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

The 11th 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, 4th 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, 4th ed. Fig 12-8

from Alberts et al., Molecular Biology of the Cell, 4th 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, 4th 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, 6th 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, 6th ed. Fig 4-31

Each cell undergoes 50,000 DNA damaging events per day

Humans possess 8-10 translesion synthesis polymerases which are error prone

The Human Genome Contains Multiple DNA Polymerases

from Loeb and Monnat, Nature Rev.Genet. 9, 594 (2008)

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 reatrictions 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, 6th 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, 6th ed. Fig 6-49

The Action of Telomerase Solves the Replication Problem

from Alberts et al., Molecular Biology of the Cell, 4th 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

Functional Telomeres Prevent Activation of the DNA Damage Response

from Azzalin and Lingner, Nature 448, 1001 (2007)

Low levels of TRF2 derepresses ATM induces senescence and permits NHEJ

Low levels of POT1 derepresses ATR

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

TRF2 recruits the MRE11 complex to promote strand invasion

Formation of the t-Loop

from de Lange, Genes Dev. 19, 2100 (2005)

TRF1 binds ds telomeric repeats

TRF1 contains DNA bending and looping activity

TIN2 enhances the architectural effects of TRF1

from Bertuch and Lundblad, Curr.Opin.Cell Biol. 18, 247 (2006)

Increased levels of shelterininhibits 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 protein accumulate at unprotected telomeres

Shelterin may contain an ATM inhibitor

T-loops are Similar to the Initiation of the RDR Pathway

Some telomere-associated proteins play a role in RDR

from de Lange, Nature Rev.Mol.Cell Biol. 5, 323 (2004)

Model for Evolution of Telomeres

The first linear chromosomes may have acquired terminal repeats

Terminal repeats may have been capped and maintained by t-loops

Telomerase may have evolved from a pre-existing retrotransposon RTase

T-loops are not used for telomere replication

from de Lange, Nature Rev.Mol.Cell Biol. 5, 323 (2004)

from Lodish et al., Molecular Cell Biology, 6th ed. Fig 25-31

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 telomeres 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 includeDirect telomerase inhibitionTelomerase immunotherapy