Downloaded 224 times
Uploaded byBahauddin Zakariya University lahore
Replication in prokaryotes
The document summarizes key aspects of DNA replication in bacteria. It describes the semi-conservative model of replication, which was experimentally proven by Meselson and Stahl using E. coli. Replication initiates at a fixed origin and proceeds bidirectionally. It involves unwinding of DNA by helicase, synthesis of new strands by DNA polymerase, and ligation of Okazaki fragments. Replication is tightly regulated to occur once per cell cycle.
More Related Content
Similar to Replication in prokaryotes
More from Bahauddin Zakariya University lahore
Replication in prokaryotes
- 1. DNA Replication Conservativereplication ◦ Intact the original DNA molecule and generate a completely new molecule. Dispersive replication ◦ Produce two DNA molecules with sections of both old and new DNA interspersed along each strand. Semi-conservative replication ◦ Produce molecules with both old and new DNA – each molecule would be composed of one old strand and one new one.
- 3. DNA Replication isSemi- conservative: Experimental Proof (1957) Matthew Meselson and Franklin Stahl They grew the bacterium Escherichia coli medium that contained 15N in the form of ammonium chloride. Conclusion: Semi-conservative model is the most appropriate for replication
- 5. John Cairns In1963, John Cairns proved the same thing by using autoradiography. His conclusions were: 1. The chromosome of E. coli is circular. 2. Replication starts from a fixed point called the origin of replication. 3. During replication the chromosome maintains its integrity or circular shape.
- 6. DNA Replication inBacteria DNA replication is bi-directional from the origin of replication DNA replication occurs in both directions from the origin of replication in the circular DNA found in most bacteria
- 7. The Mechanism of Replication Tightly controlled process: ◦ Occurs at specified times during the cell cycle ◦ Starts at a fixed point Requires: ◦ A set of proteins and enzymes ◦ Energy Two basic steps: ◦ Initiation ◦ Elongation Two basic components: ◦ Template ◦ Primer
- 8. Origin of Replication Contains a total of 245 bps. Contains tandem repeats on the left side Has initiation protein binding sites on the right side.
- 9. Enzymology of DNA Replication DNA Helicase: Unwinds DNA ahead of replication fork. DNA Polymerase: DNA synthesis and repair of gaps. DNA Ligase: Joins fragments of DNA DNA Primase: Synthesizes primers DNA gyrase: Releases torsional strain caused by helicase activity SSBs: Stabilize single-stranded regions of the replication fork
- 10. Helicase Operates in replicationfork Separates strands to allow DNA Pol to function on single strands Moves along in a single strand 5’ 3’ or 3’ 5’ direction by hydrolyzing ATP
- 11. Topoisomerase Enzyme Regulatesthe overwinding or underwinding of DNA Topoisomerases bind to either single- stranded or double-stranded DNA It also cuts the phosphate backbone of the DNA It is further categorized into a) Topoisomerase I, and b) Topoisomerase II enzyme
- 12. Single Stranded DNABinding Proteins (SSB) Maintain strand separation once helicase separates strands. Primase enzyme: It catalyzes the copying of a short stretch of the DNA template strand to produce RNA primer sequence.
- 13. The DNA PolymeraseFamily In 1955, Arthur Kornberg was the first scientist who discovered the DNA Polymerase I which is also known as the Kornberg enzyme.
- 14. A total of5 different DNAPs have been reported in E. coli DNAP I: functions in repair and replication DNAP II: functions in DNA repair (proven in 1999) DNAP III: principal DNA replication enzyme DNAP IV: functions in DNA repair (discovered in 1999) DNAP V: functions in DNA repair (discovered in 1999)
- 15.
- 16. Function of DNAPolymerase DNA Polymerase function has the following requirements: ◦ All 4 deoxyribonucleoside triphosphates: dTTP, dATP, dGTP, and dCTP ◦ Mg+2 ◦ A Template ◦ An RNA primer – a short strand of RNA
- 17.
- 18. Initiation of Replication Replication initiated at specific sites: Origin of Replication (ori) Two types of initiation: ◦ De novo – Synthesis initiated with RNA primers. Most common ◦ Covalent extension – Synthesis of new strand as an extension of an old strand (“Rolling Circle”)
- 19. De novo Initiation Binding to OriC by DnaA protein Opens strands Replication proceeds bidirectionall y
- 22. The Problem ofOverwinding
- 23. Topoisomerase Type I Precedes replicating DNA Mechanism ◦ Makes a cut in one strand, passes other strand through it. Seals gap. ◦ Result: induces positive supercoiling as strands are separated, allowing replication machinery to proceed.
- 24. Gyrase-A Type II Topoisomerase Introduces negative supercoils Cuts both strands Section located away from actual cut is then passed through cut site.
- 25. 1. Helicase denaturingDNA causes tighter winding in other parts of the circular chromosome. Gyrase relieves this tension. 2. Leading strand is synthesized continuously, while lagging strand is synthesized discontinuously, in the form of Okazaki fragments. DNA replication is therefore semi- discontinuous. 3. Each fragment requires a primer to begin, and is extended by DNA Polymerase III. 4. Okazaki data shows that these fragments are gradually joined together to make a full-length dsDNA chromosome. DNA Polymerase I uses the 3’-OH of the adjacent DNA fragment as a primer, and simultaneously removes the RNA primer while resynthesizing the primer region in the form of DNA. The nick remaining between the 2 fragments is sealed with DNA ligase.
- 27. Rolling Circle Replication Another model for replication is Rolling circle. It is used by several bacteriophages. Rolling circle replication begins with a nick (single-stranded break) at the origin of replication. The 5’ end is displaced from the strand, and the 3’ end acts as a primer for DNA Polymerase III, which synthesizes a continuous strand using the intact DNA molecule as a template. The 5’ end continues to be displaced as the circle “rolls”, and is protected by SSBs until discontinuous DNA synthesis makes it a dsDNA again.
- 29. A DNAmolecule many genomes in length can be made by rolling circle replication. During viral assembly it is cut into individual viral chromosomes and packaged into phage head. Bacteriophage λ, regardless of whether entering the lytic or lysogenic pathway, circularizes its chromosome immediately after infection.
- 30. DNA Replication Model 1.Relaxation of supercoiled DNA. 2. Denaturation and untwisting of the double helix. 3. Stabilization of the ssDNA in the replication fork by SSBs. 4. Initiation of new DNA strands. 5. Elongation of the new DNA strands. 6. Joining of the Okazaki fragments on the lagging strand.
- 31.
- 32. 2 Strands LeadingStrand Lagging Strand
- 33. Leading strand Continuouslyelongating strand In 5’ to 3’ direction Towards the replication fork
- 35. Primosome Contains Primaseand Helicase Forms RNA primer
- 36. Primase Forms RNAprimer Provides free 3’ OH end for DNA Polymerase III
- 37. DNA Polymerase III Addition of new nucleotides to 3’ end of primer About 1000 nucleotides/second
- 38. Okazaki fragments Discoveredby Reiji Okazaki 1000 to 2000 nucleotide sequence Separated by 10 nucleotide RNA primer
- 40. Lagging strand Discontinuouslyelongating strand Away from replication fork Primosome formation
- 41. DNA Polymerase I DNALigase Removes DNA Polymerase III Replaces RNA primer Joins the okazaki fragments
- 43. Speed of Elongation Due to continuous and discontinuous elongation, leading strand should elongate quickly Prevented by helicase which acts as temporary halting signal, stopping DNA unwinding
- 44. Folding of Laggingstrand For simultaneous replication of both strands by DNA Polymerase III subunits
- 46.
- 47. The Replication Trap 2 replicating forks meet at Ter region Clusters of Ter sequence repeats
- 48. Tus Protein Tusprotein binds Tus-ter complex DnaB also bound Sequence specific Bacillus subtilis: has RTP
- 49. One complex, One direction Fork arrested from one direction Why not collision? Asymmetric
- 50. Catenanes Depends onOriC number Not covalent itself Interwound Separation needed
- 51.
- 52.
- 53.
- 54. Regulation of Initiation Ratio of ATP to ADP Ratio of DnaA to number of DnaA boxes Hemimethylation of OriC Sequestering of OriC
- 55. ATP : ADP DnaA complexes with ATP and ADP Faster growth, more ATP complex Strict regulation of DnaA
- 56. Methylation and Sequestering OriC hemimethylated by Dam methylase GATC: N6 position of adenine OriC = 11/245 Whole = only 1 in 256
- 57. Right after replication… Parental strand hemimethylated OriC seq. to membrane by SeqA binding protein ◦ DnaA cannot access for a while ◦ DnaA binds better with full than hemi OriC released, fully methylated DnaA can bind
- 58. Why these two? With more DnaA, more DNA to cell mass (and vice versa). Without Dam methylase, E. coli inefficiently replicates ◦ Therefore, OriC methylation triggers, too.
Editor's Notes
- #2 In conservative replication one of the daughter DNA molecules contained both parental strands and the other contained an entirely new stand. Parent strand in this case acts as a template to synthesize the new strand. After that the parent strand reunites hence this is called conservative replication. In this case only the phosphodiester bonds break. In the second case, i.e. the semi conservative replication each daughter has one parental strand and one new strand. In this both the strands are the same. In the last case the parental DNA strands do not maintain their identity. It breaks up into tiny strands and forma an entirely new strand.
- #4 (1957) Mathew Meselson and Franklin Stahl grew the bacterium Escherichia coli on medium that contained 15N in the form of ammonium chloride. The 15N became incorporated into DNA (nitrogenous bases). The resulting heavy nitrogen-containing DNA molecules were extracted from some of the cells. When subject to density gradient centrifugation, they accumulated in the high-density region of the gradient. The rest of the bacteria were transferred to a new growth medium in which ammonium chloride contained the naturally abundant, lighter 14N isotope. The newly synthesized strands were expected to be less dense since they incorporated bases containing the lighter 14N isotope. The DNA from cells isolated after one generation had an intermediate density, indicating that they contained half as many 15N isotope as the parent DNA. This finding supported the semi-conservative model - each double helix would contain one previously synthesized strand and a newly synthesized strand. It is also consistent with the dispersive model which would yield one class of molecules, all with intermediate density. It was inconsistent with the conservative model which predicted that there would be two classes of double-stranded molecules, those with two heavy strands and those with two light strands. After another cycle of cell division in the medium with the lighter 14N isotope, two types of DNA appeared in the density gradient. One with hybrid DNA helices (one strand 15N isotope and the other strand 14N), whereas the other contained only strands of the light isotope. This finding refuted the dispersive model, which predicted that all stands should have intermediate density. It however supported the semiconservative method which predicted that each parent strand would act as a template for the synthesis of new strands.
- #6 Then in 1963 John Cairns proved the same thing by using autoradiography. It is a technique whereby he used an x-ray film to visualize molecules or fragments of molecules that had been labeled by radioactive elements. He grew E. coli on a medium containing 3H-thymine. In his conclusions he said that the chromosome of E. coli is circular. He also stated that replication starts from a fixed point which is now called as the origin of replication. An intermediate theta structure forms (topologically similar in shape to the Greek letter.) He also stated that during replication the chromosome maintains its integrity or circular shape. Therefore according to him a part of chromosome opens up during replication and after replication it again coils up over one another.
- #7 • At each origin of replication, there are two replication forks, points at which new polynucleotide chains are formed • There is one origin of replication and two replication forks in the circular DNA of prokaryotes
- #9 Replication in case of prokaryotes starts from a fixed point called the Ori-c or origin or replication. It contains a total of 245 bps. This region contains 13mer sequences also called the Tandem repeats having 13 base pairs on the left side. Along with this it also contains 4 9mer sequences on the right side that act as the initiation protein binding sites. Tandem repeats are dispersed.
- #12 Topoisomerases are enzymes that regulate the overwinding or underwinding of DNA. The winding problem of DNA arises due to the intertwined nature of its double helical structure. For example, during DNA replication, DNA becomes overwound ahead of a replication fork. If left unabated, this tension would eventually grind replication to a halt (a similar event happens during transcription.) In order to help overcome these types of topological problems caused by the double helix, topoisomerases bind to either single-stranded or double-stranded DNA and cut the phosphate backbone of the DNA. This intermediate break allows the DNA to be untangled or unwound, and, at the end of these processes, the DNA backbone is resealed again.
- #16 Processivity is basically defined as the average number of nucleotides that are added before a polymerase dissociates. Before separation, Polymerase I can add about 3-200 nucleotides to the strand, whereas polymerase II can add about 1500 nucleotides and polymerase III can add up to 50,000 nucleotides. Therefore the efficiency of the enzyme is calculated on the basis of its accuracy and processivity. Hence DNA polymerase III is considered to be a very processive enzyme.
- #32 The second step in the replication process is the elongation of the strands. As we know, there are two single separated strands termed as leading and lagging strand.
- #34 ELONGATION OF THE LEADING STRAND: It is the continuous elongating strand in which elongation moves in the direction of replication fork i.e. from 5’ to 3’. The dnaB enzyme (primase) and dnaG (helicase) form a functional unit called as primosome. On the single separated leading strand, this primosome synthesizes a RNA primer which is a short nucleotide sequence of about 10 base pairs. Another enzyme DNA polymerase III binds with this primer and add new nucleotides to the 3’ end of the primer, thus elongating it.
- #41 ELONGATION OF THE LAGGING STRAND: This strand is elongated discontinuously in 3’ to 5’ direction i.e. away from the replication fork. As in the case of elongation of leading strand, a primosome is formed, which synthesizes RNA primer. The basic purpose of the primer is to provide a free 3’ end because DNA polymerase III can add free nucleotides only at 3’ end. When DNA polymerase III reaches primer, it adds a short sequence of nucleotides of about 1000 to 2000 base pairs to the primer. These sequences are called as okazaki fragments. After the addition of okazaki fragments DNA polymerase I replaces DNA polymerase III. It removes RNA primer and replaces it with DNA sequence. Basically DNA polymerase I removes DNA polymerase III and replaces Uridine in RNA primer with Thymine. After the addition of one fragment, the primosome moves forward on thE replication fork, it synthesizes new primer and the process is repeated. DNA ligase binds the two fragments together by the formation of phosphodiester bond.
- #42 SPECIAL PROPERTY OF DNA POLYEMRASE III DNA polymerase III is a single enzyme and its subunits act on both strands i.e. two different DNA polymerase III doesn’t act on both strands. The lagging strand folds upon itself so that DNA polymerase III subunit can act as one unit and both strands can replicate simultaneously.