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CDB > Cell Nucleus I > Flashcards

Cell Nucleus I Flashcards

1
Q

which cyclin complexes control dna replication

A

E-2 and A-2

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2
Q

what forms the pre-RC

A

OCCM

ORC, cdt1, cdc6, MCM

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3
Q

when is high CDK activity required during the cell cycle

A

for origin firing in S phase and for preventing pre-RC re-assembly in S and G2 (CDK
activities are low in G1)

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4
Q

describe the cdt1 and geminin mechanism to prevent origin refiring

A

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

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5
Q

what holds together sister chromatids

A

SMCs: structural maintenance of chromosomes proteins

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6
Q

what is sister chromatid cohesion useful for

A

homologous recombination and repair, if one chromatid becomes mutated.

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7
Q

describe the process of chromatin assembly during dna replication

A

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)

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8
Q

why are more origins licensed than actually fire during dna replication

A

dormant origins act as back up - in case fork stalls. they are either fired or actively removed by repl fork during replication.

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9
Q

describe the licensing factor model

A

LF binds unreplicated chromatin, required for firing then destroyed to prevent refiring.
= cdt1+mcm

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10
Q

which proteins are involved in nucleosome assembly

A

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.

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11
Q

describe what happens in chromatin remodelling

A

remodelling factors use atp to slide nucleosomes along the dna fibre.

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12
Q

why is chromatin remodelling required

A

means chromatin is dynamic and can react to needs of transcription, repair, replication, and binding factors are able to access specific sites on dna

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13
Q

when does the nucleus disassemble in eukaryotes

A

during the prophase of mitosis

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14
Q

describe the process of nuclear disassembly and re-assembly

A
  • 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.)
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15
Q

describe npc structure

A
  • 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
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16
Q

describe models of the gate structure of npc

A

virtual gate

selective phase

  • non-saturated hydrogel
  • saturated hydrogel
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17
Q

describe nucleoporins

A

proteins ining central gate of NPC, have FG repeats which are hydrophobic amino acids, form dynamic hydrogel through interactions

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18
Q

how can remodelling factors exchange histones

A

histone chaperones can exchange histone subunits or entire nucleosome cores.

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19
Q

what is required after nuclear envelope reassembly?

A

proteins and RNA molecules

must travel across the nuclear envelope per minute during interphase

20
Q

nuclear localisation signals

A

internal peptide motifs that cause nuclear import

lys and arg: +ve amino acids. similar to histone tails, post-transl modification could help regulation.

21
Q

2 steps of nuclear import

A

rapid binding of the cargo to the cytoplasmic side of
the nuclear pores, then a slower energy-dependent translocation through
pores

22
Q

2 key proteins for nuclear import

A 
importin
ran (small GTPase)
23
Q

how are importin and ran involved in nuclear import

A

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.

24
Q

how does importin B interact with the NPC

A

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.

25
Q

ran cycle

A
  • 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.
26
Q

how was the length of DNA per nucleosome determined experimentally

A

MNase digest - releases nucleosome beads (only cuts linker DNA)
dissociate histones with high salt, run on gel.

27
Q

how was it determined whether DNA was inside or outside the histone octamer?

A

DNase I digest - gave 10-12 bp fragments corresponding to helical turn, so DNA accessible and on outside.

28
Q

what mediates nucleosome assembly in vivo?

A

CAFs, histone chaperones

29
Q

what is H1 needed for?

A

to form higher order chromatin structures

30
Q

describe the hierarchical folding of chromatin

A
  • beads on a string
  • 30nm fibre: solenoid w H1 in centre
  • stages of looping attached to protein scaffold
  • most compact = metaphase chromosome
31
Q

how to identify MARs/ SARs

A

matrix/ scaffold associated regions

  • use restriction enzymes to digest loops
  • isolate DNA
  • sequence
32
Q

stages of HiC

A
  • 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
33
Q

3 features of YACs

A

ARS, CEN, TEL

34
Q

how were ARS identified in yeast

A
  • 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.
35
Q

ARS structure, how was this determined

A
  • 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

36
Q

ARS structure, how was this determined

A
  • 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

37
Q

ini-seq

A
  • DNA replication initiated for a few mins in cells synced in late G1, with modified nucleotides
  • fragment DNA, purify for mod (immunoprecipitate)
  • sequence
38
Q

where are higher eukaryotic origins commonly found and why?

A

actively transcribed regions of euchromatin, often overlap with TSS
so replication and transcription elongation complexes move in same direction and do not collide.

39
Q

assembly of the OCCM/ inactive replicative helicase

A
  • 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
40
Q

activation of the MCM helicase

A

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]

41
Q

pol a
pol delta
pol E

A

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

42
Q

RPA

A

single strand binding protein, stabilises unwound strands and recruits pol a

43
Q

PCNA

A

sliding clamp, binds pol delta/ E, fen 1 etc

eg binding of polymerase provides a strong motile association w/ DNA

44
Q

DNA synthesis: initiation and elongation of both strands

A
  • 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
45
Q

maturation of okazaki fragments: 2 step flap processing

A
  • 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
46
Q

topo I

topo II

A

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.

CDB (2 decks)

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