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DNA Replication

DNA Replication. Nilansu Das Surendranath College. Paper III Group A: Cellular and Molecular Biology Unit I 1 . DNA Replication: (10)

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DNA Replication

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  1. DNA Replication Nilansu Das Surendranath College

  2. Paper III Group A: Cellular and Molecular Biology Unit I 1. DNA Replication: (10) DNA-Replication-Meselson-Stahl experiment as evidence for semiconservative replication; Mechanism of replication-Rolling-circle model & Theta (8) structure (bidirectional) 2. Transcription in prokaryotes: (15) Mechanisms (Initiation, elongation, termination); promoter structures, subunits of bacterial polymerases, functions and domains responsible for activity, elongation process, mechanism of termination, -dependent and independent termination; lac, trp, ara operons. 3. Mechanism of translation in prokaryotes: (15) Description of ribosomal cycle including phenomena of initiation, elongation, termination; description of factors involved in these processes; genetic code; tRNA: clover-leaf structure & function; rRNA: structure and function; role of aminoacyl tRNAsynthetases. Non-ribosomal peptide synthesis: cyclic peptide antibiotics e.g. Gramicidin etc

  3. old old new Replication

  4. The Central Dogma of Molecular Biology DNA Replication DNA Reverse Transcription Transcription RNA Replication RNA Translation Protein

  5. DNA metabolism comprises of: • Replication – the process by which copies of DNA molecules are faithfully made; • Repair and Recombination – the processes that affect the inherent structure of the information. • Two very important criteria of DNA replication: • Fidelity - DNA replication process requires an exquisite degree of accuracy. Uncorrected errors that arise during DNA synthesis can have dire consequences, not only because they can permanently affect or eliminate the function of a gene but also because the change is inheritable. • Speed – DNA molecules may contain a million of bases. The machinery (including a myriad of proteins and enzymes) that synthesizes DNA consequently do so with extraordinary speed and fidelity. • Many of the seminal discoveries in DNA metabolism are made with Escherichia coli.

  6. Map of the E.coli chromosome The map shows the relative positions of genes encoding many of the proteins important in DNA metabolism. The number 0 to 100 denote a genetic measurement called minutes. Each minute corresponds to ~40,000 bp along the DNA molecule of E. coli. The three-letter names of genes and other elements generally reflect some aspects of their functions. mut – mutagenesis dna – DNA replication pol – DNA polymerase rpo – RNA polymerase uvr – UV resistance rec – recombination dam – DNA adenine methylation lig – DNA ligase Ter– termination of replication ori – origin of replication The number of genes involved in DNA replication provides a hint of the complexity of this process.

  7. Mysteries How organisms create faithful copies of themselves? Results of landmark experiments conducted by the following scientists: 1868 - Friedrich Miescher 1944 - Oswald T. Avery, Colin MacLeod, and Maclyn McCarty 1952 - Alfred D. Hershey and Martha Chase proved that DNA is the genetic material. How the cells produce identical copies of large and complex macromolecules? Speculation – the concept of a template – a structure that would allow molecules to line up in a specific order and get joined, to create a macromolecule with a unique sequence and function. The DNA double helical structure as deduced by, 1953 - James D. Watson and Francis Crick from theX-ray diffraction pattern of DNA, demonstrated by Rosalind Franklin and Maurice Wilkins,supported the template speculation. The fundamental features of the DNA replication process have proved to be essentially identical in all species of the prokaryotic as well as the eukaryotic worlds. Many of the protein complexes are functionally and structurally conserved.

  8. Replication of DNA as suggested by Watson and Crick From their model, Watson and Crick could predict, well in advance of the actual experimental evidences, that the DNA double helical structure could be replicated by: 1) separating the two strands, and 2) synthesizing a complementary strand for each. The separated parent strands would serve as the templates.

  9. DNA Replication Follows a Set of Fundamental Rules • DNA Replication is Semiconservative • Replication Begins at the Origin and Usually Proceeds Bidirectionally • DNA Synthesis Proceeds in a 5’→3’ Direction and is Semidiscontinuous

  10. Three Hypotheses For DNA Replication

  11. SEPARATION OF DNAS BY CESIUM CHLORIDE DENSITY GRADIENT CENTRIFUGATION (a) Photo of DNA in an ultracentrifuge tube made with UV light (b) Densitometric trace of UV scan

  12. DNA Replication is Semiconservative Each DNA strand serves as a template for the synthesis of a new strand, producing two new DNA molecules, each with one new strand and one old strand. This is semi-conservative replication. • Messelson-Stahl Experiment: • Cells were grown for many generation in a medium containing only 15N (15NH4Cl) as the nitrogen source, so that all the DNA was 15N labeled and showed up as a single band (blue) when centrifuged in a CsCl density gradient. • The cells were next transferred to a medium containing only 14N and allowed to grow for one generation. The DNA isolated from these cells banded in an intermediate region (between 15N DNA and 14N DNA). • Continuation of replication for a second generation yielded two hybrid DNA and two light DNAs (red), confirming semi-conservative replication.

  13. N15 - N15 N14 - N15 N14 - N14 N15 - N14

  14. Bidirectional Replication  (Theta) Mode Replication Replication of E.coli

  15. Following the confirmation of a semiconservative mechanism of replication, some fundamental questions arose: Does replication begin at random places or at a unique point? After initiation at any point in the DNA, does replication proceed in one direction or both?

  16. E.coli ori C

  17. DnaA the Replication Initiator DnaA is a protein that activates initiation of DNA replication in bacteria. It is a replication initiation factor which promotes the unwinding of DNA at oriC.  DnaA accumulates during growth and then triggers the initiation of replication. Replication begins with active DnaA binding to 9-mer (9-bp) repeats upstream of oriC. Binding of DnaA leads to strand separation at the 13-mer repeats. This binding causes the DNA to loop in preparation for melting open by the DnaBhelicase .

  18. Binding of DnaA at ori C

  19. Unwinding of ori C

  20. Loading of Helicase (DnaB) Helicases are a class of enzymes vital to all living organisms for replication of DNA. They are motor proteins that move directionally along a nucleic acid phosphodiester backbone, separating two annealed nucleic acid strands (i.e., DNA, RNA, or RNA-DNA hybrid) using energy derived from ATP hydrolysis.

  21. The Role of Primase (DnaG) DNA primase is an enzyme involved in the replication of DNA and is a type of RNA polymerase. Primase catalyzes the synthesis of a short RNA segment called a primer complementary to a ssDNA template. Primase is of key importance in DNA replication because no known replicative DNA polymerases can initiate the synthesis of a DNA strand without an initial primer (RNA or DNA)  

  22. TOP STRAND GROWS 3’ TO 5’ BOTTOM STRAND GROWS 5’ TO 3’ BOTH STRANDS GROW 5’ TO 3’; LEADING STRAND (BLUE) GROWS CONTINUOUSLY LAGGING STRAND (RED) GROWS DISCONTINUOUSLY BOTH STRANDS GROW 5’ TO 3’; BOTH STRANDS GROW DISCONTINUOUSLY THREE POSSIBLE WAYS IN WHICH DNA MIGHT BE SYNTHESIZED AT THE REPLICATION FORK WEAVER

  23. DNA gyrase, or simply gyrase, is an enzyme that relieves strain while double-stranded DNA is being unwound by helicase. This causes negative supercoiling of the DNA.

  24. Primers (RNA) 5” 5” 5” 3” 3” 3” 5” 3” Most recently synthesized DNA strand Leading strand 3” 5” 5” Lagging strand 3” direction of fork movement 5” 3” 5” 5” 3” 3” The Progression of Replication fork

  25. DNA Synthesis Proceeds in a 5’→3’ Direction and is Semidiscontinuous (cond.) • A new DNA strand (red) is always synthesized in the 5’3’ direction. • The template is read in the opposite direction, 3’5’. The leading strand is • continuously synthesized in the direction taken by the replication fork. • The other strand, the lagging strand is synthesized discontinuously in short pieces (Okazaki fragments) in a direction opposite to that in which the replication fork moves. • The Okazaki fragments are joined together by DNA ligase. • In bacteria, Okazaki fragments are ~1000 to 2000 nucleotides long. In eukaryotic cells, they are 150 to 200 nucleotides long

  26. DNA is Synthesized by DNA Polymerases • 1955 – the search for an enzyme that could synthesize DNA began. • Arthur Kornberg and his colleagues initially purified and characterized a DNA polymerase from E. coli. • This polymerase, now called, DNA Polymerase I, is a single polypeptide enzyme – Mr 103,000 – encoded by the polA gene. • Later investigators found that E. coli contain more distinct polymerases.

  27. Search for other DNA polymerases began soon after the discovery of DNA pol I since evidence began to accumulate that it is not suitable for replication of the large E. coli chromosome on the following grounds: • the rate at which it adds nucleotides is too slow to account for the rates at which the replication fork moves in the bacterial cell; • DNA polymerase I has a relatively low processivity; • genetic studies demonstrated that many genes and therefore many proteins, are involved in replication. Hence, DNA pol I does not act alone; • in 1969 John Cairns isolated a bacterial strain with a mutation in the DNA pol I gene that produced an inactive enzyme. This strain was however, viable. • A search for other DNA polymerases led to the discovery of E. coli DNA polymerase II and III in the early 1970s and DNA polymerase IV and V in 1999. • DNA polymerase III is the principal replication enzyme in E. coli.

  28. Replication is Very Accurate (contd.) • A base can sometimes exist in the unusual tautomeric form, allowing it to hydrogen-bond with an incorrect partner. • Consequently the accuracy of the polymerization reaction itself will be insufficient to account for the high degree of fidelity in replication. • In fact, careful measurements in vitro have shown that DNA polymerase insert one incorrect nucleotide for every 104 to 105 correct ones. • A mechanism, intrinsic to virtually all DNA polymerases is a separate 3’ 5’ exonuclease activity that double-checks each nucleotide after it is added.This nuclease activity of the polymerase, also called the proofreading activity, • permits the enzyme to remove a newly added nucleotide and is highly specific for mismatched base-pairs. • Proofreading is not the opposite of polymerization reaction since pyrophosphate is not involved. • In the monomeric DNA pol I the polymerizing and proofreading activities have separate active sites within the same polypeptide. • When both selection and proofreading are combined, DNA polymerase leaves behind one error for every 106 to 108 bases added. • The measured accuracy of replication in E. coli is 109 to 1010. This additional accuracy is provided by a separate enzyme system that repairs mismatched base pairs remaining after replication.

  29. DNA polymerase III • A very complex DNA polymerase III, has ten types of subunits. • The polymerization and proofreading activities reside in its  and  subunits respectively. • The  subunits associates with  and  to form a core polymerase which can polymerase DNA but with limited processivity. • [After adding a nucleotide to a growing DNA strand, a DNA polymerase either dissociates or moves along the template and adds another nucleotide. Dissociation and Reassociation of the polymerase can limit the overall polymerase rate – the process is much faster when a polymerase adds more nucleotides without dissociating from the template. The average number of nucleotides added before a polymerase dissociates defines its processivity.]

  30. DNA polymerase III • Two core polymerases can be linked by another set of subunits, a clamp-loading complex, or-complex, consisting of five subunits of four different types, 2’. • The core polymerases are linked together by the  subunits. • Two additional subunits  and  are bound to the clamp loading complex or the -complex. • The  subunit is generated by a translational frameshifting mechanism of the  gene that leads to premature translational termination. • The entire assembly of 13 protein subunits [()22’] is called DNA polymerase III*. • DNA polymerase III* can polymerize DNAbut with a much lower processivity. • The necessary increase in processivity is provided by the addition of the  subunits, four of which complete the DNA polymerase holoenzyme. • The  subunits associate in pairs to form donut-shaped structures that encircle the DNA and act like clamps. One dimeric clamp per core subassembly.

  31. DNA Replication Requires Many Enzymes and Protein Factors • The replication process in E. coli requires along with the DNA polymerase III many other different enzymes and proteins, each performing a specific task. The entire complex is termed the DNA replicase system or replisome. • Helicases are enzymes that move along the DNA and separate the strands that are to act as templates for the synthesis of the leading and the lagging strands. ATP hydrolysis is required to accomplish this task. • Topoisomerases relieves the topological stress in the helical DNA structure created by the strand separation. • The separated strands are stabilized by the DNA-binding proteins. • Primases synthesize the primers, generally short segments of RNA, an absolute requirement of the DNA polymerases. • DNA polymerase III carries out the polymerase activity which includes three different stages – initiation, elongation and termination. • RNA primers are finally removed by the DNA polymerase I activity (5’3’ exonuclease activity) and also fills up the gap created by the primer removal by its polymerase action. • The nick created by the DNA polymerase I action (between the newly synthesized Okazaki fragments) are sealed by the DNA ligase. • All these processes require coordination and regulation, an interplay best characterized in the E. coli system.

  32. RNA polymerase requirement: • RNA could be required to read into the origins from adjacent transcription units; by terminating at sites in the origin, it could provide the 3’-OH ends that prime DNA polymerase III. (e.g. D-loops at the mitochondrial origin) • Alternatively, the act of transcription could be associated with a structural change that assists initiation. This idea is supported by observation that transcription does not have to proceed into the origin; it is effective up to 200 bp away from the origin, and can use either strand of DNA as template in vitro.

  33. The complexity in Okazaki fragment synthesis lies in the coordination of leading and lagging strand synthesis: both strands are produced by a single asymmetric DNA polymerase III dimer, which is accomplished by looping the DNA of the lagging strand, bringing together the two points of polymerization. Black arrows: direction of movement of the parent DNA through the complex. Red arrows: direction of the DNA synthesis.

  34. The DnaB helicase and DnaG primase constitutes a functional unit within the replication complex, the primosome. • The DnaB helicase unwinds the DNA at the replication fork as it travels along the lagging strand in the 5’3’ direction. • DNA primase occasionally associates with DnaB helicase and and synthesize a short RNA primer.

  35. A new  sliding clamp is then positioned at the primer by the clamp-loading complex of DNA polymerase III.

  36. When synthesis of an Okazaki fragment has been completed, replication halts, and the core subunits of DNA polymerase III dissociate from their  sliding clamp and hence, from the completed Okazaki fragment. • The core subunit then associates with the new clamp. • This initiates synthesis of a new Okazaki fragment.

  37. Once an Okazaki fragment has been completed, its RNA primer is removed and replaced by DNA polymerase I. • The nick is sealed by DNA ligase. • The entire complex responsible for coordinated DNA synthesis at a replication fork is a replisome.

  38. RNA primers in the lagging strand are removed by the 5’3’ exonuclease activity of DNA polymerase I and replaced with DNA by the same enzyme. • The remaining nick is sealed by DNA ligase. • DNA ligase catalyzes the formation of a phosphodiester bond between a 3’ hydroxyl at the end of one strand and a 5’ phosphate at the end of the other strand. • The phosphate must be activated by adenylation. • DNA ligase from bacteria uses NAD+ as a source of the activating AMP group whereas eukaryotes and viruses use ATP for the same. • DNA ligase has become an important reagent in recombinant DNA technology.

  39. Termination • Eventually, the two replication forks of the circular chromosome meet at a terminus region containing multiple copies of a 20 bp sequence called Ter. • The Ter sequences are arranged on the chromosome to create a sort of trap that a replication fork can enter but cannot leave. • Tus (termination utilization sequence) binds at the Ter sequence. • Only one Tus-Ter complex functions per replication cycle and hence, can arrest replication fork from only one direction - decided by which of the two replication forks first encounter the complex. • When either replication fork encounters a functional Tus-Ter complex it halts. The other fork halts when it meets the first arrested fork. • The final few hundred base pairs of DNA between these large protein complexes are then replicated (mechanism not known). • Replication is completed giving rise to two topologically interlinked or catenated circular cromosomes known as catenanes.

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