE 10.33 The assembly of new nucleosomes during chromosome replication requires proteins that transport histones from the cytoplasm to the nucleus and that concentrate them at the site of nucleosome assembly. PCNA proliferating cell nuclear antigen

37. ROLLING-CIRCLE REPLICATION FIGURE 10.30 The rolling-circle mechanism of DNA replication. Material for progeny chromosomes (in this case, single stranded DNA for the virus X174) is produced by continuous copying around a nicked, double-stranded DNA circle, with the intact strand serving as a template. Electron micrograph courtesy of David Dressler, Harvard University.

38. ROLLING-CIRCLE REPLICATION In used By many viruses to duplicate their genome. In bacteria to transfer DNA from donor cells to recipient cell during conjugation. The unique aspect of rolling-circle replication is that one parental circular DNA strand remains intact and rolls (thus the name rolling circle) or spins while serving as a template for the synthesis of a new complementary strand. This form of replication is initiated by a break or sequence-specific endonuclease cleaves in one strand at the origin that creates 3’-OH group and 5’-phosphate group (termini). New nucleotides are added to the 3’ end used as template. As new nucleotides are added to the 3’ end, the 5’ end of the broken strand is displaced from the template, rolling out like thread being pulled off a spool. The 3’ end grows around the circle, giving rise to the name rolling-circle model. The replication fork may continue around the circle a number of times, producing several linked around the circle, the growing 3’ end displaces the nucleotide strand synthesized in the preceding revolution. Eventually, the linear DNA molecule is cleaved from the circle, resulting in a double -stranded circular DNA molecule & single stranded linear DNA molecule. The linear molecule circularizes either before or after serving as a template for the synthesis of a complementary strand.