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CONJ 662 - Genetic Mechanisms > Test1: Lect1 Maureen Hoatlin > Flashcards

Test1: Lect1 Maureen Hoatlin Flashcards

1
Q 
Components of cellular replicase are variations on a theme.
Name eukaryote and E. Coli for:
Polymerase:
Sliding clamp:
Clamp loader:
A 
E-coli:
Polymerase: Core alpha, epsilon, and theta
Sliding clamp: Beta clamp
Clamp loader: Gamma complex
Eukaryote:
Polymerase: Pol delta/epsilon
Sliding clamp: PCNA
Clamp loader: RFC (replication factor C)
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2
Q

Inability to replicate is lethal.

Define conditional lethals:

A

A conditional lethal is a mutant which can replicate under permissive conditions (E.G. survives at 25 degrees C but not 37 degrees C)

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3
Q

Quick Stop Mutants:

  • Define:
  • Why?
A

Quick Stop Mutants:
- Define:
Immediately stop replication under permissive conditions.
- Why?
Mutated protein is likely a component of the replication apparatus

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4
Q

Slow Stop Mutants:

  • Define:
  • Why?
A

Slow Stop Mutants:
- Define:
Stop replication after they finish their replication cycle.
- Why?
The defect lies in a protein which leads up to the initiation of replication, but is not needed for replication

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5
Q

In vitro complementation:

A

in vitro replication system prepared from temperature sensitive mutant ->
different wild-type protein extracts are added until function is restored ->
proteins purified by fractionation and identified.

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6
Q

What are the two types of DNA replication?

A

Semi-conservative replication

DNA synthesis in DNA repair

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7
Q

In vitro replication system

A

a system where all the necessary components for replication are thrown in a ‘test tube’ or some other container and DNA replicates there. This is outside of a cell.

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8
Q

Semi-conservative replication:

A

Is the form we use.

Strands are split, both strands are replicated. Each DNA duplex has one completely new and one old DNA strand.

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9
Q

dnaE:

A

Gene encoding the subunit of the DNA polymerase III complex which gives 5’ -> 3’ catalytic activity to DNA polymerase 3

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10
Q

dnaQ:

- Other name for it:

A

Gene encoding the epsilon subunit of the DNA polymerase III complex which gives 3’ -> 5’ exonuclease activity to DNA polymerase 3
- Other name for it:
MutD

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11
Q

Which polymerase is responsible for the bulk of DNA synthesis?

A

Pol III

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12
Q

What is the shape of DNA polymerase III?
- How many families of the polymerase?
- What varies and what
stays the same?

A

In description, take right hand hand, palm towards you, cup hand leave thumb loose. (see image on slide 22, first lecture)

  • five families
  • Palm stays the same, fingers and thumb vary
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13
Q

Hi Fi stands for:

A

High fidelity

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14
Q

How is Hi-Fi replication maintained?

A
  • correct nucleotide has higher fidelity for active polymerase (more likely to come in)
  • An incorrect nucleotide takes longer to bind, giving it more time to diffuse out
  • If the incorrect nucleotide is added, its width will be incorrect. The ‘finger and thumb’ of the polymerase feel this incorrect width, and shift it to the exonuclease site.
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15
Q

Polymerase from T7 Bacteriophage steps:

A

Template strand takes a sharp turn to expose itself to incoming nucleotide
->
O helix grasps incoming nucleotide, creates a catalytic site by rotation of finger and thumb
->
(Reversed when nuc is incorporated)
->
Nuc moves out of site

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16
Q

How exactly is a mispair identified?

A

Fingers (with O-helix) cannot rotate towards palm to grab nucleotide. Leaves 3’ free to bind nuclease domain

17
Q

What does AZT stand for?

  • What is it?
  • Specificity?
  • Downside?
A

Azidodeoxythymidine
- What is it?
A thymidine analog which acts as a reverse transcriptase inhibitor
- Specificity?
100x to 300x affinity for rt vs DNA pol
- Downside?
Mitochondrial Pol is affected by side effects

18
Q

DNA primase:

  • Function:
  • Gene name:
A
  • Function:
    Adds RNA primer 5’ to 3’
  • Gene name:
    dnaG
19
Q

DNA helicase:

  • Function:
  • Gene name:
  • Are there multiple varieties?
A
  • Function:
    1: Expends ATP to open strands
    2: attaches lagging strand (at least dnaB does)
  • Gene name:
    dnaB (encircles lagging)
  • Are there multiple varieties?
    Yes
20
Q

DNA helicase:

  • Structure:
  • How does it work?
A
  • Structure:
    six identical subunits together in a hexamer
  • How does it work?
    The motion below propels helicase forward
    Subunits, in sync with their opposite pair, Binding ATP ->
    hydrolysizing ATP to ADP (move in bind to strand) ->
    Release ADP, release strand.
21
Q

Single Stranded Binding (SSB) Proteins:

  • Function?
  • Quick stop or slow stop?
A
  • Function?
    1: Stabilize ssDNA during replication so it cannot form hairpins
    2: Does not interfere with base pairing, just falls off when strands anneal
  • Quick stop or slow stop?
    Quick stop
22
Q

Are the leading and lagging strands synthesized by the same proteins?

  • How common are exceptions?
  • Name one?
A 
No, different enzymes may be required.
- How common are exceptions?
Yes, some proteins will work on both strands.
- Name one?
1: dnaE

(same enzymatic subunit) used on both strands
2: Note: not all organisms use the same sub unit

23
Q

E-Coli replicase (DNA polymerase III):

- Structure:

A
  • Structure:
    1: Dimeric structure
    2: each monomer has a catalytic core, a dimerization subunit and a processivity (stays on the DNA) component
    3: One catalytic core is associated with each strand
24
Q

Holoenzyme:

A

a biochemically active compound formed by the combination of an enzyme with a coenzyme.

25
Q

Dimeric structure:

A

A structure with two identical or very similar proteins which link together

26
Q

What is the processivity subunit of DNA polymerase III:

A

Specifically: beta dimer
Generally: it is a sliding clamp

27
Q

What places the sliding clamp onto the DNA strand?

A

A clamp loader (the gamma complex)

28
Q

What are the steps for beta dimer (a clamp) to bind to DNA/early initiation?

A

A beta dimer (clamp) and gamma complex (clamp loader) recognize a primer-template
->
preinitiation complex formed
->
Beta su transfered to primed template, clamp formed
->
Binding to DNA causes beta su to change and favor binding with core pol, and core is brought to DNA
->
Tau dimer binds to core, and provides dimerization with second core.

29
Q

What is the human form of beta clamp (Clamp protein)?

A

PCNA (proliferating cell nuclear antigen)

30
Q

Clamp loader cycle (e-coli):

A

ATP binds to the delta1-gamma1 interface (of the clamp loader)
->
Gamma2 and Gamma3 bind ATP
->
Gamma3 opens up
->
delta interacts with beta clamp (clamp protein) opening it
->
DNA + beta = ATP hydrolysis of gamma ATPs
->
Beta clamp shuts since gamma (clamp loader requires ATP to stay open)
->
Beta clamp released by clamp loader

31
Q

What happens with clamps on Okazaki fragments?

A

It dissociates at the end of each Okazaki Fragment, and reassembles at the primer of the next

32
Q

What initiates each okazaki fragment?

A

helicase (dnaB) interacting with dnaG (primase)

33
Q

What is DNA polymerase I doing during this time?

A

It is out about, looking for clamps assembled on the RNA primer of Okazaki fragments, not part of the replicase complex

34
Q

A third polymerase has been discovered as part of polymerase III. What is it hypothesized to do?

A

1: Act as a reserve enzyme to overcome certain replication obstacles.
Or
2: coordinate the slower lagging strand synthesis

35
Q

What is a more accurate viewpoint for “single DNA polymerases are bound continuously to their substrates”
- Implication?

A

Polymerases and repair factors are cycled and switched, predominately by PCNA (sliding clamp), as needed during replication. This occurs without dismantling of the replisome.
- Implication?
Some repair postreplicative repair processes may occur during replication when replisome pauses.

36
Q
  • What starts a point of replication?

- What comes after it is primed?

A
  • What starts a point of replication?
    Helicase and primase
  • What comes after it is primed?
    Clamp and clamp loader recognize primer RNA

CONJ 662 - Genetic Mechanisms (13 decks)

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