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Part2. Basic Genetic Mechanisms

Part2. Basic Genetic Mechanisms. MB 207 – Molecular Cell Biology. DNA replication DNA repair Recombination. DNA REPLICATION MECHANISMS. Duplication DNA. Cell division. Template S strand. DNA double helix. 5’. 3’. 3’. 5’. Enzyme-catalyzed polymerization -DNA polymerase.

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Part2. Basic Genetic Mechanisms

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  1. Part2. Basic Genetic Mechanisms MB 207 – Molecular Cell Biology DNA replication DNA repair Recombination

  2. DNA REPLICATION MECHANISMS Duplication DNA Cell division Template S strand DNA double helix 5’ 3’ 3’ 5’ Enzyme-catalyzed polymerization -DNA polymerase

  3. DNA Replication is Semiconservative • Genetic material must be reproduced accurately • A DNA replication process produces two DNA molecules — identical to each other and identical to the original. Each strand of the original • (parent) molecule remained intact as it served as the template for • the synthesis of a new complementary strand. • In the first generation, half of each new molecule of DNA is old; half is new. First generation

  4. Three models of DNA replication

  5. Semiconservative DNA replication:suggested by Watson and Crick based on proof Messelson-Stahl experiment (3 generation of growth of E. coli) Density analysis Light Hybrid Heavy daughter duplex Parental duplex

  6. Replication of DNA is semiconservative Generation 2 is hybrid + light Generation 1 is hybrid density Parental DNA is heavy density Replicate in light density medium

  7. Mechanism of DNA replication:1) Initiation of DNA Replication (replication of origin, unwinding of duplex, replication fork)2) DNA polymerization (RNA primer, addition of deoxyribonucloside triphosphate (dNTPs), leading and lagging strand)3) Proofreading (DNA repair)4) DNA assembly

  8. DNA polymerase: the enzymes that make DNA • 3 DNA polymerase enzymes in E. coli: • DNA polymerase I: repair of damaged DNA, in a subsidiary role and in semiconservative replication • DNA polymerase II: implicated in repair • DNA polymerase III: a multisubunit protein, is the replicase responsible for de novo synthesis of new strands of DNA “DNA polymerase for mammalian??”

  9. Basic steps in DNA replication: • A portion of the double helix is unwound by helicases, forming a replication fork (asymmetric structure) • A molecule of a DNA polymerase binds to one strand of the DNA and begins moving along it in the 5' to 3' direction, using it as a template for assembling a continuous leading strand of nucleotides and forming a new double helix. • Because DNA synthesis can only occur 5' to 3', a molecule of a second type of DNA polymerase (epsilon, ε, in eukaryotes) binds to the other template strand as the double helix opens. This molecule must synthesize discontinuous segments of polynucleotides (Okazaki fragments). Another enzyme, • DNA ligase I then stitches these together into the lagging strand.

  10. Helicases: Helicases are often utilized to separate strands of a DNAdouble helix or a self-annealed RNA molecule using the energy from ATP or GTP hydrolysis. Okazaki fragment: 100-200 nucleotides long. To be polymerized only in the 5’ to 3’chain direction and to be joined together after their synthesis to create long DNA chains. Leading strand: The DNA daughter strand that is synthesized continuously Lagging strand: The daughter strand that is synthesized discontinuously.

  11. DNA synthesis involve addition of dNTPs • adds a deoxyribonucleoside triphosphate (dNTP) to the 3’ end of a polynucleotide chain (primer strand) • the template strand determines which of the 4 nucleotides (dATP, dCTP, dGTP or dTTP) will be added based on base-pairing • driven by large, favorable free-energy change caused by release of pyrophosphate and its subsequent hydrolysis to two inorganic phosphate

  12. The Replication Machine Mammalian Parental DNA helix Bacteria Mammalian DNA primase is a subunit DNA polymerase Primase + Helicase = primosome (bacteria)

  13. Initiation and Speed of DNA Replication Replication origin: The position at which the DNA helix is first opened contains DNA sequence that attract initiator protein and DNA stretches that are easily opened, i.e A-T rather than G-C • Prokaryotes • The single molecule of DNA that is the E. coli genome • contains 4.7 x 106 nucleotide pairs • DNA replication begins at a single replication origin • Rate: 500-1000 nucleotides/sec (completed in ~40 min) • Include a "proof-reading" function, only about one • incorrect nucleotide for every 109 nucleotides • inserted. In other words, more often than not, the • E. coli genome is copied without error! DNA replication of a bacterial genome

  14. DNA helix is 1st opened (Initiator proteins bind to double-stranded DNA and pry the 2 strand apart, breaking the H-bonds between the bases) A-T base pair is held together by fewer H-bonds. A-T pairs are easy to pull apart. The regions of DNA enriched in A-T pairs are found at replication origins

  15. Origin of replication in Eukaryotes • The average human chromosome contains 150 x 106 nucleotide pairs • which are copied at a rate of ~ 50 nucleotides / sec, much slower • because DNA is packaged tightly in chromatin. • The process occurs usually in one hour - contain multiple Origins of • replication per chromosome. • Replication origins tend to be activated in clusters, called replication units, • individual origins are spaced at interval of 30,000 – 300,000 nt apart • New replication units can be activated at different times during the S-phase of cell cycle and the DNA is completely replicated at the end of S-phase.

  16. Replication fork in Eukaryotes • Replication fork is formed in pairs and create a replication bubble as they move in opposite directions away from the origin • Nature of origin of replication: - Highly condensed chromatin replicates late, while genes in less condensed region replicate early - Defined by specific DNA sequence which contain the binding site for the initiator protein called Origin recognition complex (ORC) as well as several auxiliary binding sites for proteins An origin of replication in yeast

  17. A replication bubble formed by replication fork initiation

  18. Initiation of DNA Replication • Before a cell can divide, it must duplicate all its DNA. In eukaryotes, this occurs during S phase of the cell cycle. • Firstly, the DNA double helix is unwound, and the 2 strands are separated into individual templates. • Two proteins involved in this process: 1) DNA helicase is the enzyme that unwinds the DNA. At each replication fork, 2 helicases are needed (one for the leading and one for the lagging strand respectively). Hydrolysis of ATP provide energy for the prying process 2) Single-stranded DNA-binding protein (helix-destabilizing proteins) keep the separated strands from coming back together. Aid helicases by stabilising the single-stranded conformation DNA helicase ssDNA binding protein

  19. Single-stranded DNA One nick (a single strand cut) is made with a DNAse. A new strand (the leading strand) grows from the 3’ hydroxyl at the nick and displaces the other end of the nicked strand, where upon lagging strand synthesis can begin on the displaced end. Thus a growing fork with leading and lagging strand synthesis is created by rolling circle replication.

  20. DNA Polymerization RNA primer synthesis • DNA polymerase is needed to start DNA polymerization once a replication fork is established • However, DNA polymerase cannot start a new polynucleotide chain by joining 2 nucelosides triphosphate together • There are one primer for the leading strand and many primers for the lagging strand (~1 at intervals of 100-200 nts) • A special nucleotide-polymerizing enzyme (DNA primase) synthesizes short RNA primer (~10 nts) on the lagging strand. • DNA primase uses ribonucleoside triphosphate to synthesize short RNA primers using the opposite strand as a template in the 5’ to 3’ direction and stops: leaving the 3’ end of this primer available for the DNA polymerase

  21. RNA primer synthesis Reaction catalyzed by DNA primase, the enzyme that synthesizes the short RNA primers made on the lagging strand using DNA as a template. This enzyme can start a new polynucleotide chain by joining 2 nucleoside trophosphates together. The primase synthesizes a short polynucleotide in 5’ to 3’ direction and then stops, making the 3’ end of this primer available for DNA POLYMERASE

  22. DNA Polymerization – Leading and Lagging strand • DNA polymerase elongates the DNA based on the sequence of the template DNA to form Leading strand • At the Lagging strand, DNA polymerase forms many short Okazaki fragments • Each Okazaki fragment ends when DNA polymerase runs into the RNA primer attached to the 5’ end of a previous fragment • Special DNA repair system acts quickly to erase the old RNA primer and replace it with DNA • DNA ligase then joins the 3’ end of the new DNA fragment to the 5’ end of the previous one: use a molecule of ATP to activate the 5’ end at the nick before forming the new bond

  23. The reaction catalyzed by DNA ligase ATP hydrolysis To activate the 5’ end at the nick (step 1) before forming the new bond (step 2). Nick-sealing reaction Phosphodiester bond was sealed by DNA ligase

  24. DNA Polymerization Types of Polymerase

  25. DNA Polymerization-topologically linked by sliding clamp • Naturally, DNA polymerase will rapidly dissociate from the DNA template after it has synthesized a short fragment of DNA • Enables it to be recycled for synthesis of next Okazaki fragment • For synthesis of the long Leading strand, a protein that act as a regulated sliding clamp is there to hold a moving DNA polymerase on the DNA molecule and releases it when it runs into a dsDNA Regulated sliding clamp holds DNA polymerase on DNA

  26. Quality Control Explanation for the 5’-to-3’ direction of DNA growth • Three levels to ensure high fidelity: 1) Unique features of DNA polymerization: base pairing and 5’3’ polymerization 2) The 3’5’exonucleolytic proofreading 3) Strand-directed mismatch repair • Replication in the 5’  3’ direction allows efficient error correction

  27. Quality Control The 3’5’ exonucleolytic proofreading • DNA polymerase require a free 3’-OH end to add further nucleotide • Mismatched nucleotide at the 3’-OH end can not be used by DNA polymerase to add on new nucleotide • 3’5’ proofreading exonuclease activity (activity performed by DNA polymerase or by another protein) clips off any unpaired residues all the way to regenerate a new 3’-OH that can prime DNA synthesis Polymerizing and editing mode by DNA polymerase

  28. Exonucleolytic proofreading by DNA polymerase during DNA replication

  29. Strand-directed mismatch repair • Detects the potential for distortion in the DNA helix that results from the misfit (errors that were missed by proofreading exonuclease). Based on 3 steps: 1) Recognition of the mismatch (only on newly synthesized DNA strand).MutS – scans and binds to mismatched bp, sites that can be readily kinked - DNA methylation in prokaryotic (for old DNA),MutH - Nicked DNA in eukaryotic (for new DNA) 2) Excision of the segment of DNA containing the mismatch.MutL – scans nearby DNA for nick and degrades nicked strand) 3) Re-synthesize segment using the old strand as template kinked

  30. Winding problem during DNA replication Every 10 base pairs replicated at the fork correspond to 1 complete turn about the axis of the parental double helix, as the fork moves forward, a “winding problem” will be created. Problem solved by DNA topoisomerase I and II DNA topoisomerase I : prevents DNA from tangling during replication by 1) Producing a transient single-stranded nick 2) DNA helix on either side of the nick to rotate freely using the phosphodiester bond in the strand opposite the nick as a swivel point 3) Tension released 4) Energy retains from the cleaved phospho-diester bond is used to rejoin the DNA

  31. The reversible nicking reaction catalyzed by a eucaryotic DNA topoisomerase I enzyme

  32. Winding problem during DNA replication DNA Topoisomerase II Separate two inter-locked DNA circles. Use when two double helices cross over each other. 1) Break 1 double helix reversibly to create a DNA “gate” 2) Allow the second double helix to pass through this break 3) Re-seal the break 4) ATP hydrolysis to provide energy to drive these reactions

  33. The DNA –helix passing reaction catalyzed by DNA topoisomerase II

  34. Assembly of newly synthesized DNA to chromatin • New histone proteins are required to assembled the replicated DNA into chromatin and then chromosome • Chromatin assembly factors(CAFs) help to packaged the newly synthesized DNA • Both newly synthesized DNA helices inherit some old histone with some newly syntheized histone proteins The additional of new histones to DNA behind a replication fork

  35. The end of the synthesis • Prokaryotes have a circular DNA – no problem • Eukaryotes have a linear DNA – problem arise when the replication reach the end of the chromosome  no place to produce a short primer to start the last Okazaki fragment • Special arrangement: - Special nucleotide sequence at the end of chromosome (Telomeres) - Telomerase an enzyme that carries a fragment RNA with it - Extend the template DNA at the 3’ end based on the RNA template - Allow a new RNA primer to be attached to the extended portion and hence complete the lagging strand DNA synthesis

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