L8, Centromeres and Telomeres Flashcards
1
Q
How are centromeres arranged?
A
- Constricted region of heterochromatin in each eukaryotic chromosome -> includes spindle attachment site
- Each chromosome must have exactly one -> correct segregation without chromosome breakage
2
Q
What 3 key components are required for kinetochore assembly -> result?
A
- DNA sequences
- Specialised histone variants
- Specific proteins
- Kinetochore -> Centromeric Chromatin
3
Q
Give the 3 types of centromere with outline for each:
A
- Point: 1 microtubule attaches, defined by DNA sequence, recruits specific histones
- Regional: Not defined by specific sequences but repetitive region. Also recruit specific histones
- Dispersed/holocentric: Recruit specific histones
4
Q
Give the 3 types of centromere with example for each and common feature between them:
A
- Point: budding yeast
- Regional: Human, mouse, fission yeast
- Dispersed/holocentric: C.elegans/nematode
- Common feature is a replacement of histone H3 with centromere-specific histone variant CENP-A
5
Q
Centromeres in budding yeast:
A
- Point -> Contain short essential DNA sequences (‘CEN’)
- CEN elements = short, conserved (CDE-I, CDE-II flank A-T rich region CDE-II) -> ~120bp total
- The central region, CDE-II wraps around alternative nucleosome (CENP-A) -> flanking regions recruit proteins
6
Q
+ Where is CDE-III vulnerable to mutation?
A
- Single mutation converting CCG->CTG in CDE-III abolishes function
7
Q
Kinetochore assembly in budding yeast:
A
- CBF3 recruited to CDE-III (‘landing pad’)
- Cbf1 homodimer binds CDE-I
- Interaction of bound proteins across CDE-I, II, III with the Ctf19 complex
- Ctf19 links the centromeric complex to kinetochore
- (Ctf19 is one of many microtubule binding proteins involved)
- Serve as an assembly platform for kinetochore -> connecting to single microtubule
8
Q
Centromeres in S. pombe:
A
- Regional -> core sequence flanked by repetitive DNA (~35-110kb in f.yeast)
- core: cnt (wraps around multiple nucleosomes)
- cnt is flanked by imr repeats which contain tRNA gene clusters in f.yeast
- Both CENP-A and H3-containing nucleosomes map to the central domain, can provide landing sites for kinetochore assembly
- Central domain flanked by left and right outer repeats (termed otr -> ‘dg’ and ‘dh’ respectively
9
Q
Write out the order of repeat sequences in S.pombe centromeres:
A
<-dh, dg->imr->[cnt]<imr<-dg,dh->
- dg and dh make up the otr
10
Q
What state are regional centromeres in? Exception and relevance?
A
- Whole structure is specified as heterochromatin
- Lacking transcription, with the exception of inner and outer repeat regions where DNA is transcribed, producing dsRNA -> siRNAs -> heterochromatin
11
Q
Detail the process of regional centromere formation:
A
- Centromeric repeats transcribed by RNA PII
- dsRNA formed via convergent transcription and RNA-dependent RNA P
- siRNAs produced by Dicer
- siRNAs loaded onto RITS (contains Argonaute)
- RITS binds to nascent repeat transcripts -> recruits histone methyl transferase and RDRP -> more dsRNA
- H3K9 methylation recruits chromodomain proteins (HP1) -> heterochromatin spreading
12
Q
Centromeres in humans:
A
- Made up of long arrays of simple tandem repeats (HOR of alpha-I satellite DNA of 171bp repeats )
- Pericentromere: divergent repetitive sequences and retrotransposons (alpha-II satellite DNA)
13
Q
What do telomeres protect against?
A
- Prevent chromosome shortening and chromosome fusion -> unprotected end may be recognised as a DSB and ‘repaired’
- Critical to maintain genome stability
- Also useful in meiosis by facilitating pairing of homologous chromosomes
14
Q
How are telomeres structured?
A
- Telomeric repeat sequences; conserved within vertebrates and ciliates respectively
- G rich 5’ to 3’ towards telomere
- C rich 5’ to 3’ towards centromere
- 3’overhang -> facilitates t-loop formation, results from post-replicative processing of C-rich strand (removing RNA primers and iDNA)
- Variable telomere length in organisms
15
Q
How are t-loops formed:
A
- 3’overhand folds back around, pairs with section of other strand -> displacement loop
- t-loop: 5-10kb
- Catalysed by TRF2 enzyme
16
Q
How are telomeres extended?
A
- Telomerase (active in stem cells and cancer cells) -> ribonucleoprotein complex made up of reverse transcriptase (TERT) and an RNA template (TERC)
- Telomerase binds to parental strand overhang, extends 3’ end (away from chromosome)
- Lagging strand is completed by DNA pol in direction pointing back into chromosome
17
Q
What protein complex protects telomeres from damage-repair pathways and regulates their length?
List key components with role
A
- Shelterin complex
- Made up of 6 proteins…
- TRF1, 2 (repeat binding)
- Rap1 (repressor and activator)
- POT1 (protection of telomeres)
- TIN2, TPP1(bridging molecules)
18
Q
How does Shelterin work?
A
- Acts to inhibit telomerase by binding repeat sequences and assembling
- -> Telomerase inhibited
- TERRA signals for repair
19
Q
Why is it important for telomeres to be kept heterochromatic?
A
- They contain highly repetitive regions
- At risk of homologous recombination -> genome instability
- Telomeric DNA is thus sequestered by being formed into heterochromatin
20
Q
3 key marks in telomere heterochromatin:
A
- H3K9 methylation
- H4K20 methylation
- HP1 binding (chromo-domain containing ‘reader’)
- …Resembles that of pericentric chromatin
21
Q
Why can telomeric DNA not be methylated?
A
- It has no CpG sequence
- Conversely, sub-telomeric DNA is methylated -> degenerated repeats
22
Q
What is the function of TERRA RNAs?
A
- Promote heterochromatin formation, facilitate DNA replication and control telomere length
- Transcribed from the sub-telomeric sequences in a centromere-to -telomere orientation
- Hoogsteen base pairing -> both C-rich and G-rich strand
- Act as a scaffold molecule, promoting recruitment of proteins and enzymatic activities at telomeres; prevent binding of telomerase
23
Q
How are telomeres involved in cell senescence?
A
- Limit cell division cycles -> Hayflick limit
- Progressive shortening due to repressed telomerase; cell senescence eventually triggered to prevent DNA loss -> tumour suppression
- Cancer cells do not senesce; telomerase often reactivated
