Telomeres:

The strands of time

Jonathan Fay
BMCB 625
June 14, 2007

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

What is a telomere?
5-8 bp G-rich tandem repeats
Repetitive noncoding DNA

http://www.phoenixbiomolecular.com/regenerative_medicine.html

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/lecturesf04am/ReplicationFork.gif

How do they get there?

Telomerase

http://www.phoenixbiomolecular.com/regenerative_medicine.html

What do they do?

Genetic Clock
telomeres are shortened each time a cell divides

Why do you care?

Telomeres gone bad.

Cancer and Age

DePinho, The age of cancer. Nature. 2000 Nov 9;408(6809):248-54.

Genetic Clock

Annu Rev Cell Dev Biol. 2006;22:531-57.

Crisis

Genomic Instability

DePinho, The age of cancer. Nature. 2000 Nov 9;408(6809):248-54.

Genetic Clock

immortalization

Annu Rev Cell Dev Biol. 2006;22:531-57.

DePinho, The age of cancer. Nature. 2000 Nov 9;408(6809):248-54.

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

What to they do?

More than a genetic clock.
Protective cap
of telomere end protection
Protect chromosomesLoss
form:
leads to genome instability

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 Jun;18(3):247-53.

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

Structure: D-loop-T-loop

Cell Vol 97 419 199

G-quadruplex (G4)

G-Tetrad

The structure of telomeric DNA.

Curr Opin Struct Biol. 2003 Jun;13(3):275-83

http://www.nature.com/embor/journal/v7/n4/images/7400661-f1.jpg

The Cap that is a lot of stuff!

Annu Rev Cell Dev Biol. 2006;22:531-57.

Diverse telomere-capping
strategies

Cell biology. Telomere capping--one strand fits all.

Science. 2001 May 11;292(5519):1075-6.

Budding Yeast
Stn1 and Ten1 bind Cdc13

The structure of telomeric DNA.

Curr Opin Struct Biol. 2003 Jun;13(3):275-83

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 Jun;18(3):247-53.

OB fold

Bind oligonucleotides
or oligosaccarides
No significant amino
acid sequence
similarity between OB
fold proteins
Notorious for absence of
primary sequence features
that can be used to predict
the domain.

Notable sequence
conservation
OB-fold domain
of Rpa2
OB fold
Notorious for absence of
primary sequence

DNA
Binding
Domain

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

Does Stn1 bind DNA?

Domain Swap
Essential function of
RPA2
Restored by OB fold
of Stn1
Further evidence Nterm of Stn1
contains an OB fold
Perhaps an
evolutionary
relationship
between Stn1 and
Rpa2

Does Ten1 bind DNA?

Rpa2,Rpa3 weak telomeric binding

Stn1 and Ten1 form a subcomplex

Domain Swap

N-terminal domains of Stn1 and Rpa2 are

sufficient for viability

Summary
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??

3
2
1

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

Discussion Points
Reverse chimera Stn1-OBRPA2 didnt work
Ten1 OB fold? Rosetta?
Oligomerization domain
Cooperatively?

G4 binding
Affinity? Is it too much to ask?

THEND

Sup 1

SUP 2

Sup 3

Supplementary Figure 3. Comparison of the domain structure of subunits of the RPA

and Cdc13-Stn1-Ten1 complexes.
(a) Cdc13 and Rpa1 share a similar domain organization. Both proteins contain a
centrally located OB-fold DNA binding domain (indicated in black), which binds with
exceptionally 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 DNA
Supplemental Figure 3. (legend, continued).
bound to the Cdc13 DBD is similar to that observed with RPA, but very distinct from that
assumed by single-stranded DNA in complex with O. nova TEBP6. Furthermore, the Pot1
protein (which exhibits weak sequence similarity with the ! subunit of the O. nova TEBP
complex) 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 of
the 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 domain
of Cdc1311 (grey box), using a sensitive sequence profile comparison program. Finally,
the N-terminal regions of Cdc13 and Rpa1 each serve as protein-protein interaction
modules. Cdc13 interacts with the Est1 subunit of telomerase, mediated through a 15 kDa
domain located at aa 211 to 331 of Cdc1312,13. Rpa1 also has a well-characterized Nterminal
120 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 is
indicated by a black box, and the boundaries of the essential domain defined by the
experiment in Fig. 5c are bracketed.
(c) The smallest subunit of the RPA complex, Rpa3, is folded into a single OB-fold
domain15, indicated by a black box. Using the bioinformatics techniques that uncovered
similarities between Stn1 and Rpa2, we were not able to detect comparable sequence
identity between Rpa3 and Ten1, and therefore we cannot conclude whether Ten1
contains an OB-fold domain or not. This may be a reflection of the fact that both proteins
have diverged rapidly at the primary sequence level, as revealed by the alignments of
Rpa3 and Ten1 sequences from fungal genomes. Although the Rpa3 alignment shown
here, composed from a collection of fungal Rpa3 proteins, reveals a modest degree of
sequence conservation, we nevertheless were unable to place more distantly related Rpa3
proteins, such as the human homolog, on this alignment. The Ten1 protein family appears
to be even more divergent; for example, we were only able to identify the A. gossypii Ten1
sequence 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 fungal
genomes, further suggesting rapid sequence divergence within the Ten1 family.