Structure and Function of Telomerase

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Structure and Function of Telomerase

• By Peng Wang Yuzhen Wang
• May 11,2006

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Overview 1. Telomere and End Replication Problem
2. Function of Telomerase 3. General structure
of Telomerase 4. Telomerase Essential
N-terminal (TEN) Domain 5. Regulation of
Telomerase Activity
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Telomeres

• Telomeres are the structure at the end of linear
  eukaryotic chromosome.

Molecular Biology of the Cell, 4th edition
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Telomere Function
Telomeres function by protecting chromosome ends
from recombination, fusion to other chromosomes,
or degradation by nuclease. Allow cell to
differentiate between natural chromosome ends and
damaged DNA. Provide Mechanism for Replication
of Linear DNA Ends
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Telomere Function
Telomeres function by protecting chromosome ends
from recombination, fusion to other chromosomes,
or degradation by nuclease. Allow cell to
differentiate between natural chromosome ends and
damaged DNA. Provide Mechanism for Replication
of Linear DNA Ends
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Structure of Telomeres
Telomere are composed of tandem repeats of
GC-rich DNA sequence and telomere associated
protein. In human, the repeat is TTAGGG, up to
10,000bp in length. Telomeres have a 3 G-rich
overhang
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t-loop of telomere
Cell 1997, 97419-422
Molecular Biology of the Cell, 4th edition
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Replication Problem of End of Linear Chromosomes

Molecular Biology of the Cell, 4th edition
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http//users.rcn.com/jkimball.ma.ultranet/BiologyP
ages/T/Telomeres.htmlReplication_of_linear_chromo
somes_presents_a_special_problem.
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Telomerase
Telomerase is a specialized reverse
transcriptase, contains both RNA and Protein
Science 1997, 276 561-567
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Annu.Rev.Biochem. 2006, 75493-517
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Unique features of telomerase Two
components---- telomerase reverse
transcriptase(TERT) protein and telomerase
RNA Internal RNA serves as template for reverse
transcription by TERT Ability to translocate
along the DNA
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Structure and Evolution of TERT
Annu.Rev.Biochem. 2006, 75493-517
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RT domain of TERT
Mediate the addition of nucleotide.
Three Asp residues in RT motifs A and C of TERT
are important for catalysis of nucleotide
addition, two-metal catalysis mechanism. Sizable
insertion between motif A and B(IFD) invovle in
telomerase activity.
Science 1997, 276561-567
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C-terminal domain of TERT
Consistute the thumb domain of telomerase, Weak
consevation of sequences Multiple mutations in
this domain of both yeast and human Might be
involved in nucleotide addition and processivity
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Telomerase RNA
The template region Main TERT-binding
region Template boundary element(TBE) TRE,
Template recognition element Low affinity TERT
binding sites
Annu.Rev.Biochem 2006, 75493-517
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Crystal Structure of the Essential N-terminal of
the telomerase reverse transcriptase (TEN)
Nature structureal molecular biology 2006, 13
218-225

• Recombinant TEN (2-191 A.A.) from Tetrahymena ,
  with a HIS6 tag, was purified from E.coli and
  crystalized with 3 molecules in crystallographic
  asymmetric unit.
• Telomerase activity assay, DNA binding assay, RNA
  binding assay to compare the wild type, TEN
  deleted or TEN mutant TERT
• 3. A conceptual model

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Telomerase Essential N-terminal (TEN) Domain of
TERT from Tetrahymena
(a) Location of TEN within TERT (b)Stereo ribbon
diagram of the TEN domain a core ßsheet, 4
anti-parallel ßstrands, a ßhairpin and 7 ahelix,
2 long loops. (c) Topology diagram of the TEN
domain. H-bonds betweenß 2 and ß5, a3 and a6, a2
and a5 (d) Superposition of backbone atoms of the
three TEN molecules found in the crystallographic
asymmetric unit. Dashed lines, disordered
portions of the C termini of chains B and C.
Flexible? Need partener?
Nature structureal molecular biology 2006, 13
218-225
Recombinant TEN (2-191 A.A.), with a HIS6 tag,
was purified from E.coli and crystalized with 3
molecules in crystallographic asymmetric unit.
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Conservation of TEN domain structure
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In vitro telomerase activity of TEN mutants
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Interaction of TEN with telomeric iodinated
DNAphoto-cross-linking
IU 5-iodouridine substitutions far from the 3
end (a) Specific binding between purified TEN
domain protein and telomeric DNA specific to
telomeric DNA (b) w/wo RNA subunit of TERT or
TERT with the TEN deletion (equal mixture) TEN
and TR increase the binding (c) Site-specific
mutant of TERT TEN increase binding , mutation
decrease binding (d) Wild type/ mutant TERT mixed
with TEN deleted TERT Q168, F178, W187 (e)
Quantification of the amount of telomeric DNA
cross-linked to each mutant TEN domain (f)
Surface representation of TEN domain with the
three residues essential for telomeric DNA
cross-linking in red
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RNA-binding activity of the TEN domain (a) RNA
binding of TERT2-191 and TERT13-184 non-specific
RNA binding but require full length 2-191 (b)
Electrostatic surface potential of the TEN domain
, positively charged C-terminal (c) Binding of TR
to the TEN domain protects the N and C termini
for limited proteolysis by Lys-C. The Ends of
TEN are disordered in solution and subject to
Lys-C digestion.
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Conceptual model of contributions of the TEN
domain to the telomerase mechanism
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Regulation of Telomerase Activity 1.
Transcriptional level hTERT instead of TR is the
rate-limiting factor for assembly of an active
telomerase complex Myc/Mad, Estrogen and
androgen upregulate TERT expression Tumor
suppressor gene(WT1) downregulate TERT expression
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• 2. Postranslation
• Folding p23, Hsp90
• Dimerization believed to be more active
• Modification
• Phosphorylation by PKC, AKt, upregulate
  activity
• Ubiquitination by MKN1, a ubiquitin
  ligase, degradation
• Translocation cytoplasm to nucleus A.A. 1-15,
  326-620
• Direct protein binding Ku?, hEST1?, Kip?and
  PinX1?
• Telomere length Shortening of telomere activate
  telomerase

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Telomerase activity
Hsp90
Adrogen
AKt
PinX1
PKC
Estrogen
WT1
MKN1
Aging
Cancer