DNA Replication, Telomeres, and Cancer

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DNA Replication, Telomeres, and Cancer
Kathy Friedman
Stevenson Center 2-417
322-5143
katherine.friedman_at_vanderbilt.edu
Reading C.W. Greider (1998) Telomeres and
senscence The history, the
experiment, the future. Current Biology 8,
178-181.
C.W. Greider (1999) Telomeres do D-loop-T-loop.
Cell 97, 419-422.
T.M. Bryan and T.R. Cech (1999) Telomerase and
the maintenance of chromosome ends. Cur. Opin.
in Cell Biol. 11, 318-224.
As in my last lecture, many of the notes will be
filled in during class.
Part I History of telomere biology.
Maintenance of chromosome ends by telomerase.
Part II The capping function of telomeres.
Emerging roles of telomerase in cell
immortality and cancer. Modeling
telomere loss in a transgenic mouse system.
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What Makes a Chromosome?
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First Discovery of Telomeres
The terminal gene must have a special function,
that of sealing the end of the chromosome, so
to speak, and that for some reason a
chromosome cannot persist indefinitely without
having its ends thus sealed. This gene may
accordingly be distinguished by a special term,
the telomere.
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Corroboration in Maize
If a chromosome, broken at the previous meiotic
anaphase, is delivered to the primary endosperm
nucleus, the breakage-fusion- bridge cycle will
continue in the successive nuclear divisions
during the development of the endosperm tissues.
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DNA end-replication problem
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5
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Is there something different about the chromosome
end?
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Use an organism with more ends!!
Ciliated protozoa
Tetrahymena thermophila
Micronucleus
Macronucleus
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Telomere sequence in ciliates
Blackburn and Gall (1978)
CCCCAA/GGGGTT
Tetrahymena
Oxytricha
CCCCAAAA/GGGGTTTT
(Oka et al. 1980 Klobutcher et al. 1981)
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Is this a common theme among organisms?
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Two models of telomere maintenance
1)
5
3
5
3
2)
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Distinguishing between the models
(Szostak and Blackburn 1982)
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Again, turn to the ciliates as a source of
Telomerase
J. Brown and J. Gaertig
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Telomerase activity detected in Tetrahymena
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RNA component was purified and sequenced
McCormick-Graham and Romero (1995)
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Model Telomerase is a Reverse Transcriptase
that uses an intrinsic RNA molecule to template
synthesis
Watch animation at http//faculty.plattsburgh.ed
u/donald.slish/Telomerase.html
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Now that the RNA is known, what about the protein?
Genetics
Biochemistry
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Purification of Euplotes telomerase
(Lingner and Cech 1996)
Avidin
Biotin
template
5
3
5
3
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Two proteins of 120 and 43 kDa copurify with
telomerase RNA
(Lingner et al, 1997)
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Genetics and Biochemistry meet again...
(from Nakamura et al., 1997)
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The catalytic aspartates are essential for
telomerase function
(Lingner et al, 1997)
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Telomerase activity can be reconstituted in
vitro from TERT and telomerase RNA
TERT
- TERT
TERT
Beattie et al. 1998
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There are some alternatives to telomerase...
Transposition
HeT-A
Recombination
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The capping function of telomeres
Recognition site for HO endonuclease
Sandell and Zakian (1993)
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How do telomeres evade recognition as broken DNA?
b
Oxytricha nova
a
3
Horvath et al., 1998
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What about human cells?
TRF1
TRF2
WT
NLS
Dimerization

DNA binding
TAG
Dimerization
NLS
TRF2-DN
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Cells expressing dominant-negative TRF2
green TRF2-DN
pink endogenous TRF2
van Steensel, Smogorzewska and de Lange, 1998
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Telomeric DNA is retained at the fusion site
van Steensel, Smogorzewska and de Lange, 1998
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TRF2 promotes the formation of telomere loops!
100-200 nt
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Griffith et al. 1999
Bound TRF2
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T loops also form in vivo
Griffith et al. 1999
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Model of T Loop Structure
Griffith et al. 1999
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The model is not resolved yet...
Pot1 (Protection of Telomeres) identified in
human cells
POT1
TRF2
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Telomerase and cell immortality
Hayflick noted that fibroblast cultures (skin
derived) have a finite capacity to divide.
HAYFLICK LIMIT
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Can cellular lifespan be increased by lengthening
telomeres?
TERT-
TERT
robust
senescing
Bodnar et al. 1998
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Telomerase plays a role in tumor formation
germ cells
somatic cells
Telomere length
Crisis
Senescence (Hayflick limit)
population doublings
from Bryan and Cech, 1999
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gt90 of human tumors have telomerase activity
The remaining 5-10 maintain telomeres by an
alternate mechanism
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Inhibition of telomerase can kill tumorigenic
cells
no DN-TERT
with DN-TERT
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Modeling telomerase dysfunction in the mouse
Create mice lacking telomerase RNA (mTR-)
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Telomeres progressively shorten as mTR- mice are
bred
telomere fluorescence
Blasco et al. (1997)
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Defects in late-generation mTR- mice
decreased testis size
WT intestine
mTR- intestine
Herrera et al. (1999)
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Mixed effects on tumor formation
Some tumor types INCREASE in late generation mTR-
mice
Some tumor types DECREASE in late generation mTR-
mice
Gonzalez-Suarez et al. (2000)
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Telomerase defects in humans? Dyskeratosis
congenita
Normal
Affected
Not sure
Vulliamy et al (2001) Sept. 27 Nature
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Effects of DKC remarkably similar to mTR- mice!
Bone marrow failure
Nail dystrophy
Abnormalities of the skin
Liver/gut disease
Age of onset seems to decrease with every
generation
Generation I
Diagnosed at 29, 40, 46, 48 years
Generation II
Generation III
Diagnosed at 17, 22 years
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The Double-Edged Sword
Telomeres
Genomic Stability
telomerase
- telomerase