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DNA Replicates by a Semiconservative Mechanism

DNA Replicates by a Semiconservative Mechanism. Grow cells in 15 N and transfer to 14 N. Analyze DNA by equilibrium density gradient centrifugation. Presence of H-L DNA is indicative of semiconservative DNA replication. from Lodish et al ., Molecular Cell Biology, 6 th ed. Fig 4-29.

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DNA Replicates by a Semiconservative Mechanism

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  1. DNA Replicates by a Semiconservative Mechanism Grow cells in 15N and transfer to 14N Analyze DNA by equilibrium density gradient centrifugation Presence of H-L DNA is indicative of semiconservative DNA replication from Lodish et al., Molecular Cell Biology, 6th ed. Fig 4-29

  2. The 11th Commandment

  3. The Replicon Model Sequence elements determine where initiation initiates by interacting with trans-acting regulatory factors from Aladjem, Nature Rev.Genet.5, 588 (2007)

  4. Mechanics of DNA Replication in E. coli Leading strand is synthesized continuously and lagging strand is synthesized as Okazaki fragments The 5’ to 3’ exonuclease activity of Pol I removes the RNA primer and fills in the gap DNA ligase joins adjacent completed fragments from Lodish et al., Molecular Cell Biology, 4th ed. Fig 12-9

  5. Initiation of DNA Replication in E. coli DnaA binds to high affinity sites in oriB DnaA facilitates the melting of DNA-unwinding element DnaC loads DnaB helicase to single stranded regions DnaB helicase unwinds the DNA away from the origin from Mott and Berger, Nature Rev.Microbiol.5, 343 (2007)

  6. DnaB is an ATP-dependent Helicase DnaB unwinds DNA in the 5’-3’ direction DnaB uses ATP hydrolysis to separate the strands SSB proteins prevent the separated strands from reannealing from Lodish et al., Molecular Cell Biology, 4th ed. Fig 12-8

  7. RNA Primer Synthesis Does Not Require a 3’-OH Primase is recruited to ssDNA by a DnaB hexamer from Alberts et al., Molecular Biology of the Cell,4th ed., Fig 5-12

  8. Coordination of Leading and Lagging Strand Synthesis Two molecules of Pol III are bound at each growing fork and are held together by t The size of the DNA loop increases as lagging strand is synthesized Lagging strand polymerase is displaced when Okazaki fragment is completed and rebinds to synthesize the next Okazaki fragment from Lodish et al., Molecular Cell Biology, 4th ed. Fig 12-11

  9. Interruption of Leading Strand Synthesis by RNA Polymerase Most transcription units in bacteria are encoded by the leading strand Natural selection for co-directional collisions in the cell from Pomerantz and O’Donnell, Nature456, 762 (2008)

  10. Replisome Bypass of a Co-directional RNA Polymerase from Pomerantz and O’Donnell, Nature456, 762 (2008)

  11. Replisome Bypass of a Co-directional RNA Polymerase Replication fork recruits the 3’- terminus of the mRNA to continue leading-strand synthesis The leading strand is synthesized in a discontinuous fashion from Pomerantz and O’Donnell, Nature456, 762 (2008)

  12. Bidirectional Replication of SV40 DNA from a Single Origin from Lodish et al., Molecular Cell Biology, 6th ed. Fig 4-32

  13. Replication of SV40 DNA T antigen binds to origin and melts duplex and RPA binds to ss DNA Primase synthesizes RNA primer and Pol a extends the primer PCNA-Rfc-Pol d extend the primer from Lodish et al., Molecular Cell Biology, 6th ed. Fig 4-31

  14. Initiation of DNA Synthesis ORC serves as a platform for the assembly of the preRC CDKs phosphorylate MCM components to recruit additional proteins to form the preIC Initiation proteins are inactivated after the ori has initiated from Aladjem, Nature Rev.Microbiol.5, 588 (2007)

  15. Replication Origins in Eukaryotes DNA replication in metazoans initiate from distinct confined sites or extended initiation zones Selection of initiation regions occurs via restrictions by other metabolic processes that occur on chromatin from Gilbert, Science294, 96 (2001)

  16. Replication Origins are Licensed in Late M and G1 Origins are licensed by Mcm2-7 binding to form part of the pre-RC Mcm2-7 is displaced as DNA replication is initiated Licensing is turned off at late G1 by CDKs and/or geminin from Blow and Dutta, Nature Rev.Mol.Cell Biol.6, 476 (2005)

  17. Control of Licensing Differs in Yeasts and Metazoans CDK activity prevents licensing in yeast Geminin activation downregulates Cdt1 in metazoans from Blow and Dutta, Nature Rev.Mol.Cell Biol.6, 476 (2005)

  18. Telomeres are Specialized Structures at the Ends of Chromosomes Telomeres contain multiple copies of short repeated sequences and contain a 3’-G-rich overhang Telomeres are bound by proteins which protect the telomeric ends initiate heterochromatin formation and facilitate progression of the replication fork from Gilson and Geli, Nature Rev.Mol.Cell Biol. 8, 825 (2007)

  19. Functions of Telomeres Telomeres protect chromosome ends from being processed as a ds break End-protection relies on telomere-specific DNA conformation, chromatin organization and DNA binding proteins from Gilson and Geli, Nature Rev.Mol.Cell Biol. 8, 825 (2007)

  20. The End Replication Problem Leading strand is synthesized to the end of the chromosome Lagging strand utilizes RNA primers which are removed The lagging strand is shortened at each cell division from Lodish et al., Molecular Cell Biology, 6th ed. Fig 6-49

  21. Solutions to the End Replication Problem 3’-terminus is extended using the reverse transcriptase activity of telomerase Dipteran insects use retrotransposition with the 3’-end of the chromosome as a primer Kluyveromyces lactis uses a rolling circle mechanism in which the 3’-end is extended on an extrachromosomal template Telomerase-deficient yeast use a recombination- dependent replication pathway in which one telomere uses another telomere as a template Formation of T-loops using terminal repeats allow extension of invaded 3’-ends from de Lange, Nature Rev.Mol.Cell Biol. 5, 323 (2004)

  22. Telomerase Extends the ss 3’-Terminus Telomerase-associated RNA base pairs to 3’-end of lagging strand template Telomerase catalyzes reverse transcription to a specific site 3’-end of DNA dissociates and base pairs to a more 3’-region of telomerase RNA Successive reverse transcription, dissociation, and reannealing extends the 3’-end of lagging strand template New Okazaki fragments are synthesized using the extended template from Lodish et al., Molecular Cell Biology, 6th ed. Fig 6-49

  23. The Action of Telomerase Solves the Replication Problem New Okazaki fragments are synthesized using the extended template from Alberts et al., Molecular Biology of the Cell, 4th ed. Fig 5-43

  24. Shelterin Specifically Associates with Telomeres Shelterin subunits specifically recognize telomeric repeats Shelterin allows cells to distinguish telomeres from sites of DNA damage from de Lange, Genes Dev. 19, 2100 (2005)

  25. Telomere Termini Contain a 3’-Overhang A nuclease processes the 5’-end POT1 controls the specificity of the 5’-end from de Lange, Genes Dev. 19, 2100 (2005)

  26. Structure of Human Telomeres Telomeres consist of numerous short dsDNA repeats and a 3’-ssDNA overhang The G-tail is sequestered in the T-loop Shelterin is a protein complex that binds to telomeres TRF2 inhibits ATM-dependent DNA damage response Shelterin components block telomerase activity from O’Sullivan and Karlseder, Nature Rev.Mol.Cell Biol. 11, 171 (2010)

  27. Telomerase Action is Restricted to a Subset of Ends Telomere length is regulated by shelterin Increased levels of shelterin inhibits telomerase action Telomerase is inhibited by increased amounts of POT1 Elongation of shortened telomeres depends on the recruitment of the Est1 subunit of telomerase by Cdc13 end-binding protein from Bertuch and Lundblad, Curr.Opin.Cell Biol. 18, 247 (2006)

  28. Dysfunctional Telomeres Induce the DNA Damage Response Shelterin may contain an ATM inhibitor Telomere damage activates ATM DNA damage response proteins accumulate at unprotected telomeres ATM activates p53 and leads to cell cycle arrest or apoptosis from de Lange, Genes Dev. 19, 2100 (2005)

  29. Loss of Functional Telomeres Results in Genetic Instability Dysfunctional telomeres activate DSB repair by NHEJ Fused chromosomes result in chromatid break and genome instability from O’Sullivan and Karlseder, Nature Rev.Mol.Cell Biol. 11, 171 (2010)

  30. Loss of Telomeres Limits the Number of Rounds of Cell Division Stem cells and germ cells contain telomerase which maintains telomere size Somatic cells have low levels of telomerase and have shorter telomeres Loss of telomeres triggers chromosome instability or apoptosis Cancer cells contain telomerase and have longer telomeres from Lodish et al., Molecular Cell Biology, 6th ed. Fig 25-31

  31. Telomerase-based Cancer Therapy Telomerase is widely expressed in cancers 80-90% of tumors are telomerase-positive Strategies include Direct telomerase inhibition Telomerase immunotherapy

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