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Chapter 25

Chapter 25. DNA Replication. Chapter 25 Outline :. Early Insights into DNA Replication DNA Polymerases: Enzymes Catalyzing Polynucleotide Chain Elongation A Brief Review of Microbial Genetics Multiple DNA Polymerases Other Proteins at the Replication Fork

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Chapter 25

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  1. Chapter 25 DNA Replication

  2. Chapter 25 Outline: • Early Insights into DNA Replication • DNA Polymerases: Enzymes Catalyzing Polynucleotide Chain Elongation • A Brief Review of Microbial Genetics • Multiple DNA Polymerases • Other Proteins at the Replication Fork • Proteins in Eukaryotic DNA Replication • Replication of Chromatin • Initiation of DNA Replication • Replication of Linear Genomes • Fidelity of DNA Replication • RNA Viruses: The Replication of RNA Genomes

  3. Early Insights into DNA Replication • Three central features of DNA replication were predicted in 1953 from Watson and Crick’s model: • DNA replication is semiconservative—each of the two identical daughter DNA molecules contains one parental strand and one newly synthesized strand. • This prediction was confirmed in 1957 by the elegant experiment of Meselson and Stahl. • Parental strand unwinding and synthesis of new DNA occur simultaneously, in the same microenvironment. In other words, replication occurs at a fork, in which parental strands are unwinding and both daughter strands are undergoing elongation, as suggested by the diagram. • Replication begins at one or more fixed sites—replication origins—on a chromosome.

  4. Early Insights into DNA Replication • Biosynthesis of nucleic acids and proteins is carried out through the processes of replication, transcription, and translation. • Most regulation occurs at the level of initiation.

  5. DNA Replication Is Semiconservative.

  6. Replication Forks may Move Either Unidirectionally or Bidirectionally

  7. Demonstration of bidirectional replication by radioautography: • Initiation of a new round of replication before completion of the preceding round: • Labeling with [3H]-thymidine was started when the first round of replication began. • The pattern of grain density shows that one of the reinitiated branches contains two labeled strands, while the other contains only one. • Solid lines denote radiolabeled DNA, and dashed lines denote unlabeled DNA. Black arrowheads depict first-generation replication forks, and blue arrowheads depict second-generation replication forks.

  8. Bidirectional replication from several fixedorigins (O) on a linear eukaryotic chromosome:

  9. DNA synthesis is catalyzed by DNA polymerases in the presence of (i) primer, (ii) template, (iii) all 4 dNTP, and (iv) a divalent cathion such as Mg++. • The DNA polymerase reaction: • Each incoming dNTP is positioned by base pairing with the appropriate template nucleotide, and a phosphodiester bond is created by nucleophilic attack of the primer strand 3’ hydroxyl group on the phosphate of the dNTP.

  10. DNA Polymerase I: • DNA polymerase I is the first DNA polymerase discovered by Arthur Kornberg in 1959. • The DNA polymerase Imolecule is a single polypeptide that contains three enzymatic activities: polymerization, 3’-5’exonuclase, and 5’-3’ exonuclease. • Large (Klenow) fragment of DNA polymerase I retains polymerization and proofreading (3’ to 5’ exo) activities.

  11. DNA substrates that can be acted upon by purified DNA polymerase: • Each blue arrowhead marks a 3’ hydroxyl terminus at which chain extension is occurring. • Nicked duplex (e) can only be a DNA substrate by certain DNA polymerase (such as DNA polymerase I).

  12. Identifiction of genes involved in DNA replication: A Brief Review of Microbial Genetics • Bacterial conjugation: • Male E. coli contains fertility factor (a plasmid) which produces pili to make contact with female bacteria (see next figure). • Hfr (High frequency recombination) strain of E. coli has F plasmid integrated into the chromosome and can transfer donor chromosome into female. • Later, the transferred DNA strand can undergo recombination with the recipient chromosome, thereby transferring genetic markers to the female. • Through such a genetic study, genes involved in DNA replication can be identified and mapped.

  13. Conjugation in bacteria Hfr

  14. Partial genetic map of E. coli: • Genes whose products are involved in DNA replication or repair are shown. • Gene product names and function are given in blue. • The dna genes play essential roles in DNA replication. • The mut genes specify proteins that, when mutated, can cause elevated rates of spontaneous mutation. • ori and ter are sites of initiation and termination of genome replication, respectively.

  15. Partial map of the T4 genome: • Genes whose products participate in DNA metabolism are shown on the outside of the circle. • The inner numbers represent distances in kbp. • The reference point (0) represents the divide between two genes not shown—rIIA and rIIB.

  16. Multiple DNA Polymerases

  17. DNA Synthesis Can’t be Continuously on Both Strands (because the DNA duplex is antiparallel and all DNA polymerases synthesize DNA in a 5’ to 3’ direction) What is the source of primer used for lagging strand synthesis?

  18. A model of Semi-discontinuous DNA replication • Semi-Discontinuous DNA Synthesis: • The first two questions were answered primarily through the work of Reiji Okazaki, who proposed that DNA replication could be discontinuous. • In principle one parental strand (the leading strand) could be extended continuously, with polymerase moving from the 5’ terminus to the 3’ terminus in the same direction as fork movement. • Synthesis on the lagging strand would be discontinuous; chain extension along the leading strand would expose single-strand template on the lagging strand. • This template could be copied in short fragments (later named Okazaki fragments), with polymerase moving opposite to the direction of fork movement. • Thus, lagging strand synthesis would occur in short pieces. These could then be joined to high-molecular-weight DNA by the enzyme DNA ligase.

  19. DNA Replication may involve: • Initiation. • Elongation. • Termination.

  20. Proteins involved in Elongation of DNA

  21. Elongation:Synthesis of Okazaki fragments

  22. Helicases are multimeric proteins that bind preferentially to one strand of a DNA duplex and use energy of ATP hydrolysis to actively unwind the duplex. • A model for helicase action: • In this model a homodimeric enzyme, such as the E. coli Rep helicase, shows 3’5’ polarity. The DnaB helicase is a hexamer and translocates in the 5’ to 3’ direction.

  23. A model of T7 gp4 helicase action: • The protein is rotating along the blue DNA strand, excluding the red strand from the central channel.

  24. Single-strand DNA binding(SSB) proteins andPrimase • Primase, a special class of RNA polymerase, synthesizes short RNA molecules as primers for lagging strand DNA replication. • Single-strand DNA binding(SSB) proteins: stabilizes separated DNA strands to maintain optimal template conformation.

  25. DNA polymerase III is the replicating enzyme • Subunit structure of the E. coli DNA polymerase III holoenzyme: • DNA polymerase III holoenzyme, a complex bacterial enzyme containing at least 10 subunits, plays the predominant role in replicative chain elongation.

  26. PolIII*consists of two cores, a clamp-loading complex (g complex) consisting of t2gdd’, and two additional proteins c and y. Holoenzyme is PolIII* plus b subunits.

  27. Structure of the sliding clamp: • Each protein forms a “doughnut” that can completely surround double-stranded DNA and thus keep polymerase associated with its DNA templates. • The a-helices on the inner surface of the subunit contact DNA but do not bind tightly enough to retard movement of the protein. The E. coli protein has two identical subunits with two DNA-associating domains, while the human and RB69 proteins have three subunits, each with two DNA-associating domains.

  28. Scheme for action of the E. coli clamp loader • The complex contains five protein subunits: three copies of g(B, C, and D in the figure) and one each of dand d’ (A and E, respectively).

  29. Topoisomerases: relieves topological stress • Action of type I and type II topoisomerases, as shown by gel electrophoresis: • Lane 1 shows a relaxed circular DNA. • Lane 2 shows the pattern from treatment of supercoiled DNA with type I topoisomerase. • Lanes 3–5 show relaxed circles treated with DNA gyrase, a type II topoisomerase, for different lengths of time. • Note that more different topoisomers can be seen in topoisomerase I reaction mixtures, as expected if changes in the linking number (L) occur in units of 1, whereas gyrase changes L in units of 2. • Type I topoisomerases break and reseal one DNA strand • Type II topoisomerases catalyze double-strand breakage and rejoining. • Hence, type I and type II enzymes change DNA linking number in units of 1 and 2, respectively

  30. Action of a type I topoisomerase: • The enzyme breaks one strand and immobilizes the 5’ end by a covalent bond between the DNA phosphate and a tyrosine residue (in E. coli topoisomerase I). • Rotation of the 3’ end is followed by resealing. • The linking number is increased by 1 in the example shown (an underwound DNA). • Action of a type I topoisomerase on overwound DNA would decrease the linking number, by essentially the same mechanism.

  31. Action of a type II topoisomerase • DNA gyrase of E. coli; the example shown is a tetrameric protein with two A and two B subunits. • The enzyme is shown introducing two negative turns and changing the linking number from +1 to -1. • The enzyme catalyzes a double-strand break, and the two DNA ends are bound by A subunits, which move the DNA ends apart so that the unbroken duplex can pass through the gap. • Resealing converts the positive supertwist to a negative one, giving the overall molecule a DL of -2. • Type II topoisomerases can relax underwound duplexes by the reverse of the above pathway.

  32. The types of topological interconversionscatalyzed by type II topoisomerases Relaxation. Catenation and decatenation. Knotting and unknotting.

  33. DNA Pol I: removes RNA in Okazaki fragments and fills the gaps between Okazaki fragments. • Nick translation in removal of RNA primers by coordinated action of the 5’ exonuclease and polymerase activities of DNA polymerase I: • The figure shows replacement of base paired UMP in the RNA primer by dTMP in the growing DNA chain. • The template DNA is the lagging strand.

  34. Ligase: seals nicks The reaction catalyzed by DNA ligase:

  35. The E. coli replisome • Note that the clamp loader, via the protein, serves as a link to dimerize the pol III holoenzyme.

  36. Schematic view of a replication fork: • Note that polymerases catalyzing leading- and lagging-strand replication are linked together.

  37. Model for the synthesis of DNA on the leading and lagging strands by the asymmetric dimer of PolIII The primase-polymerase switch during lagging strand synthesis: DnaB helicase encircles the lagging strand, and primase has synthesized an RNA primer. Primase must contact SSB to remain bound. Core polymerase on the lagging strand is forcing that strand and the daughter strand to loop out. The c subunit of the complex interacts with SSB, leading to primase displacement. The complex is opening the clamp, and a newly completed Okazaki fragment is being released, along with its template. Primase rebinds to single-strand template DNA to begin a new primer.

  38. Eukaryotic DNA polymerases

  39. Eukaryotic DNA replication proteins

  40. Model for chromatin replication: • Nucleosomes on parental DNA are dissociated as the replication fork approaches and are reformed on newly synthesized daughter strands, with both old and newly synthesized histones being used. • Maturation occurs slowly, with full organization not being re-established until many kilobases behind the moving fork.

  41. Termination of DNA replication: circular DNA • Polarized replication termination in E. coli: • Replication initiates bidirectionally from oriC. • The orientation of Ter sites insures that both replisomes arrive at the same site (between TerA and TerC)before termination can begin. • When the two opposing forks meet in a circular chromosome. Replication of the DNA separating the opposing forks generated catenanes, or interlinked circles.

  42. Type II topoisomerase (TopoIV) decatenate interlinked circles • In the absence of topoisomerases, steric forces would prevent gyrase from unwinding DNA as replication forks approached each other. • Topoisomerase allows the circles to decatenate. • It is not known whether decatenation occurs before or after the completion of replication. • Both possibilities are shown here.

  43. Termination: End-Replication problem of linear genomes • The problem of completing the 5 end in copying a linear DNA molecule: • Telomerase adds repeated short DNA segments to the ends of chromosomes.

  44. Strategies for replicating linear genomes: To overcome the end-replication problem associated with replicating linear genomes. Terminal redundancy: Recombination between redundant chromosome ends of phage T4 and T7 can generate concatemers. Cohesive ends of lambda phage allow it to convert to circular DNA upond infection. Terminal protein: Using a dCMP bound to protein serine hydroxyl as a primer (such as phage f29 or adenovirus). Telomere and telomerase: Telomeric DNA repeat sequences and extension of telomeric DNA by specialized reverse transcriptase.

  45. Terminal Proteins Enable Initiation at the Ends of Viral DNAs strand displacement – A mode of replication of some viruses in which a new DNA strand grows by displacing the previous (homologous) strand of the duplex.

  46. Telomeric repeat sequences

  47. Extension of telomeric DNA by telomerase: • The overall reaction: telomerase adds simple repeat sequences to the 3’ end of telomeric DNA, by the mechanism shown in part (b). • Addition of an RNA primer allows lagging-strand synthesis, followed by ligation and RNA removal. • Proposed action of telomerase: the RNA carried by the telomerase matches the 3’ DNA end, and allows its extension.

  48. Initiation of DNA Replication: A model for E. coli • A model for initiation of E. coli DNA replication at oric: • HU and IHF are double-strand DNA-binding proteins that facilitate DNA bending at the origin.

  49. Initiation of DNA Replication in yeast • Preparing a fission yeast replication origin for initiation: • Steeper steps in this process represent sites of regulation. • At each step, proteins that were bound in earlier steps are shown as fainter images.

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