DNA & Disease- Prokaryotic Replication

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What are the two main recognition components?
Replicator sequence (oriC) and DnaA (an oriC binding initiator protein)
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What is the oriC?
a highly conserved, 250bp segment that supports bi-directional replication
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How many monomers of DnaA bind the __bp repeats in oriC?
5, 9
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What do they do?
Recognise and bind 9bp repeats in oriC
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What is the consensus sequence of the 9bp repeats?
5'-TTATCCACA-3'
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To form what?
a complex of supercoiled DNA wrapped around a central core
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What do the histone-like HU or IHC proteins do?
facilitate the formation of the complex by helping to bend DNA
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What is the consensus sequence of the three tandemly repeated AT rich segments to the left of the oriC?
5'-GATCTNTTNTTTT-3'
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In what state can this 45bp segment be cut by the __ nuclease?
Single stranded (open complex) . P1.
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How does DNA melting occur?
At 37C and 5mM ATP (and DnaA)
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What does this single stranded state require the presence of?
DnaA and ATP
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What is the single-stranded DNA now sensitive to?
P1 Nuclease
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What does the P1 nuclease do?
It cleaves the AT-rich 13bp sequences that have been melted by DnaA to make room for the replisome
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What does the DnaA protein complex recruit? To where? To form? What does this allow? What is then released?
DnaB6:DnaC6 complex to melted region to form prepriming complex. This allows 5 more DnaA monomers to bind the bound DnaA to form DnaA dimers. DnaC6 is released.
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What type of enzyme is DnaC6?
An ATPase that promotes loading
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What type of enzyme is DnaB6? What does it do? What is necessary for its activity?
A helicase. It unwinds the DNA in the prepriming complex in both directions. SSB (** binding) proteins and gyrase
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What do the SSB's do?
prevent reannealing
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What does the gyrase do? (2 things)
relieves torsional stress by negatively supercoiling the DNA, and further unwinds the helix to allow entry of RNA polymerase
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What does the RNA polymerase do?
Activates the primase to begin RNA primer synthesis
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How does bidirectional replication then progress?
by recruitment of the replisome complex
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How many bp's per chromosome of E.coli?
4.6x106
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What is the constant rate of fork movement?
1000nts/s
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What is the chromosome replication time for E.coli?
40 minutes
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What is the segregation time?
20 minutes
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What does this indicate about cells with doubling times of less than 60 minutes?
that chromosome replication must be initiated before the end of the preceding cell division cycle (i.e. multiple replication forks)
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Where do the multiple replication forks initiate from?
The same origin
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After initiation, chain elongation occurs at ____ rate, suggesting:
Uncontrolled. post-initation oriC site is prevented from interacting with replication invitation machinery
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What 4 things does dna synthesis require?
DNA pol, SS template, nucleoside triphosphates, that the strand they're extending forms a double helix with template strand
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What is the leading strand synthesis catalysed by?
pol III holoenzyme
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What is the lagging strand synthesis catalysed by?
also by pol III holoenzyme, but after priming by primosome
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What does the replisome consist of?
2 pol III cores (made up of a, E and theta subunits linked by tau subunits) + primosome
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What happens when DNA pol III holoenzyme reaches old OKZ fragments?
the bound template strand is released
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how is the next OKZ fragment synthesised?
by synthesis of short segment of RNA inserted by primosome
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What must the primosome do when synthesising this RNA primer?
reverse the direction of travel
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What happens when this RNA primer finds its way into the empty holoenzyme?
the strand is extended to give the next OKZ fragment
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How are the RNA primers now in the lagging strand removed?
By DNA polymerase, which replaces RNA with DNA.
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What are the OKZ fragments joined by?
DNA ligase
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The replication terminus is a ____kb fragment flanked by what?
350kb. 6 homologous non-palindromic 23bp ter loci
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What ter loci would replication by terminated by if the replication fork is travelling a) clockwise b) anticlockwise
a) Ter F/B/C b) E/D/A
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What does Tus stand for?
Terminator Utilisation Substance
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Tus: what does it do? how big is it? pH?
interacts with helices to inhibit action... 36kd (306 residues), highly basic
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In what form of bacteria is this highly conserved?
Gram negative
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How can we tell it isn't essential?
Deletion of ter loci doesn't affect DNA replication in E.coli
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Card 2

Front

What is the oriC?

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a highly conserved, 250bp segment that supports bi-directional replication

Card 3

Front

How many monomers of DnaA bind the __bp repeats in oriC?

Back

Preview of the front of card 3

Card 4

Front

What do they do?

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Preview of the front of card 4

Card 5

Front

What is the consensus sequence of the 9bp repeats?

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