BIOL253 Lecture 3

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  • Created by: Katherine
  • Created on: 29-03-17 19:20
What was the Meselson-Stahl experiment?
Proved that DNA was replicated in a semi-conservative fashion.
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What type of DNA replication is DNA replication?
Semi-conservative - each strand of the parental double helix acts as a template for synthesis of a new daughter DNA strand.
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What ensures that each daughter double helix is identical to the parental double helix, and each daughter cell will receive identical DNA molecules.
The base on the template strand must be identified and complementary base added.
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What are the sites of replication called?
Origins of replication.
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What direction does DNA replication go from initiation?
Both directions - it is bidirectional - forming a replication bubble.
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Where is the DNA double helix opened?
It is opened at the origin of replication and unwound to form replication forks.
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What happens at replication forks?
Single stranded DNA is exposed, DNA synthesis can occur.
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What direction does DNA synthesis occur?
5' to 3' direction
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Is synthesis continuous on both strands?
No, it is continuous on one strand (leading strand) and discontinuous on the other strand (lagging strand).
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DNA synthesis has 3 stages, what are these?
Initiation, elongation and termination.
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What happens during initiation? Step 1-2
Origin of replication is recognised by an initiatior protein that opens up the double helix and recruits helicases. DNA helicases unwind the helix to expose single stranded DNA, which is coated with ssDNA binding proteins.
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What happens during initiation? Step 3-4
Initiation is controlled to occur once per cell cycle. DNA synthesis needs a primer - DNA polymerases can only add nucleotides to an existing 3' end. The primer is a short RNA strand synthesised by primase (2).
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What happens during Elongation?
After RNA primer is synthesised, sliding clamp is recruited. DNA polymerase is associated with DNA via the sliding clamp. The replication machinery moves along the DNA, copying the strands. Each base in the parental DNA is read by DNA polymerase,.
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What does the DNA polymerase do?
It adds complementary bases to the growing strand in a 5' to 3' direction.
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What happens during Termination?
The replication complexes are disassembled. RNA primers are removed and replaced with DNA. DNA ligase connects adjacent strands.
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When does Termination occur?
When two different forks meet, the fork reaches the end of a linear chromosome, DNA polymerase meets the previously replicated strand.
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DNA synthesis is catalysed by...
DNA polymerase
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What does DNA synthesis require?
dNTPs, Primer: Template Junction, DNA pol - catalyses the addition of a new nucleotide to the 3'OH of the last nucleotide of the growing strand.
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What is the orientation of the template strand?
As DNA strands are antiparallel, template strand has opposite orientation to newly synthesised strand.
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What is the main replicative polymerase of bacteria?
DNA polymerase III (leading and lagging strand synthesis).
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What is the main replicative polymerase of Eukaryotes?
DNA polymerase δ (lagging strand synthesis), DNA polymerase ε (leading strand synthesis).
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Are DNA polyermases highly conserved?
Yes, and they are composed of multiple subunits.
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How long for DNA polys remain attached to DNA?
For long stretches, before dissociation.
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DNA polymerase structure resembles a...
Right hand.
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Which region of the polymerase is the most highly conserved?
The catalytic site for nucleotide addition is the most highly conserved region of DNA polymerase.
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What are the 3 domains of DNA polymerases described as?
Thumb, fingers and palm.
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Where does the growing double stranded DNA fit into?
The palm
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Where does the ssDNA fit?
It winds through the fingers
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What does the finger domain do?
It helps position the incoming nucleotide; the thumb domain holds the elongated dsDNA.
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The active site of DNA polymerase catalyzes a...
phosphoryl transfer reaction.
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The active site does this by.
Linking the 5' phosphate of the incoming nucleotide to the 3' OH of the growing DNA to form a phosphodiester bond.
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What type of reaction is the mecahnism of catalysis by DNA polymerase?
Nucleophilic attack by the 3'OH on the α-phosphate of the incoming dNTP releasing two phosphates as pyrophosphate
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The hydrolysis of released pyrophosphate provides...
energy
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Is the active site of DNA polymerase selective?
Yes for correct base pairing - only the correct nucleotide fits precisely in the active site. Mismatches have a different shape to correctly matched bases and don't fit in the active site well.
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What removes bases from the 3' end of mismatched DNA?
Proofreading exonuclease activity
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What happens to the DNA polymerisation active site when incorrect nuleotides are present?
It has reduced affinity for 3'OH.
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What does the exonuclease active site do when there are incorrect nucleotides?
It removes them.
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The 3' end of the new strand is returned to...
the polyerase active site and DNA synthesis s resumed.
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What is required to removed the incorrect nucleotide?
Energy
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Most DNA polymerases are processive, what does this mean?
Replicative polymerases stay attached for many 1000s of nucleotides - they are processive syntheis.
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Some DNA pols only add a few nucleotides before falling off, what does this make them?
Distributive.
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DNA pols are grouped into families according to what?
According to the evolutionary lineage of the rest of the protein.
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What is indicative of early divergence of DNA pol groups?
The fact that DNA pols are more similar within groups than within organisms.
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What unwinds double stranded DNA?
DNA helicases catalyse the unwinding - they travel along the DNA and unwind at the replication fork.
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What do helicases bind to?
They bind to and move directionally along ssDNA desplacing the complementary strand.
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What are DNA helicases?
They are hexameric ring proteins
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dhfdfjfdsfsd
Direction moved on the strand that is bound.
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How many identical subunits (coded for by the dnaB gene), does the E.coli helicase (DNAB) have?
6
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What structure does E. coli helicase, DNAB exist as?
As a ring structure in cells (loaded on to DNA by the DNAC helicase loader complex.
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In Eukaryotes, the core of the replicative helicate is called the...
MCM complex (Mini Chromosome Maintenance)
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What is DNA replication?
The complete, faithful copying of the DNA comprising the cell's chromosomes.
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What direction does DNA synthesis occur?
5' to 3'
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DNA syntheis starts at...
the Origin of Replication.
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Helicases are...
6 membered ring proteins. The structure is conserved.
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What do the helicases do?
Move along, grabbing the bases and separating DNA
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What is DNA b?
The bacteria helicase - it has to be loaded onto the DNA by DNA C
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How do you get a ring on DNA?
You have to open it up.
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What is the purpose of DNA c?
To open up DNA B and put it on the DNA strand.
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Single strand DNA in itself can form...
secondary structures if it is exposed by helicase activity
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What is the single stranded DNA binding protein in bacteria called?
Single stranded DNA binding protein (SSB)
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What is the single stranded binding protein called in Eukaryotes?
Replication protein A (RPA)
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What do the binding proteins do?
Keeps unwould DNA strands open, and protect them from the activity of nucleases.
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What do Topoisomerases do?
Assist assist helicases by removing supercoils from DNA.
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How are Supercoils made?
As DNA is unwound, the helical twist from the DNA is pushed into positive supercoils ahead of the fork as it moves, and creates torsional stress.
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Why is supercoiling bad?
It makes the DNA ahead of the form more difficult to unwind
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How to topoisomerases help helicases?
They release the overwould DNA by transiently breaking DNA and allowing supercoils to relax
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Where are topoisomerases found?
In all organisms and most have several different types of topoisomerases
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What is topology?
The state of supercoiling
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What are the 2 types of topoisomerases?
Type 1 (cut one strand of DNA) or Type 2 )cut both DNA strands)
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While type I and II, always refers to the number of strands cut, in terms of topoisomerases, eukaryottes have type 1a,1b and II, while bacteria have 1a and II - what do the a and b mean?
They refer to the difference in mechanisms by which the topoisomerases relieve the supercoiling.
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How do type 1B topoisomerases work?
They cut one strand of DNA and become transiently covalently attached to the 3' end of the cleaved DNA. The free end swivels to release supercoils before the DNA ends are rejoined and the topoisomerase is released..
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What do type 1B topoisomerases rely on?
The tension of the DNA to drive relaxation of supercoils
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How do type 1A topoisomerases work?
They cut one strand of DNA and have a cleave and pass mechanism. They become tranisently covalently attached to the 5' end of the cleaved DNA. The second DNA strand is passed through the transient break, and the ends rejoin and topoisomerase released
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How do type 11 topoisomerases work?
Cleave and pass mechanism - cut botrh strands of DNA helix and become transiently covalently attached to the 5' end of the cleaved DNA. A double stranded segment of DNA strand is passed through the break, and the ends rejoined and the topoisomerase r
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What does type II topoisomerase require?
ATP hydrolysis.
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What is DNA gyrase and what does it do?
A bacterial type II topoisomerase that introduces negative supercoils (no equivalent in eukaryotes). It maintains the bacterial chromosoe in negatively supercoied state.
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What does negative supercoiling allow for?
Easier strand separation.
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Card 2

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What type of DNA replication is DNA replication?

Back

Semi-conservative - each strand of the parental double helix acts as a template for synthesis of a new daughter DNA strand.

Card 3

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What ensures that each daughter double helix is identical to the parental double helix, and each daughter cell will receive identical DNA molecules.

Back

Preview of the front of card 3

Card 4

Front

What are the sites of replication called?

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Card 5

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What direction does DNA replication go from initiation?

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