Telomeres:

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Telomeres
The strands of time
Jonathan Fay BMCB 625 June 14, 2007
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Background

• What is a telomere?
• Why do we have them?
• How do they get there?
• What do they do?
• Why do you care?
• Gao et al. Nat Struct Mol Biol.
• 2007 Mar 14(3)208-14

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What is a telomere?
What is a telomere?
What is a telomere?

• 5-8 bp G-rich tandem repeats
• Repetitive noncoding DNA

http//www.phoenixbiomolecular.com/regenerative_me
dicine.html
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Why do we have them?

• Replication problem
• Lagging strand synthesis
• Unable to replicate the 3 ends faithfully
• Loose chromosomal DNA

• Evolutionary development of telomere

http//www.uic.edu/classes/bios/bios100/lecturesf0
4am/ReplicationFork.gif
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How do they get there?

• Telomerase

http//www.phoenixbiomolecular.com/regenerative_me
dicine.html
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What do they do?Genetic Clock

• telomeres are shortened each time a cell divides

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• Limited capacity of the cell to replicate
• Telomere length serves as intresnsic biological
  clock at regulationg life span of the cell
• Hayflick limit maximal number of cell division
  that a cell can achieve in vitro
• When cells reach this limit they undergo
  morphological and biochemical changes that
  eventually lead to arrest of cell proliferation a
  processes called cell senescence

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Why do you care?

• Telomeres gone bad.

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Cancer and Age
DePinho, The age of cancer. Nature. 2000 Nov
9408(6809)248-54.
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• Cancer rises exponentially in the final decades
  of life
• Age-dependent escalation in caser risk
• Cumulative mutational load, increaed epigenetic
  gene silencing and telomere dysfunction
• Exposure to eDNA damaging agents
• Mutations from proof-reading and mismathc errors
  during DNA replication
• Mutator phenotype
• Inherent instability of turmor cell genomes

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Genetic Clock
Annu Rev Cell Dev Biol. 200622531-57.
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• Bypass cell-cycle arrest checkpoint pathway
• Tummor Supressor genes
• p53, also known as tumor protein 53 (TP53), is a
  transcription factor that regulates the cell
  cycle and hence functions as a tumor suppressor.
  I
• RB Retinoblastoma critical for cell cycle exit
  when dividing retinal progenitors differentiate
  into postmitotic transition cells.
• Continue to divide very short telomers
• No longer protect the ends
• Cells enter secondary proliferative block
• Called crisis
• Characterized by short telomeres, end0to0end
  fusion, anaphase bridges and apoptosis
• I.E rampant genomic instability wide spread
  cell death

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CrisisGenomic Instability
DePinho, The age of cancer. Nature. 2000 Nov
9408(6809)248-54.
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• Leads to chromosomal fragmentation and
  no-reciprocal tranlocation
• Spectral karyotype profeile of mouse turmor cells
  with function telomerase on left and
  disfunctional on right
• Widespeard changes in gene dosage

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Genetic Clock
immortalization
Annu Rev Cell Dev Biol. 200622531-57.
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• However 1 in 10 million chance of immortal cell
• Overcome barrier for senescence and the criis
  phase M2 (mortality stage normoally undergo
  apoptosis) have the ability to proliferate
  indefinitely
• Express telomerase escape for crisis requres
  telomerase maintenance functions
• Telomerase is expressed in 80-90 of all cancers
  analyzed, and it is lacking in most somatic
  tissue

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DePinho, The age of cancer. Nature. 2000 Nov
9408(6809)248-54.
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• Summary continual renewal and somatic mutations
  that disable checkpoint allow for unrestrained
  growth and telomere attrition
• Culminating in agenomic instability
• After reactivation of telomerase cancer cell
  opulation incurs additional mujtations essential
  for progression towards full transformation.
• Malignant Invasive tumors.
• Dysfunctional telomere-induced genomic
  instability
• model of epithelial carcinogenesis. Continuous
  epithelial
• turnover during ageing is thought to lead to
  telomere
• shortening. When coupled with somatic mutations
  inactivating
• retinoblastoma/INK4a/p53 checkpoints, the
  Hayflick limit
• (mortality stage 1 (M1) or replicative
  senescence) can be
• bypassed. Continuous proliferation beyond the
  Hayflick limit
• results in progressive telomere attrition and
  subsequent
• fusionbridgebreakage cycles in cells with
  dysfunctional
• telomeres. This process culminates in aneuploidy
  and complex

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Summary

• Replication Problem
• Evolutionary development of telomere
• Telomere 5-8 bp G-rich noncoding repetitive DNA
• Telomerase adds telomere to end of chromosome
• Telomere dysfunction can lead to cancer

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What to they do?

• More than a genetic clock.
• Protective cap
• Protect chromosomes form
• recombination, exonuclease degradation and
  end-to-end fusion
• Distinguish telomeres from DNA ds breaks
• That would hinder progression into G2 phase
• Inappropriate recombination events
• Prevent Oncogenesis

Curr Opin Cell Biol. 2006 Jun18(3)247-53.
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• NHEJ occuing between telomere ends
• Polycentric chromosomes
• Arrested growth and attempts by the cell to
  repair the ends

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What is the Cap?

• Nucleoprotein Complex
• Number of different proteins that bind to
  telomeres
• ssDNA dsDNA coat and protect the telomere
• Telomeric silencing
• Structure protects ends

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Genetic Clock
Medscape.com
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Structure D-loop-T-loop
Cell Vol 97 419 199
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G-quadruplex (G4)
G-Tetrad
The structure of telomeric DNA.Curr Opin Struct
Biol. 2003 Jun13(3)275-83
http//www.nature.com/embor/journal/v7/n4/images/7
400661-f1.jpg
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• Proteins bind G4 TEBP-a/b block telomerase
  activity removed by phosophorylation in S phase
  prevent assocation
• Unfold G4 in S-phase
• The G-quads inhibit tolemerase (active in
  stem,germ, and cancer cells).
• The would be a rare structure in the cell too.
• So the ideas is that drugs that mimic them or
  bind to them (stabalize the G-quartet) would
  inhibit tolemerase

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The Cap that is a lot of stuff!
Annu Rev Cell Dev Biol. 200622531-57.
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• Double strand break repair
• NHEJ and HR have been shown to play a role in
  survival and alternative telomere maintenance in
  cels that are delted for protiens involved in
  telomere maintenance/elongation and protection
• Removal of Trt1 in yeast leads to complete loss
  of telomeric DNA and viability
• Taz1 is the fission yeast ortholog of both TRF2
• Taz1 loss renders telomeres vulnerable to the two
  DSB repair pathways
• G1 NHEJ
• Past S phase (diploid) HR
• two modes of repair vary through the cell cycle
  by a factor of 10, with NHEJ being higher in G1
  and HR being higher in G2.

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Diverse telomere-capping strategies
Cell biology. Telomere capping--one strand fits
all.Science. 2001 May 11292(5519)1075-6.
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• Common them in telomere capping
• Signle-stranded telomeric DNA binding protein
• TRF telomeric repeat binding factor
• Loss of TRF2 leads to cycle cycle arrest and
  end-to-end ligation of telomeres
• Inhibition of TRf2 induces immediate activation
  of ATM/p53 DNA damage checkpoint pathway and cell
  cycle arrest
• Taz1 ortholog of TRF

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Budding Yeast Stn1 and Ten1 bind Cdc13
The structure of telomeric DNA.Curr Opin Struct
Biol. 2003 Jun13(3)275-83
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• RAP1 telomere dsDNA binding protein
• conditional allele of RAP1 and show that Rap1
  loss results in frequent fusions between
  telomeres
• Since the presence of Rap1 at telomeres has been
  conserved through evolution, the establishment of
  NHEJ suppression by Rap1 could be universal.
• Cdc13 recruits capping proteins STn1p and ten1 p
  removal of any of these three leads to degradtion
  of 5 crhosomsome ends and cycle cycle arrest

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Stn1 and Ten1 bind Cdc13

• Role of Cdc13
• Cell cycle arrest mutant
• Dual function protein
• Capping yeast telomeres
• Recruit telomerase
• Unknown biochemical function of stn1 and ten1

Curr Opin Cell Biol. 2006 Jun18(3)247-53.
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• Consensus structure prediction methods
• 3d-jury rmodels generated using ab initio folding
  simulations
• Beta barrel capped by and a-helics located
  between the third and forth strands
• Amino acid swqueces show no significat similarity
• Bind oligonucleotides or oligosaccarides

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Notable sequence conservation

• OB-fold domain of Rpa2
• OB fold
• Notorious for absence of primary sequence

DNA Binding Domain
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Notable sequence conservation

• OB-fold domain of Rpa2
• RPA (Replication protein A)
• Heterotrimer
• RPA2,RPA3, RPA1
• Core component of DNA replication repair and
  recombination
• Binds ssDNA stabilizing unwound DNA and
  facilitates assembly of the complex through
  protein protein interactions

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Does Stn1 bind DNA?
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Domain Swap

• Essential function of RPA2
• Restored by OB fold of Stn1
• Further evidence N-term of Stn1 contains an OB
  fold
• Perhaps an evolutionary relationship between Stn1
  and Rpa2

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Does Ten1 bind DNA?
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Rpa2,Rpa3 weak telomeric binding
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• Flag-rpa2 and rpa3-flag affinity purified from E.
  coli
• Tested for DNA binding
• Weak binding
• No random binding

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Stn1 and Ten1 form a subcomplex
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• Rpa2 and rpa3 stable subcomplex in vitro
• Stn1 and ten1 also interact as assesed by COIP
  and Yeast two hybrid assays
• Stoichiometry?
• FLAG-STn1 naked Ten1 rabbit reticulocyte lystate
  as S35 labled protiins
• Anti Flag IP
• Quanify radioactivity
• About 11

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Domain Swap

• N-terminal domains of Stn1 and Rpa2 are
  sufficient for viability

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Summary

• Cdc13, Stn1 and Ten1 form RPA-like complex that
  binds telomeres

• Cdc13, Stn1 and Ten1 form RPA-like complex that
  binds telomeres

Rpa 2 Rpa3 have specificity for telomeric
DNA Not just any ssDNA Rpa can localize to
chromosome ends Competition??

• Rpa2 and Stn1 have similar biochemical activity
• (Chimera)
• Ten1 is like Rpa3
• It forms a complex with Stn1 or Rpa2
• It is the smallest subunit of RPA(like) complex

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• Dimer of tetramers
• Oligmerize as tetramer
• Stn1 is the bridge

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Discussion Points

• Reverse chimera Stn1-OBRPA2 didnt work
• Ten1 OB fold? Rosetta?
• Oligomerization domain
• Cooperatively?
• G4 binding
• Affinity? Is it too much to ask?

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THEND
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Sup 1
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SUP 2
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Sup 3
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• Supplementary Figure 3. Comparison of the domain
  structure of subunits of the RPAand
  Cdc13-Stn1-Ten1 complexes.(a) Cdc13 and Rpa1
  share a similar domain organization. Both
  proteins contain acentrally located OB-fold DNA
  binding domain (indicated in black), which binds
  withexceptionally high affinity to
  single-stranded DNA substrates of similar size
  (10-11 nt)1-5.As noted by Wuttke and colleagues,
  the extended conformation of single-stranded
  DNASupplemental Figure 3. (legend,
  continued).bound to the Cdc13 DBD is similar to
  that observed with RPA, but very distinct from
  thatassumed by single-stranded DNA in complex
  with O. nova TEBP6. Furthermore, the Pot1protein
  (which exhibits weak sequence similarity with the
  ! subunit of the O. nova TEBPcomplex) has a
  different domain structure from that of Cdc13 and
  Rpa1 most notably,high affinity binding is
  mediated through two OB-folds located in the
  extreme N-terminus ofthe Pot1 protein7,8. Rpa1
  contains an additional OB-fold in its C-terminal
  region9,10(indicated by a grey box), and an
  OB-fold has also been detected in the C-terminal
  domainof Cdc1311 (grey box), using a sensitive
  sequence profile comparison program. Finally,the
  N-terminal regions of Cdc13 and Rpa1 each serve
  as protein-protein interactionmodules. Cdc13
  interacts with the Est1 subunit of telomerase,
  mediated through a 15 kDadomain located at aa
  211 to 331 of Cdc1312,13. Rpa1 also has a
  well-characterized Nterminal120 amino acid
  domain that interacts with several different
  protein complexes,including the p53 tumor
  suppressor protein14.(b) Both Stn1 and Rpa2
  contain a single OB-fold, in the N-terminal half
  of the protein,which performs essential roles in
  each protein. The OB-fold domain shown in Fig. 1
  isindicated by a black box, and the boundaries
  of the essential domain defined by theexperiment
  in Fig. 5c are bracketed.(c) The smallest
  subunit of the RPA complex, Rpa3, is folded into
  a single OB-folddomain15, indicated by a black
  box. Using the bioinformatics techniques that
  uncoveredsimilarities between Stn1 and Rpa2, we
  were not able to detect comparable
  sequenceidentity between Rpa3 and Ten1, and
  therefore we cannot conclude whether
  Ten1contains an OB-fold domain or not. This may
  be a reflection of the fact that both
  proteinshave diverged rapidly at the primary
  sequence level, as revealed by the alignments
  ofRpa3 and Ten1 sequences from fungal genomes.
  Although the Rpa3 alignment shownhere, composed
  from a collection of fungal Rpa3 proteins,
  reveals a modest degree ofsequence conservation,
  we nevertheless were unable to place more
  distantly related Rpa3proteins, such as the
  human homolog, on this alignment. The Ten1
  protein family appearsto be even more divergent
  for example, we were only able to identify the A.
  gossypii Ten1sequence based on its syntenic
  position in the genome, rather than by a BLAST
  search.We have so far been unable to recover
  additional Ten1 proteins, even from other
  fungalgenomes, further suggesting rapid sequence
  divergence within the Ten1 family.