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Yeast Has Defined Origins. ARS directs autonomous replication of plasmid DNA. S. cerevisiae ARS contains a conserved 11 bp ARS consensus sequence and multiple B elements. The ORC complex binds to the ARS during most of the cell cycle. The S. pombe origin is larger and

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Yeast Has Defined Origins

ARS directs autonomous

replication of plasmid DNA

S. cerevisiae ARS contains a

conserved 11 bp ARS consensus

sequence and multiple B elements

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

Initiation can influence

initiation at an adjacent site

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


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


Different Classes of Replication Origins in Metazoans

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

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


Chromatin Structure Influences ORC Binding

Chromatin remodelling complexes

can facilitate HAT binding

preRC proteins can be modified by HATs

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


Influence of Distal Elements on Initiation

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 b-globin LCR

prevents initiation within the locus

Deletion of the CNS1 sequence

in the Th2 cluster do not

initiate within the IL13 gene

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


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

Mcm2-7 helicase is inactive until S phase

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


Origins Are Activated at Different Times

preRCs are formed during G1 on origins

Heterochromatic regions replicate

later than euchromatic regions

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


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

preRC is formed during G1

by recruitment of Mcm2-7

Phosphorylation of MCM proteins

by DDK recruits GINS and

stabilizes Cdc45 association

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


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

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


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

from Botchan, Nature445, 272 (2007)

11-3-2 promotes helicase activation


Initiation of Chromosome Replication Sld3

DDK phosphorylates Mcm proteins

CDK phosphorylates Sld2 and

Sld3 to interact with Dpb11

GINS and Pol e are recruited

to form the RPC (replisome progression complex)

Activation of the helicase allows priming by Pol a

Pol e extends the leading strand and

Pol d extends each Okazaki fragment

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


Replication Origins are Licensed in Late M and G1 Sld3

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 Sld3

CDK activity prevents licensing in yeast

Geminin activation downregulates

Cdt1 in metazoans

from Blow and Dutta, Nature Rev.Mol.Cell Biol.6, 476 (2005)


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 Chromosomes

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 Chromosomes

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 Chromosomes

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)


Telomerase Extends the ss 3 Chromosomes’-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 Chromosomes

New Okazaki fragments are

synthesized using the extended template

from Alberts et al., Molecular Biology of the Cell, 4th ed. Fig 5-43


Shelterin Specifically Associates with Telomeres Chromosomes

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)


Telomere Termini Contain a 3 Chromosomes’-Overhang

A nuclease processes the 5’-end

POT1 controls the specificity of the 5’-end

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


Structure of Human Telomeres Chromosomes

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)


Telomerase Action is Restricted to a Subset of Ends Chromosomes

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)


Dysfunctional Telomeres Induce the DNA Damage Response Chromosomes

Shelterin contains 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)


Loss of Functional Telomeres Results in Genetic Instability Chromosomes

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)


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


Telomerase-based Cancer Therapy Division

Telomerase is widely expressed in cancers

80-90% of tumors are telomerase-positive

Strategies include

Direct telomerase inhibition

Telomerase immunotherapy


Endogenous DNA Damage Division

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


Biological Molecules are Labile Division

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 Division

deamination

oxidation

depurination

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


Hydrolysis of the N-glycosyl Bond of DNA Division

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

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

Causes formation of an AP site – not mutagenic


Deamination of Cytosine to Uracil Division

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


5-Methyl Cytosine Deamination is Highly Mutagenic Division

Deamination of 5-methyl

cytosine to T occurs rapidly

- base pairs with A

5-me-C is a target for

spontaneous mutations

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


Deamination of A and G Occur Less Frequently Division

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


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