Test1: Lect1 Maureen Hoatlin Flashcards
Components of cellular replicase are variations on a theme. Name eukaryote and E. Coli for: Polymerase: Sliding clamp: Clamp loader:
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)
Inability to replicate is lethal.
Define conditional lethals:
A conditional lethal is a mutant which can replicate under permissive conditions (E.G. survives at 25 degrees C but not 37 degrees C)
Quick Stop Mutants:
- Define:
- Why?
Quick Stop Mutants:
- Define:
Immediately stop replication under permissive conditions.
- Why?
Mutated protein is likely a component of the replication apparatus
Slow Stop Mutants:
- Define:
- Why?
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
In vitro complementation:
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.
What are the two types of DNA replication?
Semi-conservative replication
DNA synthesis in DNA repair
In vitro replication system
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.
Semi-conservative replication:
Is the form we use.
Strands are split, both strands are replicated. Each DNA duplex has one completely new and one old DNA strand.
dnaE:
Gene encoding the subunit of the DNA polymerase III complex which gives 5’ -> 3’ catalytic activity to DNA polymerase 3
dnaQ:
- Other name for it:
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
Which polymerase is responsible for the bulk of DNA synthesis?
Pol III
What is the shape of DNA polymerase III?
- How many families of the polymerase?
- What varies and what
stays the same?
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
Hi Fi stands for:
High fidelity
How is Hi-Fi replication maintained?
- 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.
Polymerase from T7 Bacteriophage steps:
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
How exactly is a mispair identified?
Fingers (with O-helix) cannot rotate towards palm to grab nucleotide. Leaves 3’ free to bind nuclease domain
What does AZT stand for?
- What is it?
- Specificity?
- Downside?
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
DNA primase:
- Function:
- Gene name:
- Function:
Adds RNA primer 5’ to 3’ - Gene name:
dnaG
DNA helicase:
- Function:
- Gene name:
- Are there multiple varieties?
- 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
DNA helicase:
- Structure:
- How does it work?
- 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.
Single Stranded Binding (SSB) Proteins:
- Function?
- Quick stop or slow stop?
- 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
Are the leading and lagging strands synthesized by the same proteins?
- How common are exceptions?
- Name one?
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
E-Coli replicase (DNA polymerase III):
- Structure:
- 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
Holoenzyme:
a biochemically active compound formed by the combination of an enzyme with a coenzyme.
Dimeric structure:
A structure with two identical or very similar proteins which link together
What is the processivity subunit of DNA polymerase III:
Specifically: beta dimer
Generally: it is a sliding clamp
What places the sliding clamp onto the DNA strand?
A clamp loader (the gamma complex)
What are the steps for beta dimer (a clamp) to bind to DNA/early initiation?
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.
What is the human form of beta clamp (Clamp protein)?
PCNA (proliferating cell nuclear antigen)
Clamp loader cycle (e-coli):
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
What happens with clamps on Okazaki fragments?
It dissociates at the end of each Okazaki Fragment, and reassembles at the primer of the next
What initiates each okazaki fragment?
helicase (dnaB) interacting with dnaG (primase)
What is DNA polymerase I doing during this time?
It is out about, looking for clamps assembled on the RNA primer of Okazaki fragments, not part of the replicase complex
A third polymerase has been discovered as part of polymerase III. What is it hypothesized to do?
1: Act as a reserve enzyme to overcome certain replication obstacles.
Or
2: coordinate the slower lagging strand synthesis
What is a more accurate viewpoint for “single DNA polymerases are bound continuously to their substrates”
- Implication?
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.
- What starts a point of replication?
- What comes after it is primed?
- What starts a point of replication?
Helicase and primase - What comes after it is primed?
Clamp and clamp loader recognize primer RNA
