BIOL253 - L4

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  • Created by: Katherine
  • Created on: 06-04-17 11:44
What do sliding clamps do?
Increase DNA polymerase processivity
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What do processive polymerases do?
Get onto the DNA and keep synthesising it.
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What is the basic feature that makes polyermases porcessive?
Their association with the sliding clamp - this keeps the polymerase attached to the DNA it's synthesising.
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What is the bacterial sliding clamp?
B protein - ring consists of two B Subunits (homodimer) each with three similar domains.
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What is the eukaroytic sliding clamp?
Proliferating cell nuclear antigen (PCNA) - ring consists of 3 PCNA polpeptides (homotrimer) each with two similar domains.
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What do Sliding clamps do?
Increase DNA polymerase processivity
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How do sliding clamps work?
During DNA replication, the clamp binds to DNA polymerase, slides along the DNA with the polymerase keeping it tethered to the DNA. If the polymerase releases the 3'OH of the nascent strand it cannot dissociate - rebinds the 3'OH.
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Which types of polymerases do not associate with sliding clamps?
Distributive polymerases.
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How do PCNA interact with eukaryotic DNA polymerases?
Through a motif of 8 amino acids in each PCNA subunit.
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The B protein ahs a short peptide motif that interacts with...
bacterial DNA polymerase III
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What is the structure of the sliding clamp ?
It is a rind and it must be opened up
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What opens up the sliding clamp?
The sliding clamp loader.
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What is the structure of the eukaryotic clamp loader?
It is a 5 ring subunit structure. Comprised of 5 different but related polypetides.
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In Eukaryotes, what is the clamp loader called?
It's called replication factor C 9RFC) -
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In bacteria, what is the clamp loader called?
Y complex
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How do clamp loaders typically exist?
They will form a ring, but typically in a spiral configration
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What is the function of the clamp loader?
To open up the clamp and put it on the DNA. To recognise the 3' hydroxyl and put on a new clamp
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What are the feautres of the clamp loader?
Low affinity for the cclamp until the loader is bound to ATP. It must be bound to ATP to bind to the clamp.
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The clamp loader sliding clamp complex has a high affinity with the...
primer template junction.
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What does binding of the clamp loader sliding complex stimulate?
ATPase activity, which closes the sliding clamp and releases the clamp loader. The sliding clamp remains associated with DNA. Sliding clamp recruits DNA pol to begin elongation.
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How does the clamp loader assemble the sliding clamp onto template
ATP binding leads to conformational change that allows clamp binding and opening. 3' end of primer enters clamp loader. ATP hydrolysis, clamp loader release. Clamp recruits DNA polymerase.
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What happens to the clamp on binding with ATP?
The clamp loader binds to the sliding clamp, forming a spiral shape and opening the clamp.
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What type of affinity does the clamp loader sliding clamp complex have with the primer template junction?
High
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Binding of the complex to the primer template junction stimulates what?
ATPase activity, which closes the sliding clamp and releases the clamp loader..
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What does the sliding clamp recruit to begin elongation?
DNA polymerase
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What is required to initiate DNA replication?
Origins of replication. (ori).
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What are origins?
Regions where the dsDNA is unwound and separated ready for replication proteins to attach.
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How many origins do bacteria and eukaryotes have?
Bacteria have one origin per chromosome, eukaryotes have many (there is more DNA to replicate).
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What is an origin defined by?
By the ability to bind initiator proteins.
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Some organisms have origins of replication with a defined DNA sequence, close to...
AT rich elements.
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In E.coli what is the initiator protein?
DnaA.
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In Eukaryotes, what is the initiator protein?
Origin Recognition Complex (ORC) consisting of 6 subunits.
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It E.coli, chromosome replication is initiated by which protein?
Dna A
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How does DnaA binding at oriC initiate DNA replication in E.coli
DNA initiator protein binds to DnaA boxes and bends the DNA at the replication origin. Bending of the DNA allows local unwinding of the AT rich region.
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Why is the AT rich region easy to unwind?
Because A-2 have only 2 hydrogen bonds.
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What happens when DnaA binding occurs at ori C?
DNA helicase is recruited to unwind the DNA.
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DNA wrapping about Dna A complex induces...
local unwinding of AT rich region.
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After the unwinding of the AT rich region, what happens?
Dna C assembles onto the Dna B helicase and binds to Dna A.
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After Dna C assembles onto the Dna B helicase and binds to Dna A, what happens?
Dna C loads the Dna B helicase onto the DNA and then disassembles.
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What is known about eukaryotic replication originas?
origins are defined by binding of the origin recognition complex (ORC).
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Where are ORC binding sites found?
Adjacent to AT rich sites, B2 and B3.
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What does ORC recruit?
It recruits cdc6 and cdt1, which are helicase loading proteins.
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What do cdc6 and cdt1 recruit?
They recruit the MCM helicase proteins
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Where is replication initiated?
At specif chromosomal regions, but the DNA sequences are not conserved.
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No DNA polymerase can inittte DNA synthesis..
Denovo, they can only extend a primer to then carry out DNA synthesis
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What does DNA primase do?
Produces a short RNA molecules (Primer).
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Where must primers be synthesised?
On the leading strand (infrequently) and lagging strand (frequently)
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How many subunits does Eukaryotic polymerase a primase complex have?
4
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Howdoes the polymerase a primase complex work?
It starts by making an RNA of 10-30 bases. The polymerase a subunit then adds a short piece of DNA- iDNA
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What is an okazaki frament?
DNA is made in short discontinuous fragments on the lagging strand - Okazaki fragments.
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What is the name of the polymerase to synthesise DNA on the lagging strand?
DNA pol III
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What happens when the DNA pol III reaches the next primer?
It disassociated. The sliding clamp remains attached but DNA pol I is recruited and removes RNA primer (5' - 3' exonuclease activity), fills the gap with DNA.
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What does DNA pol I leave in the DNA?
A nick
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What seal the DNA nick in the lagging strand?
DNA ligase I
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In eukaryotes what is the name of the polymerase to synthesise DNA on the lagging strand?
DNA polymerase delta.
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What does DNA polymerase delta do?
Instead of stopping at the RNA primer on the lagging strand, it carries on, displacing it at DNA from the template strand, making a flap.
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What cleaves the flap DNA?
Flap endonuclease (Fen 1).
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In eukaryotes what seals the DNA nick?
DNA ligase I.
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What is the function of DNA ligase I?
To join up the phosphodiester back bone.
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All ligases use...
Nucleotide co-factors - Bacteria use NAD+, Eukaryote use ATP
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What is the mechanism of DNA ligase reaction?
AMP covalently attaches to a ligase, this is transferred onto the 5' phosphate in the DNA. The 3' hydroxyl attacks the phosphate resulting in the loss of the AMP
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Where are the leading and lagging strand sythesised?
At the replication fork
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Do the strands move in the same direction from the replication fork?
No
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How is the syntheis of the lagging strand and the leading strand coordinated?
DNA on the lagging strand is looped around. The lagging strand DNA pol faces the same was as leading strand DNA pol.
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How would you draw coordination of the leading and laggins strand synthesis?
A Trombone model loop.
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What is a replisome?
Something that coordiantes replication.
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What are the core DNA pols linked by?
By 2 subunits of a flexible linker protein, tau which associates with y complex clamp loader and Dna B helicase.
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Other cards in this set

Card 2

Front

What do processive polymerases do?

Back

Get onto the DNA and keep synthesising it.

Card 3

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What is the basic feature that makes polyermases porcessive?

Back

Preview of the front of card 3

Card 4

Front

What is the bacterial sliding clamp?

Back

Preview of the front of card 4

Card 5

Front

What is the eukaroytic sliding clamp?

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