Cell Nucleus I Flashcards
which cyclin complexes control dna replication
E-2 and A-2
what forms the pre-RC
OCCM
ORC, cdt1, cdc6, MCM
when is high CDK activity required during the cell cycle
for origin firing in S phase and for preventing pre-RC re-assembly in S and G2 (CDK
activities are low in G1)
describe the cdt1 and geminin mechanism to prevent origin refiring
geminin binds and inactivates remaining cdt1 in S and G2 - preventing reassembly of new pre-RCs after origin firing. geminin is degraded during mitosis so cdt1 can assemble new pre-RCs in G1
what holds together sister chromatids
SMCs: structural maintenance of chromosomes proteins
what is sister chromatid cohesion useful for
homologous recombination and repair, if one chromatid becomes mutated.
describe the process of chromatin assembly during dna replication
before replication fork, chromatin partly disassembles. original nucleosomes are transferred to the daughter dna and new histones which were synthesised during s phase are assembled onto daughter dna by assembly factors. (CAFs)
why are more origins licensed than actually fire during dna replication
dormant origins act as back up - in case fork stalls. they are either fired or actively removed by repl fork during replication.
describe the licensing factor model
LF binds unreplicated chromatin, required for firing then destroyed to prevent refiring.
= cdt1+mcm
which proteins are involved in nucleosome assembly
Xenopus: N1 and nucleoplasmin
humans: CAF1 interacts w PCNA and targets new H3H4 to the replication fork.
old H3H4 tetramers remain together, when transferred to repl dna, they associate w new or old H2A H2B dimers. H1 associate later.
describe what happens in chromatin remodelling
remodelling factors use atp to slide nucleosomes along the dna fibre.
why is chromatin remodelling required
means chromatin is dynamic and can react to needs of transcription, repair, replication, and binding factors are able to access specific sites on dna
when does the nucleus disassemble in eukaryotes
during the prophase of mitosis
describe the process of nuclear disassembly and re-assembly
- nucleus disassembles in the prophase of mitosis
- protein phosphorylation (mostly by cyclin B-CDK1) result in nuclear envelope
breakdown - lamina depolymerises into soluble lamin A/C and membraneassociated lamin B.
- Nuclear pore complexes (NPCs) disassemble into soluble
nucleoporin subcomplexes - nuclear membranes fragment
chromatin condenses (til metaphase) and is now freely accessible in the cytoplasm - fall in kinase activity in anaphase, lamins and NPCs become dephosphorylated
- telophase - nuclei reassemble
(chromosome decondensation,
membrane assembly from vesicles, lamina polymerisation and NPC assembly from
soluble nucleoporin subcomplexes.)
describe npc structure
- ring of 8 subunits surrounds central pore
- fibrils from surfaces
nuclear size has extending basket/ gate
pore diameter ~60nm - this would not be selective. effective pore diameter ~9nm based on what is able to passively diffuse
describe models of the gate structure of npc
virtual gate
selective phase
- non-saturated hydrogel
- saturated hydrogel
describe nucleoporins
proteins ining central gate of NPC, have FG repeats which are hydrophobic amino acids, form dynamic hydrogel through interactions
how can remodelling factors exchange histones
histone chaperones can exchange histone subunits or entire nucleosome cores.
what is required after nuclear envelope reassembly?
proteins and RNA molecules
must travel across the nuclear envelope per minute during interphase
nuclear localisation signals
internal peptide motifs that cause nuclear import
lys and arg: +ve amino acids. similar to histone tails, post-transl modification could help regulation.
2 steps of nuclear import
rapid binding of the cargo to the cytoplasmic side of
the nuclear pores, then a slower energy-dependent translocation through
pores
2 key proteins for nuclear import
importin ran (small GTPase)
how are importin and ran involved in nuclear import
aB dimer of importin: a subunit binds protein nls, B binds to pore and enables translocation through. Nuclear ran-gtp causes importin to dissociate from the cargo’s NLS. B then a dissociate.
how does importin B interact with the NPC
hydrophobic patches on the B cause local melting of the hdrogel through interaction with FG repeats.
allows B to cross the hydrogel so is the mediator through the gate.
ran cycle
- ranGTP:nuclear
nuclear nucleotide exchange factor RCC1 (binds histones) promotes exchange of GDP for GTP. - ranGDP: cytosolic.
cytosolic GTPase Activating Protein (GAP) stimulates GTPase so GTP==> GDP.
The gradient of ranGTP/GDP stores energy which can be used to drive movement in the right direction.
how was the length of DNA per nucleosome determined experimentally
MNase digest - releases nucleosome beads (only cuts linker DNA)
dissociate histones with high salt, run on gel.
how was it determined whether DNA was inside or outside the histone octamer?
DNase I digest - gave 10-12 bp fragments corresponding to helical turn, so DNA accessible and on outside.
what mediates nucleosome assembly in vivo?
CAFs, histone chaperones
what is H1 needed for?
to form higher order chromatin structures
describe the hierarchical folding of chromatin
- beads on a string
- 30nm fibre: solenoid w H1 in centre
- stages of looping attached to protein scaffold
- most compact = metaphase chromosome
how to identify MARs/ SARs
matrix/ scaffold associated regions
- use restriction enzymes to digest loops
- isolate DNA
- sequence
stages of HiC
- crosslink DNA
- cut with restriction enzyme
- fill ends, mark with biotin
- ligate
- purify, shear DNA, pull down biotin
- sequence - see which sequences are in contact
3 features of YACs
ARS, CEN, TEL
how were ARS identified in yeast
- put random fragments from yeast plasmid into HIS marker plasmid
- introduce plasmid into his- cells on medium lacking his
- get a high transformant frequency if DNA fragment had ARS: plasmids able to replicate freely of host chromosome
- otherwise - get rare transformants due to integration into chromosome.
ARS structure, how was this determined
- essential consensus A box
- flanking B elements also affect efficiency of ARS function
- determine by point mutation and look at % origin function
Footprinting: A and Belements protected from digest by associated proteins.
A: ORC
B: eg cdc6, cdt1, MCM
ARS structure, how was this determined
- essential consensus A box
- flanking B elements also affect efficiency of ARS function
- determine by point mutation and look at % origin function
Footprinting: A and B elements protected from digest by associated proteins.
A: ORC
B: eg cdc6, cdt1, MCM
ini-seq
- DNA replication initiated for a few mins in cells synced in late G1, with modified nucleotides
- fragment DNA, purify for mod (immunoprecipitate)
- sequence
where are higher eukaryotic origins commonly found and why?
actively transcribed regions of euchromatin, often overlap with TSS
so replication and transcription elongation complexes move in same direction and do not collide.
assembly of the OCCM/ inactive replicative helicase
- ORC bound at origin
- recruit 1st MCM-cdt1, MCM gate opens so wraps around DNA
- ATP hydrolysis, displacement of help factors
- recruitment of 2nd MCM-cdt1
helicase = inactive
activation of the MCM helicase
in S phase:
- protein kinase activity from CDK, DDK
- association of cdc45, GINS
CGM complex
- local DNA strand unwinding, separation of MCM double hexamers so have 2 emerging replication forkss.
[CGM helicase = on lagging strand, 3’ to 5’, displaces complementary strand]
pol a
pol delta
pol E
a: primase, makes RNA primer and initial DNA extension for both strands. no proofreading activity - error prone
delta: lagging strand
E: continuous replication of leading strand
both these have proofreading 3’ to 5’ exonuclease activity
RPA
single strand binding protein, stabilises unwound strands and recruits pol a
PCNA
sliding clamp, binds pol delta/ E, fen 1 etc
eg binding of polymerase provides a strong motile association w/ DNA
DNA synthesis: initiation and elongation of both strands
- RPA binds single strand, recruits pol a
- pol a synthesises RNA primer and starts DNA synthesis
- pol a replaced by RF-C
- RF-C binds PCNA
maturation of okazaki fragments: 2 step flap processing
- PCNA binds polymerase delta
- processive strand synthesis, continues to displace RNA and pol a DNA of next okazaki fragment
- Dna2 shortens flap
- Fen-1 cleaves shortened flap, leaving only a nick which is sealed by DNA ligase
topo I
topo II
removal of superhelical stress
topo I: cuts single strand, rotate around free end, religates.
topo II: cut both strands, allow one section of DNA duplex to pass through other, eg for separation of interlocking DNA rings/ catenanes.
