BMS2002 - cell cycle Flashcards
what needs to happen to carry out a cell cycle
chromosomes duplicated
other organelles copied
cells grow
chromosomes segregated correctly
cell physically divides
G1 phase
Gap 1
- decides if conditions are right for full cycle
- growing, prep for DNA synthesis
S phase
synthesis
- replicating DNA and centrosomes
G2 phase
Gap 2
- decide if conditions are right for mitosis
M phase
chromosome segregation and cytokinesis
G0 phase
resting state - cells not in the cell cycle
- terminally differentiated cells, quiescent cells, senescent cells
CDKs
Cyclin Dependent Kinases
- master regulators
- activated by cyclin proteins (influence substrate specificity)
APC/C
ubiquitin ligase
- ubiquitylation of M- cyclin to tag them for degregation
CKIs
CDK inhibitory proteins
SCF
ubiquitin ligase
- signals degradation of CKIs to promote G1-S transition
G1-S checkpoint
- checks nutritional conditions
- is cell recieving proliferation signals?
- has DNA damage been repaired?
once passed, cell is committed to entire cell cycle
G2-M checkpoint
- has DNA damage been repaired?
- is DNA replication complete?
- is the cell big enough?
Metaphase-Anaphase checkpoint
spindle assembly checkpoint
- are chromosomes properly attached to spindle?
satisfied -> APC/C activated -> cyclin B degraded -> cells exit metaphase into anaphase
if checkpoint is not satisfied
cells withdraw from cycle
- senescence, allows cell to remain in tissue but not proliferate
apoptosis
- removes cell from organism
G1 CDKs and cyclins
CDK4 and 6
cyclin D
G1/S CDKs and cyclins
CDK2
cyclin E
S CDKs and cyclins
CDK2, CDK1 (CDC2)
cyclin A
G2/M CDKs and cyclins
CDK1 (CDC2)
cyclin B
4 ways cells control CDK-cylin kinase activity
- transcription
- CDK inhibitors - CKIs
- antagonized phsophorylation and dephosphorylation
- ubiquitin mediated proteolysis
early G1 genes that determine G1/S transition
Myc
- transcriptional factor
- can react to mitigens -> cyclin D
SCF ubiquitin ligase for protein proteolysis
where does DNA replication begin
at oriC
- recognised by ORC (origin recognition complex) for DNA unwinding
- multiple in euk
- can only be activated once per cycle to preserve genome integrity
formation of precipitation complex in vertebrates
geminin binds Cdt1 -> prevents loading MCM complex onto origin DNA
formation of precipitation complex in mitosis
APC/C degrades geminin -> ubiquitin proteolysis -> Cdt1 released -> ORC-CDC6-Cdt1 complex recruits MCM 2-7 complex on origin DNA
replication starts when..
CDK2-cyclinA phosporylates MCM2-7 -> forms CMG helicase with GINS complex and CDC45
Helicase in DNA rep initiation
breaks h-bonds -> unwinding -> DNA replication bubble and replication forks
SSBPs and RPA
Single stranded binding proteins
Replication protein A
- bind to unwound strands -> stabilises and prevents base pairing
type IIA topomerase
DNA relacation at the front of the replication fork in eukaryotes
gyrase
DNA relacation at the front of the replication fork in prokaryotes
RNA primase
during elongation:
uses ribose NTPs as a nucleotide source -> produce a short RNA primer -> provides attachment to original DNA template
DNA polymerase delta/III
adds dNTPs to 3’ end
- continuous synthesis on leading strand
- discontinuous synthesis on lagging strand -> okasaki fragments that need to be joined by DNA ligase
have endonuclease activity -> can proof read sequence and correct it
DNA sliding clamp
- scaffold, keeps polymerase attached
- processivity-promoting factor in DNA replication
telomeres
form T-loop in eukaryotes -> prevents genes from being deleted
- shortened with each division, division will stop once at a critical length
Hayflick limit
number of times a normal cell can divide before cell division stops
- doesnt apply to stem cells bc of telomerase activity in germ cells/ adult stem cells
DNA damaging agents
- DNA replication stress
- polyaromatic hydrocarbons
- UV light
- oxygen radicals
- ionizing radiation
- chemotherapeutic reagents
DNA damage types
- base mismatch, insertion, deletion
- single strand breaksm base oxidation
- DNA adducts oyramidine dimers
- intrastrand crosslinks
- double strand breaks
DNA repair mechanisms
MMR = mismatch repair
BER = base exclusion repair
NHEJ = non-homologous end joining
NER = nucleotide excision repair
HR = homologous recombination repair
consequenses of DNA damage
transient cell cycle arrests
apoptosis
cancer
aging
inborn disease
cells that never divide
mature muscle cells (e.g. cardiac muscle), nerve cells
cells arrested in G0 but can resume proliferation
skin fibroblasts, smooth muscle cells, blood vessel endothelial cells, epithelial cells in liver/pancreas/kidney/lung/prostate/breast
stem cells that need continuous cell renewal
blood cells, intestinal epithelial cells
6 sages of mitosis
Prophase
Prometaphase
Metaphase
Anaphase
Telophase
Cytokinesis
which cyclin drives entry into prophase
M-CDK - cyclinB-CDK1
- directly phosphorylates key substrate proteins
- regulates downstream mitotic kinases to phosphorylate additional substrates
prophase - kinteochore assembly
M-CDK (cyclin B- CDK1) and aurora B kinases recruit kintochores
kinetochore
microtubule binding site on chromosome, large macromolecular complex that assembles on the centromere
prophase - chromosomes condense
cohesin is removed
condensins I and II co-operate to condense the chromosomes
what happens during prophase?
- interphase chromosome structure is lost
- chromosomes condense
- kinetochore assembly begins
- microtubule dynamics change -> spindle starts to form
what happens during prometaphase
- nuclear envelope breaks down -> microtubules can access chromosomes
- spindle assembles
- microtubules attach to chromosomes
- MAPs and motor proteins become active -> chromosomes can be moved on the spindle
Microtubule structural properties
- nucleated at minus end
- dynamically unstable at plus end -> can grow and shrink from plus end
MAPs
Microtubule Adapter Proteins
- allow cell components to bind microtubules
- modulate stability of microtubules e.g. NDC80/Nuf2 at kinetochores
Spindle motor proteins
- allow cell components to move along microtubules
e.g. kinesin-5 (walks to plus end using ATP), Dynein (walks to minus end) - can carry various cargo proteins
mitotic spindle assembly
- nucleation of microtubules at centrosome (minus end)
- formation of interpolar microtubules -> sliding moves centrosome apart
- nuclear envelope breakdown -> microtubules can capture kinteochores
processes in chromosomes bi-orienting on the spindle
- making correct attatchment + error correction
- spindle checkpoint - check attachments are corrrect
- means of attachment changes once kinetochore reaches the plus end
- end-on attachments require Ndc90-Nuf2 complex
kinase Aurora B
detects bi-orientation
- localises to centromeres
- detects tension
- phosphorylates Ndc80 to remove microtubules from kinteochores
metaphase
- all chromosomes have bi-orientated + no unattached kinteochores remain
- spindle checkpoint stops singnalling -> APC/C activated
- M cyclin degraded -> exit from mitosis
- securin is degraded to separase -> separates sister chromatids
spindle checkpoint
chromosomes not properly attached -> error correction produces unattached kinetochores (detected by spindle)
unattached kinetochores -> MCC (mitotic checkpoint complex) -> inhibits APC/C -> prevents M-cyclin (CyclinB) degredation -> cells stay in mitosis
Anaphase A
chromosomes move towards spindle poles
- driven largely by microtubule/kinteochore shrinking at plus end
Anaphase B
spindle poles move apart
- driven largely by microtubule motors (E.g. kinesin-5, dynein)
telophase
- nuclear envelope refroms, nuclear pores inserted
- chromosomes decondense
condensins disassociate, cohesins re-associate
-> enables formation of chromome looping structures for gene expression
cytokinesis
- contractile ring (Actin and myosin) -> cleavage furrow formation -> pinching of the dividing cell into two
Cancer - when cell can’t exit the cycle
- cell keeps dividing even if rejected at checkpoints
- forced into the cell cycle
mutated protooncogenes
become oncogenes
-> mimic a stuck accelerator
-> excessive growth and division
2 tumour supressors
p53 - DNA damage response
Rb (retinoblastoma protein) - stop cell cycle entry (E2F transcription pathway)
CIN (chromosomal instabilities) in cancer
- generation of abnormal chromosome numbers (aneuploidy) due to chromosome mis-segregation
- chromosome rearrangements (translocations) due to abberant repair of DSB - fusion of wrong ends
Causes of aneuploidy in mitosis
- inappropriate kinetochore-MT attachments
- compromised SAC
- centrosome overduplication (cancer and microcephaly)
- problems with chromosome cohesion
- cytokinesis failure, cell fusion, endoreduplication (too many times through interphase) -> tetraploidy 4n
aneuploidy at a cellular level ->
substantial fitness loss
- impaired proliferation
- metabilic alterations
- defective stress response
Cancer’s mechanisms to tolerate aneuploidy
- lowering DNA damage response (p53)
- mistakes in cell division -> p53 activation -> increases cancer cell’s tolerance to insult inculding CIN
- increasing stress tolerance
- prolonged mitosis (SAC)
Aneuploidies that benefit tumorigenesis
- trisomy Ch12 -> increased proliferation and tumorigenesy of hESC
- in colorectal cancer: single trisomies -> advantage and increased tumorigenic behaviour upon stress
ABL
- tyrosine kinase that becomes over activates due to reciprocal translocation
ABL protooncogene activation
operates in the G1-S transition in response o mitogens
agents that promote DSB
DNA topomerase II poisons
Radiation
CIN
what causes chromosome translocations
arise from abberant DSB repair in interphase
what guides DSB misrepair
- sequence homology at chromosome breakpoint
- 3D chromosomal organisation in interphase
ABL kinase is activated by…
chromosome translocation
- releases SH3 SH2 internal inhibition
- becomes cytoplasmic
- promotes oligomerization and autophorphorylation
chromosome rearrangements ->
gene fusion -> hybrid/chimeric gene targeting transcription factors and tyrosine kinase
Rb
retinoblastoma protein - first tumour suppressor identified
- operates at G1-S transition
- loss of Rb function from mutation -> retinal tumours in children
- Rb inactivation -> predisposition to cancer
Retinoblastoma
rare childhood eye tumour that arises in precursors to photoreceptor cells
- Rb loss
- sporadic (no fam history): unilateral, no increased risk of other cancers
- familial: bilateral, high risk of other cancers
