Telomeres Flashcards
why have linear chromosomes
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
natural ends vs breaks
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)
mixing up telomeres and broken DNA ends
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
Telomere structural function
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
basic classes of Telomere binding proteins TBPs
bind only the telomeres due to their unique telomeric sequences
2 classes in almost every organism:
- recognise telomeric DNA in double stranded form
- 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)
budding yeast TBPs
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
human TBPs
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
Fission yeast TBP
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
Plants TBPs
Blunt end telomere
no overhang
Trf-Like protein dimers bind the dsDNA
no ssDNA binders as no ssDNA
dsBreak checkpoint activation + repair
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
Mec1 kinase DNa damage checkpoint activation
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
budding yeast: telomere binding - TBPs vs RPA
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
Human TBP protection of telomeres
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
long v short telomeres
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
short telomeres and BFB cycles
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
end replication problem
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
Tetrahymena telomere model organism
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
telomere-telomere recombination
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
discovering telomerase
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
Telomerase structure basic
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
telomere functions
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
catalytic core of telomerase
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
Telomerase complex deletions phenotype
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
Telomere GC rich problems
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)
telomerase telomere preference
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
senescence activation
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
SASP
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
advantage of senscence over apoptosis
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
senescence and aeging
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
senescent cells and lifespan
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
telomerase in larger animals
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)
telomerase ahploinsufficiency
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
extra telomerase
there is a family known to have 2x telomerase than normal
causes early onset cancers in 20s
telomere dynamics and cancer
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
cancer restoration of telomerase
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
ALT
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
telomerase immortalisation
can immortalise cell lines by transforming them with an actively expressed telomerase catalytic subunit
allows endless propagation of human cells in culture
telomerase proliferative advantage
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
telomerase expression and cancer prognosis
high telomerase expression correlates with poorer survival in cancer patients
telomerase is important target for anticancer drugs
90% of cancers are telomerase+
telomerase inhibition drugs
GRN163L oligo
competes with the telomeric ends for telomerase TNA template binding
-works well in vitro
-drug delivery and behaviour in situ is shakey
other telomerase inhibition methods
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)
