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Molecular Biology - 3550 > 11.3 exam 2 slides > Flashcards

11.3 exam 2 slides Flashcards

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1
Q

What is the DNA Pol III core made up of?

A 
Heterotrimer
• 3 parts:
• α = polymerase activity
• ε = Proofreading (3’  5’ exonuclease activity)
 • θ = unknown
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2
Q

What is the DNA Pol III holoenzyme made up of?

A

The entire Pol III enzyme
• 3 cores
• 3 sliding clamps
• 1 clamp loader (ATP dependent)

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3
Q

What is the β clamp?

A
  • Allows for processive synthesis

* The Pol III core stays in the proximity of DNA

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4
Q

What is the clamp loader?

A 
• Loads and unloads β clamps to DNA
• ATP dependent activity
1. Opens β clamp
2. DNA binding of clamp loader
• ATP hydrolysis causes dissociation of clamp loader
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5
Q

What does DNA helicase do?

A

NTP dependent (usually ATP)
• 5’  3’
• (Opposite in eukaryotes!)
• Ring-shaped hexamers of DnaB

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6
Q

Does helicase improve in speed with the aid of Pol III?

A

Yes

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7
Q

What is the bps/s of helicase before and after Pol III?

A
  • Helicase without Pol III = 35 bps/s

* Helicase with Pol III = 700 bp/s

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8
Q

Primase in prokaryotes:

A
  • DnaG primase
  • RNA primer
  • Interacts with helicase
  • Primer length: 11 to 13 nt
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9
Q

Pol I and ligase in prokaryotes:

A 
Pol I
• Previous section slides
• Interacts with β sliding clamp
Ligase
• ATP dependent
• Repairs covalent bond
• Acts on 5’ DNA terminus
• Interacts with β sliding clamp
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10
Q

RNAseH function:

A
  • RNAseH can also assist in removing RNA

* Cannot remove the last nucleotide • Still needs the assistance of Pol I

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11
Q

Can RNAseH remove the last nucleotide?

A

No

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12
Q

What does SSBPs stand for?

A

single stranded binding proteins

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13
Q

What do SSBPs do?

A
  • Prevent hydrogen bonds from reforming in separated DNA
  • Prevent action of endonucleases from attacking opened DNA
  • Remove hairpin structures
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14
Q

What does the replisome do?

A
  • Replisome = Pol III holoenzyme + DnaB helicase + primase

* 700 bp/s

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15
Q

What does the trombone model explain?

A

Loop formation involves:

1) An RNA primer via primase
2) A clamp

Polymerization occurs until the start of a new Okazaki fragment.

Loops are traded between the 2 lagging strand polymerases

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16
Q

How many polymerases are involved in the trombone model?

A

3 polymerases

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17
Q

What is clamp recycling?

A
  • β clamps are left behind on each completed Okazaki fragment
  • DNA Pol III hops to the next β clamp
  • Okazaki fragments to β clamp ratio is 10:1  Clamps must be recycled!
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18
Q

Do clamps have to be recycled?

A

YES

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19
Q

EUKARYOTES

A

Eukaryotes after this

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20
Q

What is the MCM in eukaryotes?

A
  • Helicase is called MCM
  • MCM requires the assembly of multiple proteins to become an “active helicase”
  • All together, these proteins are called CMG
  • Hexamer
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21
Q

What is helicase called in eukaryotes?

A

MCM

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22
Q

What does MCM require to become an active helicase?

A

multiple proteins

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23
Q

All together what is the name of the multiple proteins that the MCM requires to become an active helicase?

A

CMG

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24
Q

What are Eukaryotic polymerases?

A
  • α = primase

* δ = chromosomal replication • ε = chromosomal replication

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25
Q

What is the CMG complex?

A 
Opens 3’  5’
• Opposite prokaryotes 
• Cdc45
• MCM (helicase) 
• GINS
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26
Q

What is DNA poly α?

A
  • Primers of 25-40 nt

* No 3’5’ exonucleases activity (no proof reading)

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27
Q

What polymerases complete chromosomal replication in eukaryotes?

A

Chromosomal replication is completed by Pol δ and Pol ε
• δ lagging
ε leading
• Both have DNA polymerase and 3’5’ exonuclease activity

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28
Q

What is the eukaryotic replisome called?

A

The RPC

29
Q

What does RPC stand for?

A

Replisome Progression Complex

30
Q

What does replication protein a or (RPA) act like in eukaryotes?

A

The SSBps previously discussed with prokaryotes

31
Q

clamp in eukaryotes:

A
  • Sliding clamp = PCNA
  • No sequence homology to β clamp • But nearly superimposable, cool!
  • Clamp loader = RFC (Replication Factor C)
32
Q

How are okazaki fragments removed in eukaryotes?

A
  • RNA primer is “lifted” by the DNA Pol δ

* RNA primer is degraded by FEN1 and Dna2 5’  3’ activity

33
Q

Where does replication originate?

A

At an origin of replication

34
Q

What is a replicon?

A

The total DNA replicated from 1 origin

35
Q

Generally in bacteria 1 _____ and 1 ______ = entire chromosome

A

origin ; replicon

36
Q

initiator protein in prokaryotes:

A

DnaA

37
Q

Origin must be inactivated to prevent second round of _____ in prokaryotes?

A

replication

38
Q

Initiation regulation in prokaryotes:

A
  1. HemimethylationatGATCattracts SeqA
  2. SeqA blocks DnaA
  3. DnaAhydrolyzesATPafter initiation
  4. Can bind DNA but unable to open it
  5. HdainsuresDNA-ATPhydrolysis
  6. WhenRNApolymeraseisabsent —the DNA doesn’t open as well
39
Q

Eukaryotic initiation:

A
  • Multiple origins of replication
  • Each origin fires once
  • Occurs in S phase
  • Discrete origins have been identified in yeast, but not easily in other organisms
40
Q

How many times do eukaryotic origins fire?

A

once

41
Q

in eukaryotes, if you weren’t marked in ____ you won’t fire in ____

A

G1 ; S

42
Q

When do prereplication complexes form in eukaryotes?

A

During S

43
Q

Describe Eukaryotic initiation:

A
  • G -phase 1
  • ORC binds
  • Licensing proteins attract the 2 MCM helicase • Cdt1
  • CDC6
  • Prereplication complex (preRC) = ORC + 2 x MCM + licensing proteins
  • S-phase
  • Kinases trigger replication through phosphorylation
44
Q

How is replication triggered in the eukaryotic S phase?

A

Kinases trigger replication through phosphorylation

45
Q

What does termination accomplish in bacteria?

A

prevent collisions of polymerases

46
Q

What does termination accomplish in eukaryotes?

A

how do you replicate the ends of linear chromosomes

47
Q

Describe E, coli termination sites:

A
  • 23 bp sequences = termination sites
  • 2 clusters in either direction
  • Tus proteins bind Ter sites
  • Tus-Ter complexes has polarity (2 directions)
  • Nonpermissive direction = blocks replication
  • Permissive = permits replication
48
Q

What does non permissive direction do?

A

blocks replication

49
Q

What does permissive direction do?

A

Permits replication

50
Q

Where do Tus proteins bind?

A

Ter sites

51
Q

E. coli Ter sites have how many clusters in either direction?

A

2

52
Q

Do tus-ter complexes have polarity?

A

Yes, 2 directions

53
Q

What are the 2 directions of tus-ter complexes?

A
  • non permissive (block replication)

- permissive (permits replication)

54
Q

Where are tus-ter complexes found?

A

E. coli

55
Q

T/F Replication forks are not allowed to travel past her sites oriented in the opposite direction

A

True

56
Q

What happens in equal fork speed in E. coli?

A

Replication forks meet in terminus region.

57
Q

What happens in unequal fork speed in E. coli?

A

Clockwise fork blocked by her cluster

58
Q

Describe E. Coli replication and transcription:

A
  • usually in the same direction
  • Prevents collision of DNA pol and RNA pol
  • Codirectional collisions do not cause impedence
59
Q

What is the end replication problem in linear chromosomes?

A
  • After removing the primers on the ends of linear chromosomes there is a 3’ overhang
  • That is, there is no free 3’ –OH group to finish the chromosome ends
  •  Telomeres shorten with every cell division
  • Shorten may encroach in genic regions
60
Q

What does telomerase do?

A
  • Telomerase reverse transcriptase = creates DNA complete from RNA
  • Telomerase RNA = RNA template
  • Telomerase = TERT-TR holozenzyme
61
Q

Even WITH telomerase, what will happen?

A

there is a still a 3’ overhang

62
Q

What is the telomere capped with?

A

Shelterin

63
Q

What happens to the overhang of the telomere?

A

It is looped back as a t-loop

64
Q

Shelterin and t-loop prevents ________

A
  • cell cycle arrest
  • telomeres from joining other chromosomes
  • overzealous telomerase activiity
65
Q

What do shorter telomeres correlate with?

A

More age related diseases

66
Q

Longer telomere are not necessarily correlated with longer life but ARE correlated with _____-

A

a healthier aging life

67
Q

Adding telomerase to mouse models does what?

A

Shorten lifespan in terms of increases cancer-related deaths

68
Q

Telomerase is extinguished during what?

A

Embryonic differentiation in most somatic cells—- remains active in few cell lines like germ cells

Molecular Biology - 3550 (8 decks)

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