Transcript DNA Replication

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”
from Méchali, Nature Rev.Mol.Cell.Biol. 11, 728 (2010)
Inactive or dormant origins are
only used during replication stress
or during certain cellular programs
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, Nature 445, 272 (2007)
11-3-2 promotes helicase activation
Initiation of Chromosome Replication
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
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
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
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
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 recombinationdependent 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’-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
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
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’-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
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
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
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
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
Telomerase is widely expressed in cancers
80-90% of tumors are telomerase-positive
Strategies include
Direct telomerase inhibition
Telomerase immunotherapy
Endogenous DNA Damage
from Marnett and Plastaras, Trends Genet. 17, 214 (2001)
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
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
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
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
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
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