Yeast Has Defined Origins S. cerevisiae ARS contains a conserved 11 bp ARS consensus sequence and...
-
Upload
anya-mitchel -
Category
Documents
-
view
214 -
download
1
Transcript of Yeast Has Defined Origins S. cerevisiae ARS contains a conserved 11 bp ARS consensus sequence and...
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