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

The 11 th Commandment

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

Leading strand is synthesized continuously and lagging strand is synthesized as Okazaki fragments Mechanics of DNA Replication in E. coli 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, 4 th ed. Fig 12-9

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

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

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

Coordination of Leading and Lagging Strand Synthesis Two molecules of Pol III are bound at each growing fork and are held together by  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, 4 th ed. Fig 12-11

from Pomerantz and O’Donnell, Nature 456, 762 (2008) 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, Nature 456, 762 (2008) Replisome Bypass of a Co-directional RNA Polymerase

from Pomerantz and O’Donnell, Nature 456, 762 (2008) Replication fork recruits the 3’- terminus of the mRNA to continue leading-strand synthesis The leading strand is synthesized in a discontinuous fashion Replisome Bypass of a Co-directional RNA Polymerase

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

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

Initiation of DNA Synthesis from Aladjem, Nature Rev.Microbiol. 5, 588 (2007) 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

Replication Origins in Eukaryotes from Gilbert, Science 294, 96 (2001) 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 Blow and Dutta, Nature Rev.Mol.Cell Biol. 6, 476 (2005) 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) Control of Licensing Differs in Yeasts and Metazoans CDK activity prevents licensing in yeast Geminin activation downregulates Cdt1 in metazoans

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)

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)

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, 6 th ed. Fig 6-49

Solutions to the End Replication Problem from de Lange, Nature Rev.Mol.Cell Biol. 5, 323 (2004) 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

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, 6 th ed. Fig 6-49

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

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

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

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) Structure of Human Telomeres

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

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

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

from Lodish et al., Molecular Cell Biology, 6 th ed. Fig 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 Loss of Telomeres Limits the Number of Rounds of Cell Division

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