GER Year2 TB1 > Week 2 > Flashcards

Week 2 Flashcards

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1
Q

How many copies of DNA must be made during eukaryotic replication?

A

ONLY one copy

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2
Q

What is the danger of incomplete replication for eukaryotes?

A

Makes chromosomes vulnerable to breaking or having the wrong amount of DNA

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3
Q

What is an origin?

A

Where DNA is unwound and the replisome is assembled

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4
Q

If a eukaryotic chromosome has multiple origins, how many will be fired per replication?

A

Only one origin will fire
Some are just passively copied

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5
Q

What stops a fired/passively copied origin refiring during S phase?

A

Controlled by CDKs

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6
Q

What does CDK stand for and what deos it do?

A

Cyclin-Dependent Kinases, are a family of enzymes that regulate the cell cycle and other cellular processes like transcription, DNA repair, and metabolism

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7
Q

How does CDK activity vary throughout cell cycle?

A

G1=none/low
S=increasing
G2=furtherincrease
M=peak, then starts to decrease

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8
Q

WHat are intitators called in eukaryotes?

A

ORC (origin replicstion complex)/ Cdc6

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9
Q

In g1 stage of cell cycle, what happens with ORC/cdc6?

A
  1. ORC is bound to cdc6
  2. ORC/cdc6 is bound to ds DNA
  3. ORC/cdc6 recruits MCM2-7/ Cdt1
  4. Cdc6 and Cdt1 fall off
  5. A second MCM2-7 is recruited by the first MCM2-7
  6. The two MCM2-7s are bidirectionally oreintated (face each other, MCM2-7 helicase pair)
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10
Q

As the cell enters S phase, what phosphorylation steps follow?

A
  1. Sld3 and Cdc45 are bound
  2. S-CDK and DDK are activated
  3. Sld2, Dpb11, GINS, Pol epsilon and MCM10 bind
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11
Q

What is Pol epsilon?

A

leading strand polymerase

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12
Q

After Pol epsilon etc. binds, what happens to create the replisome?

A
  1. conformatoional change occurs
  2. Helicases now bound to ssDNA
  3. Pol epsilon on each helicase are ready to start replication
  4. Pol alpha/primase bind to the leading strand
  5. Pol delta binds tp the lagging strand
    REPLISOME IS COMPLETE= REPLICATION BEGINS
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13
Q

How is replication finsihed in linear DNA?

A

-DNA polymerases need an RNA primer to synthesise DNA
-lagging strand synthesis can’t copy the ends of linear chromosomes as it would repeatedly leave out information if it copied
-to prevent this, chromosomes are protected by telomeres and the enzyme telomerase

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14
Q

What organism contains lots of telomeres?

A

Tetrahymena (nobel peace prize 2009)

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15
Q

What’s the difference between telomeres and telomerase?

A

Telomeres= repeated sequence at the end of the genomme that moves important information away from the disappearing end
Telomerase= enzyme that can synthesize the repeat

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16
Q

What’s the difference between TERT and TER?

A

TERT (Telomerase Reverse Transcriptase): The protein subunit, a specialized reverse transcriptase that synthesizes DNA.
TER (Telomerase RNA component / TERC): The RNA component that carries the template for the DNA sequence telomerase adds

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17
Q

Who discovered telomeres and how?

A

Muller and mcClintock
Came to the conclusion that chromsomes repair themslevesif broken, but ends won’t join

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18
Q

What is Shelterin?

A

The proteins that recognise telomere repeat sequences

They bind dsDNA, can blokc or activate telomeres
Shelterin stops broken DNA recognition, and the length of telomeres

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19
Q

Which proteins normally recognise broken DNA?

A

ATM or ATR kinases

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20
Q

What kind of cells have active telomerase vs silenced telomerase?

A

Stem/germ cells= active telomerase=telomere maintenance

Somatic cells= silenced telomerase= telomere shortening

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21
Q

What is the hayflick limit?

A

Due to telomere shortening
(divisions have reached max and telomere length has reahced lower limit)

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22
Q

How does loss of telomeres cause cell cycle arrest?

A

Loss of telomeres=shelterin cant bind
Then it’s recognised as a dsdNA break
casues senescence or apoptosis

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23
Q

What is teh telomere sequence in humans?

A

TTAGGG

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24
Q

What is disadvantage to over/under sufficient telomerase?

A

Over= dangerous when hijacked by cancer
Under=essential for stem cell renewal

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25
How do preoteins read DNA?
Through chemiacl interactions
26
How are long DNA sequences read by proteisn?
Multiple chemical interactioins are needed (DNA binding proteins shape/ folding is crucial)
27
WHat does CAP stand for?
Catabolite Activator protein a.k.a cyclic AMP receptor protein
28
What is CAP?
A transcription factor in bacteria The first activator protein to be understood 2 identical subunits (symmetrical)
29
What does CHIP stand for?
Chromatin Immunoprecipitation
30
WHat does CHIP seq do?
reveals the binding pattern of cyclic AMP receptor across the e.coli genome
31
How does a CHIP seq work?
1. use an antibody to bind to what you want 2. gather antibodies (magnets etc) 3. Now you can count how many CAP proteins were bound to the e.coli genome and their genomic position
32
Where will CAP proteins bind most tightly to?
regulatory sections on DNA (although there will be lots ofbackground noise as it's very promiscuous ie. low specificity)
33
Define affinity for a DNA binding protein?
How tightly bound the protein is to the DNA
34
What is Kd?
The dissosciation constant
35
What does a small Kd mean?
Tightly bound
36
What is the equation for Kd?
Kd= [P][D]/[PD]
37
What units is Kd?
Moles
38
Define specificity in terms of proteins binding to DNA
Ratio of how tightly a protein binds to a specific sequence compared to all other DNA sequences.
39
What would high specificity look like in terms of Kds for target sequence vs Kd for non specific sequence?
Kd for target sequence is much much smaller than Kd for non specific sequence
40
Why do histone proteins have low specificity?
Because they need to be able to bind and package all DNA. (however HIGH AFFINITY)
41
What are the other names for Bandshift assays?
Gel shift assay Electrophoretic mobility shift assay (EMSA)
42
Describe a Bandshift assay
Vertical assay With dye, radioactive or fluorescent label on teh DNA
43
What's something you might change in a bandshift assay to see how tightly the protein has bound?
Singular bases CHemical cofactors amino acids
44
What kind of gel would you use in a bandshift asssay?
Non denaturing acrylamide gel
45
IF you are putting DNA and a binding protein into a bandshift assay, what would the top band represent?
Top band= protein-DNA complex Bottom band=free unbound DNA
46
How tightly does CAP bind?
A tightly as you can get (1nm is 1 molecule per cell) The difference between specific and non specific KDs for CAP is about 10,000 fold
47
What is a base pairs chemical signature characterised by?
The pattern of functional groups exposed in the DNA grooves
48
WHat kind of interaction happens between a protein and DNA if they are close enough/ the right shape, even if there is little to no specificity?
V de W (weak) mainly in the major groove
49
WHat kind of interaction happens between a protein and DNA if the binding protein is positively charged?
Ionic (DNA is -vely charged)
50
WHat kind of interaction happens between a protein and DNA if there is a proton donor and acceptor present?
Hydrogen bonds ( depends on distance and directionality- 3 atoms must be in a straight line)
51
Between which bases are H bonds very common?
Arg and Gua
52
Which DNA groove gets more action?
Major groove is more accessible and contains more information
53
What are some other common H binding components?
Lysine, threonine, asparginine, glutamine,side chains protein backbone
54
How are proteins adapted to recognise rare sequences?
DNA must be held in a very specific way single amino acid sometimes reads more than one base at a time/ a tfunny angles
55
Why do alpha helices make for such good DNA binding proteins/
They fit really well in the major groove Side chains protrude from alpha helices at regular intervals, each a.a residue can interact wwith bases
56
Out of TATA and CAP, which protein-DNA binding compelx will cause DNA to be most deformeed?
TATA= DNA needs to be very deformed CAP= slightly deforemd DNA
57
Fill in the blank. DNA binding proteins use recurring _________ _________ to recognize DNA
structural motifs
58
List some common DNA binding motifs
Helix-turn-helix zinc finger leucine zipper helix-loop-helix winged helix
59
WHy are so many DNA binding proteins dimers?
Palindromic sequences are common regulatory sites Dimers double the recognition capacity+ increases specificity Increases stability and affinity
60
Direct vs indirect readout
Direct= amino acid side chains contact specific bases in the major groove Indirect= protein senses DNA shape, flexibilty or minor groove width
61
Sequence specific vs non specifiv binding
Sequence specific= recognize motifs, indirect or direct readout non specific= bind broadly to DNA backbone (package or process DNA)
62
What specific motif does CAP display?
2 x Helix-turn-helix motifs recognition and irrelevant helices R= makes specific H bonds in the major groove I=may make non specific bonds to backbone Interaction between helices maintains structure
63
What is CAPs target sequence?
22 bps (2x 11)
64
How does CAP interact with its target sequence?
Both strands of DNA Multiple interaction need to happen (including adjacent a.a.s) eg. Arginine at 180 interacts with G residue
65
How are the two helices facing in CAP?
Rotational symmetry (inverted repeats)
66
Describe T4 DNA ligase as a binding protein
Low specificity interactions Makes extensive sequence independent interactions with DNA Lysine, Arginine, Histidine are all to accomodate -ve DNA Large contact area to increase VdeW and ionic interactions
67
How long is the DNA binding motif in a zinc finger domain?
about 30 aa long
68
What 3 general sections make up a zinc finger domain?
Beta hairpin Alpha helix (contains residues that interact with bases) Zinc(structural role)
69
How many base pairs cna a zinc finger bind?
3
70
WHy will zinc fingers normally occur in tandem array?
allows reading of longer sequences
71
Which groove will a zinc finger bind to ?
major
72
How does a zinc finger wrap itself around DNA?
in a right handed helix
73
Why will zinc fingers normally be found connected to larger proteins/other domains?
So that the protein can bind to DNA and have a useful finction
74
WHat varies within binding protein's zinc fingers that allows different DNA seuences to bind?
a.a composition varies
75
What does TALE stand for?
Transcription Activator like Effector
76
How many repeats of TAL are there?
11
77
What do the 11 repeats of TAL look like when assembled?
spokes of a wheel
78
WHat does each TAl repeat consist of?
2 helicesand a linker (NOT A HTH MOTIF)
79
Where does the DNA contact TAL?
thru the hole... linkers stick into hole, linkers contact bases in the DNA
80
What direction does TAL go in?
Follows the major groove on the DNA ie right handed
81
Which parts of TAL are highly conserved and which parts are hypervaribale?
highly conserved= helices hypervaribale= Links
82
which amino acid residues specifically in the loops have functions?
12= structural, ensures 13 is correctly positioned 13= contacts the DNA bases
83
How many base pairs can a single TAL repeat recognise?
1 base pair
GER Year2 TB1
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