Yeast Has Defined Origins

S. cerevisiae ARS contains a conserved 11 bp ARS consensus sequence and multiple B elements

ARS directs autonomous replication of plasmid DNA

The ORC complex binds to the ARS during most of the cell cycle

The S. pombe origin is larger and binds ORC by a distinct mechanism

from Bell, Genes Dev. 16, 659 (2002)

Replication Origins in Metazoans

DNA replication initiates from distinct confined sites or extended initiation zones

The potential to initiate is modulated by sequence, supercoiling, transcription, or epigenetic modifications

from Aladjem, Nature Rev.Genet. 8, 588 (2007)

Initiation can influence initiation at an adjacent site

Some Features of Eukaryotic Replication Origins

from Méchali, Nature Rev.Mol.Cell.Biol. 11, 728 (2010)

Certain characteristics are common at metazoan replication origins but are not present at all origins

Different modules contribute to the selection of a given origin

Only a small subset of origins are active during a given cell cycle

Constitutive origins are used all the time and are relatively rare

Flexible origins are used to a different extent in different cells and follow the Jesuit Model “Many are called but few are chosen”

Inactive or dormant origins are only used during replication stress or during certain cellular programs

Different Classes of Replication Origins in Metazoans

from Méchali, Nature Rev.Mol.Cell.Biol. 11, 728 (2010)

Chromatin Structure Influences ORC Binding

from Méchali, Nature Rev.Mol.Cell.Biol. 11, 728 (2010)

Chromatin remodelling complexes can facilitate HAT binding

preRC proteins can be modified by HATs

Influence of Distal Elements on Initiation

from Aladjem, Nature Rev.Genet. 8, 588 (2007)

Deletion of DHFR promoter allows initiation to occur within the gene

Truncation of the DHFR gene confines initiation to the far end of the locus

Deletion of the -globin LCR prevents initiation within the locus

Deletion of the CNS1 sequence in the Th2 cluster do not initiate within the IL13 gene

The Formation of the preRC

Mcm2-7 is loaded as a double hexamer by ORC, Cdc6 and Cdt1

Sld3 and Cdc45 bind weakly to Mcm2-7

from Labib, Genes Dev. 24, 1208 (2010)

Mcm2-7 helicase is inactive until S phase

Origins Are Activated at Different Times

from Méchali, Nature Rev.Mol.Cell.Biol. 11, 728 (2010)

preRCs are formed during G1 on origins

Heterochromatic regions replicate later than euchromatic regions

The Replicative Helicase

Mcm2-7, Cdc45, and GINS (CMG complex) form the replicative helicase

from Moyer et al., Proc.Nat.Acad.Sci.USA 103, 10236 (2006)

Assembly of the Replicative Helicase

from Sheu and Stillman, Mol.Cell 24, 101 (2006)

preRC is formed during G1 by recruitment of Mcm2-7

Phosphorylation of MCM proteins by DDK recruits GINS and stabilizes Cdc45 association

from Remus and Diffley, Curr.Opin.Cell Biol. 21, 771 (2009)

Helicase Loading and Activation in DNA Replication

DnaA and ORC are structural homologs

Replication competence is conferred by Mcm2-7 loading and is prevented by inhibition of pre-RC proteins

CDKs prevent Mcm2-7 loading and are required for helicase activation

Activation of Helicase Requires Phosphorylation of Sld2 and Sld3

G1 CDKs allow Dbf4 to accumulate

DDK phosphorylates Mcm2-7 and promotes Cdc45 association

CDK phosphorylates Sld2 and Sld3 and promotes association with Dpb11

11-3-2 promotes helicase activationfrom Botchan, Nature 445, 272 (2007)

DDK phosphorylates Mcm proteins

CDK phosphorylates Sld2 and Sld3 to interact with Dpb11

GINS and Pol are recruitedto form the RPC (replisome progression complex)

Activation of the helicase allows priming by Pol

Pol extends the leading strand and Pol extends each Okazaki fragment

from Labib, Genes Dev. 24, 1208 (2010)

Initiation of Chromosome Replication

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

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 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 proteins accumulate at unprotected telomeres

Shelterin contains 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, 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 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 includeDirect telomerase inhibitionTelomerase immunotherapy

from Marnett and Plastaras, Trends Genet. 17, 214 (2001)

Endogenous DNA Damage

Biological Molecules are Labile

RNA is susceptible to hydrolysis

Reduction of ribose to deoxyribose gives DNA greater stability

N-glycosyl bond of DNA is more labile

DNA damage occurs from normal cellular operations and random interactions with the environment

Spontaneous Changes that Alter DNA Structure

from Alberts et al., Molecular Biology of the Cell, 4th ed., Fig 5-46

depurination

deamination

oxidation

Hydrolysis of the N-glycosyl Bond of DNA

Spontaneous depurination results in loss of 10,000 bases/cell/day

Causes formation of an AP site – not mutagenic

from Alberts et al., Molecular Biology of the Cell, 4th ed., Fig 5-47

from Alberts et al., Molecular Biology of the Cell, 4th ed., Fig 5-47

Cytosine is deaminated to uracil at a rate of 100-500/cell/day

Uracil is excised by uracil-DNA-glycosylase to form AP site

Deamination of Cytosine to Uracil

5-Methyl Cytosine Deamination is Highly Mutagenic

from Alberts et al., Molecular Biology of the Cell, 4th ed., Fig 5-52

Deamination of 5-methyl cytosine to T occurs rapidly- base pairs with A

5-me-C is a target for spontaneous mutations

Deamination of A and G Occur Less Frequently

A is deaminated to HX – base pairs with C

G is deaminated to X – base pairs with C

from Alberts et al., Molecular Biology of the Cell, 4th ed., Fig 5-52