Telomeres

Question 1

why have linear chromosomes

Answer 1

meiosis
big advantage for genetic diversity
>recombination
>independent assortment of chromosomes

crossover forms between non-sister homologues in meiosis I

circular chromosomes in meiosis:
1 CO:
>makes one big circle out of the two smaller circle homologues
>will produce random breaks in separation
>deletion/duplication on chromosomes, free DNA ends - problems

needs to have an even number of crossovers
but distinguishing if even or odd no. of chromosomes is mechanistically hard

easier to have linear chromosomes that can be fine with any number of crossovers as their ends are not connected

Question 2

natural ends vs breaks

Answer 2

intact circular chromosome - no free ends
so any free DNA ends in cell would be detected as DNA damage

linear chromosomes - natural ends
dont want to recognise these as DNA damage
should not repair these
and if there is a break in addition to these -need to distinguish the natural end telomeres and the break

telomeres dont activate DNA damage response
and are protected from the repair enzymes (keeps them stable)

Question 3

mixing up telomeres and broken DNA ends

Answer 3

eg
broken fork that doesnt replicate all the way to end
have one shorter sister as result - doesnt end in telomere sequences

correct repair:
>invade sister and use as template to correctly repair up to end - restores telomere at end and any missing genetic info

incorrect repair:
>fork doesnt reach end - broken end
>and a telomere is recognised as a DNA end for repair
>telomere ligated to broken end via NHEJ
>new technically dicentric chromosome structure (see dia)
>the two centromeres can be pulled to separate poles in anaphase
>creates random break
>these new broken ends can be attached to something random again
>BFB cycles
>big genetic instability

other wrong option:
>broken end from broken fork is treated as a telomere and acter upon by telomerase (can be active in cancers too)
>will create a terminal deletion that will never be repaired
>permanent loss of genetic info

Question 4

Telomere structural function

Answer 4

give the telomeres a v different sequence to any other location on the chromosome
usually repeats
>inverted repeats
>short tandem repeats

very end structure examples:
-hairpin loop
-5’ end protein covalently bound
-3’ ss overhang
-5’ ss overhang

evolve proteins to recognise this unique telomeric sequence and bind it (only at the telomeres)
TBPs
this keeps only the telomeres hidden from DNA repair machinery

Question 5

basic classes of Telomere binding proteins TBPs

Answer 5

bind only the telomeres due to their unique telomeric sequences
2 classes in almost every organism:

1. recognise telomeric DNA in double stranded form
2. recognise telomeric DNA in single stranded form

the telomeric proteins that directly bind DNA are essential as w/out them the DNA cannot be hidden at all from repair enzymes (even if the 2’ binding proteins are all present)

Question 6

budding yeast TBPs

Answer 6

3’ overhang at end

trimeric TBP complex on the ssDNA
-Ten1
-Stn1
-Cdc13

on the dsDNA before the 3’ overhang:
Rap1 binds DNA
Rif1 and 2 bind Rap1

Question 7

human TBPs

Answer 7

3’ overhang (present in basically all vertebrates and budding yeast)

homology for the ss 3’ overhang exists within the dsDNA earlier in sequence
get invasion of the overhang into the dsDNA
forms a T-loop
POT1 complex on ssDNA

shelterin complex on the dsDNA
unlike B yeast Rap1 doesnt bind the DNA directly
instead TRF2 dimer binds the DNA
-Tln2 dimer
-Trf1 dimer
forms shelterin hexamer
Rap1 binds TRF2

Question 8

Fission yeast TBP

Answer 8

3’ overhang too
like humans -Rap1 doesnt directly bind DNA
instead Taz1 does
Rif1 and Rap1 bind Taz1

like humans Pot1 binds ssDNA
no invasion/T-loop though

Question 9

Plants TBPs

Answer 9

Blunt end telomere
no overhang
Trf-Like protein dimers bind the dsDNA
no ssDNA binders as no ssDNA

Question 10

dsBreak checkpoint activation + repair

Answer 10

NHEJ
Ku + Lig4 act to ligate broken DNA ends back together

or HR
more complex/faithful
>nuclease digest 5’ ends
>gives ss 3’ overhangs
>RPA binds ss DNA (binds any ssDNA in cell)
>ssDNA bound RPA normally only exists v transiently during replication but then is replicated to dsDNA vfast
>in DNA damage - RPA bound longer - triggers DNA damage response and cell cycle arrest via Mec1 kinase

Question 11

Mec1 kinase DNa damage checkpoint activation

Answer 11

Ddc1-Mec3-Rad17 trimer ring
and Mec1 kinase

both localise to same place on damage via RPA
Rad9/Rad53 complex recruited and phosphorylated - triggers HR response and cell cycle arrest

v important that RPA does not bind telomeres to trigger this
or cell will get stuck in arrest forever

Question 12

budding yeast: telomere binding - TBPs vs RPA

Answer 12

TBP complexes (eg Ten1-Stn1-cdc13) have much higher affinity to the ssDNA at the telomere than RPA does
will always outcompete it
>inhibits RPA recruitment of Mec1/Ddc1-Mec3-Rad17
>inhibits triggering of repair/arrest

TBP complexes cannot bind ssDNA at breaks
RPA has no competition here
free to bind it

Question 13

Human TBP protection of telomeres

Answer 13

TBP hiding of the end prevents NHEJ machinery

shelterin on the dsDNA prevents 5’ resection to form 3’ ssOverhang

RPA outcompeted by Pot complex at the ssDNA

Trf2 - dsDNA binding component of shelterin
delete Trf2
- telomeres become ligated together via NHEJ
trains of chromosomes fused end to end
multiple centromeres on these
BFB cycles begin after next mitosis

Question 14

long v short telomeres

Answer 14

long telomeres have more sequence for more TBPs to bind
better protection

if telomere v short
not many TBPs bound
not v well protected
>TBPs bind dynamicallly (as any other non-covalent protein interaction)
>some protein coming off at any given time
>with more proteins present - some come off but many still present to protect
>with fewer - TBP comes off - less TBP left on as much shorter telomere - better chance for repair machinery to recognise the telomere and create new telomere-telomere fusions

shorter telomeres have higher change of repair machinery recognising them than longer telomeres
dont necessarily need all of telomere to be gone to lose protection

Question 15

short telomeres and BFB cycles

Answer 15

have two sisters w short telomeres
since v close together - 2 badly protected telomeres close together

repair machinery recognises telomere
needs something to fuse it to
sister v nearby
more likely fuses sister telomeres

anaphase:
dicentric stucture
sister chromatids attemptef to be pulled apart
anaphase bridge forms between poles
DNA decompacts and random dsBreak
-material lost on one chromosome
-duplicated on other one

these random ends are not telomeres
are sticky ends
can randomly fuse by repair machinery to another dsBreak/Telomere
creates another dicentric structure
BFB cycle

characteristic of cancer cells and generates
-deletions
-translocations
-duplications
-aneuploidy

Question 16

end replication problem

Answer 16

problem of complete DNA replication of a linear dsDNA molecule

leading strand replicated completely to end

lagging strand done via okazaki fragments
fragments can begin a bit further from exact end
but even if on end directly - there is an RNA primer which must be removed
-leaves a 3’ overhang as lagging strand 5’ doesnt reach end of template

this would cause shortening of chromosomes over time

but has to be mechanism against it because eukaryotes w linear chromosomes have existed for bnyrs

Question 17

Tetrahymena telomere model organism

Answer 17

replication and transcription done separately
(unlike other cells)

replicating DNA localised to micronucleus
replcated DNA cut into fragments
sent to macronucleus for transcription

these fragments capped w telomeres
1000s of genome fragments
as opposed to ~16 linear yeas chromosomes

so tetrahymena has v high proportion of telomeres/cell

Question 18

telomere-telomere recombination

Answer 18

thought to be solution to end replication problem

since all telomeres have the same T/G repeats
share homology
shorter telomere can invade longer one and replicates w it as template to restore telomere length

contested as believed to be an enzyme that does this

Question 19

discovering telomerase

Answer 19

take oligo of tetrahymena telomeric DNA sequence repeats TTGGGGTTGGGG…

high fraction of telomeres in tetrahymena means must have lots of telomerase

took diff oligos
TTGGGG
AACCCC (other strand)
yeast telomere
non telomeric seq
no oligo

incubate in cell extract with radiolabelled telomere sequences
separate in chromatography that makes longer fragments go further
products in the TTGGGG and yeast telomere experiments that had extended products
other oligos no extension seen

Question 20

Telomerase structure basic

Answer 20

reverse transcriptase
contains an RNA component that is used as template for template elongation

extends 1 repeat at a time
(6 nucleotides in vertebrates)
TTAGGG - human

telomerase associates with 3’ overhang at telomere
extends it using its RNA template
then dissociates and reassociates further along
can extend another repeat

Question 21

telomere functions

Answer 21

1/ protect chromosome ends from DNA repair activity (nuclease, ligase, recombinase)

2/ prevent DNA loss due to incomplete DNA replication problem
instead of losing useful DNA sequence from genome
lose telomeric sequence instead whrn lagging strand doesnt reach end
Telomere structure and telomerase co-evolved to ensure telomerase dependent telomere synthesis compensates for incomplete DNA replication

Question 22

catalytic core of telomerase

Answer 22

the RNA in the complex is folded in complex structure

different regions in it
-template
-hairpin
-pseudoknot - 3 strands annealed together

telomerase recognises the template and binds the RNA via the pseudoknot

template boundary exists to prevent telomere from replicating past the template and adding non-telomeric sequences (bad as TBPs cannot bind these)

not much sequence similarity between different organisms (ciliates vs verts vs saccharomyces)
but v close structural similarity
other sections bind diff proteins impirtant for structure

Question 23

Telomerase complex deletions phenotype

Answer 23

hTERT catalytic subunit in humans
Est2p in yeast (ever shorter telomeres mutant)

essential in yeast
delete one of 4 genes from complex (3 proteins 1 RNA)
yeast cells die in about 50 gens as telomeres become v Short and uncapped

DELAYED lethality
very telomere associated phenotype

if remove other essential cellular components
see problems arise straight away

delay is unique property of telomerase defects

Question 24

Telomere GC rich problems

Answer 24

2/3 nucleotides in the yeast telomeres is GC pair
telomeres enriched for GC base pairs

GC base pairs bind tighter thanAT
GC rich DNA harder to unbind

also forms a G-quartet structure
>4 strands in parallel or antiparallel
>from forming H-bonds with neighbouring Gs to form quartet (see dia) because 1 strand is G rich

when G rich strand is replicated as lagging strand (hence stays unwound as ssDNA)
can form the quartets
forms problems as need to be single stranded to replicate them
so makes it harder to replicate through telomeres

need to stop and start replication
and specialised helicases to unwind the quartets
(and the T-loop that is part of the usual telomere end)

Question 25

telomerase telomere preference

Answer 25

there is v little telomerase in cells
not all telomeres are replicated every cell cycle
shorter ones are preferred

shorter telomeres have less TBPs
TBPs act as inhibitors for telomerase recruitment
shorter telomere = fewer TBPs bound = less inhibition = more likely for telomerase to bind

unknown exactly how this inhibition works mechanistically
but know its the TBPs as can fake the telomere counts by tethering extra TBPs to non-telomeric seqs

Question 26

senescence activation

Answer 26

response to stresses
(shortening telomeres, oncogene activation, ROS…)
>p53 branch activates
>causes reversible G1 arrest

can either be reversed
OR
lead to apoptosis or senescence
senescence more common than apoptosis

if cell stays in reversible G1 arrest for some time - cant resolve problem stressing them
>trigger second p16 branch of pathway
>causes irreversible arrest in G1 - will never re-enter cell cycle

Question 27

SASP

Answer 27

senescence associated secretory phenotype
activated upon senescence entry

secrete cytokines + other signals
signals 2 things:
>signals for macrophages to eliminate senescent cell - senescence triggered by unresolvable problems that may lead to cancer
>proliferative signals to other nearby cells - so they can replace the eliminated cell

Question 28

advantage of senscence over apoptosis

Answer 28

cell gets ocogene activated
on pathway to cancer
>apoptosis:
-its progeny from before apoptosing still remain with the oncogene and can grow a tumour
>senescence:
-triggers immune cells to come in and clear out all problematic cells

tissue remodelling and embryonic development:
>apoptosis - leaves an unfilled gap
>senescence - when replacing some cells with others in the embryo - senescence allows time for the other cells to replace the removed ones

Question 29

senescence and aeging

Answer 29

younger:
-acute senescence
-cells cleared pretty quickly after entering senescence
hard to find senescent cells in these individuals

older:
-more senescent cells
-they accumulate
-either more being made or less being cleared by immune cells
-SASP of these cells is an inflammatory phenotype - can be harmful

Question 30

senescent cells and lifespan

Answer 30

clearing senescent cells prolongs lifespan

rewire mice genetically so that when p16 is triggered for senescence - apoptosis is also triggered
removes senescent cells

look at longevity
trigger the apoptisis in senescent cells w a drug
mice live longer across sexes

persistent cells signalling senescence shortens their lifespan
animals lived longer after removing them
senescent cell cleared mice also looked healthier at same age as control

Question 31

telomerase in larger animals

Answer 31

in humans and other large animals
telomerase is transcriptionally inactivated
catalytic subunit not expressed starting in early embryo

only germline and ESCs have fully active telomerase
adult stem cells have lowered activity - so cant fully maintain telomeres

so in non germline/ESC - telomeres erode over many divisions
this activates the senescence pathway

if mutate p16/p53
cells keep dividing
erosion continues into non-telomere DNA
-kills majority of cells
-or become cancerous (reactivate telomerase or activate ALT)

Question 32

telomerase ahploinsufficiency

Answer 32

remove an allele for a component of telomerase
halves the amount of functional telomerase in cells
telomeres shorten quicker

faster telomere shortening can cause issues in tissues that need to divide a lot
(bone marrow, skin, myeloproliferative cancer, early mortality)

telomerase amount v tightly regulated in cells
having too little cause issues
same w/ too much

Question 33

extra telomerase

Answer 33

there is a family known to have 2x telomerase than normal

causes early onset cancers in 20s

Question 34

telomere dynamics and cancer

Answer 34

abberant proliferation of cell
telomeres are shortened faster
so reach senescence

if too much telomerase
cancer more likely to progress too far before telomeres shorten enough for senescence to kick in
>downregulation of telomerase is an anti cancer mechanism
>as in larger animals:
-have more cells - more likely for 1 to escape proliferative control
-live longer - more probablity of cancer arising in lifetime
-makes sense to have this mechanism in large animals

Question 35

cancer restoration of telomerase

Answer 35

many cancers are p53-LOF
means they dont enter senescence and can continue to progress
but then telomeres shorten and most of the cells die

BUT some reactivate telomerase
and some go via ALT

Question 36

ALT

Answer 36

alternative lengthening of telomeres
different methods

-invasion of telomere in-cis
use same telomere as template for its own extension

-invasion of telomere in-trans - use another chromosomes telomere as a template

-cancer cells that use recombination for telomere maintenance can have circular telomeric DNA present - invade circle and rolling replication to endlessly extend telomere
>some yeast have circular telomeric DNA - can introduce other circular telomeric DNA and that seq ends up on yeast telomeres - proves its the circles that are used for maintenance

Question 37

telomerase immortalisation

Answer 37

can immortalise cell lines by transforming them with an actively expressed telomerase catalytic subunit

allows endless propagation of human cells in culture

Question 38

telomerase proliferative advantage

Answer 38

at certain telomere length
cells enter crisis and die

need either telomerase reactivation/transformation ot change to ALT (recombination) to immortalise

telomerase reactivation confers proliferative advantage only if pre-cancer cell has short telomeres
if telomeres are long - once proliferative block is removed - following TERT promoter mutation - doesnt yet give advantage as doesnt matter yet - telomeres fine
if telomeres are short - get the TERT mutation advantage straight away

Question 39

telomerase expression and cancer prognosis

Answer 39

high telomerase expression correlates with poorer survival in cancer patients

telomerase is important target for anticancer drugs
90% of cancers are telomerase+

Question 40

telomerase inhibition drugs

Answer 40

GRN163L oligo
competes with the telomeric ends for telomerase TNA template binding
-works well in vitro
-drug delivery and behaviour in situ is shakey

Question 41

other telomerase inhibition methods

Answer 41

use a mechanism that targets Telomerase+ cells for death:
>tert promoter driven suicide gene therapy
>block telomerase expression/biogenesis(assembly)
>TERT immunotherapy (have immune cells recognise TERT peptides presented on MCH class I)