DNA Replication 1 & 2 Flashcards
Lecture 3 (Completed) Lecture 4 (Completed except final few slides - they are in next deck :D)
Define DNA replication
The complete, faithful (accurate) copying of the DNA comprising the cell’s chromosomes
True or False:
DNA replication is semi-conservative
True
True or False:
DNA replication is completely conservative
False:
DNA replication is semi-conservative
What does semi-conservative replication mean?
Each strand of the parental double helix acts as a template for synthesis of a new daughter DNA strand
Fill in the gaps:
Chromosome replication starts at specific sites called ______ __ ________. Here the DNA double-helix is _________ __ / _______ to expose _______ _______ DNA at _________ _____.
DNA synthesis occurs at these __________ _____, and proceeds ___________.
Chromosome replication starts at specific sites called origin(s) of replication. Here the DNA double-helix is opened up and unwound to expose single stranded DNA at replication forks.
DNA synthesis occurs at these replication forks, and proceeds bidirectionally.
True or False:
DNA synthesis during chromosome replication occurs in a 5’ to 3’ direction on both strands.
True
True or False:
DNA synthesis during chromosome replication occurs in a 5’ to 3’ direction on one strand and 3’ to 5’ on the other.
False:
DNA synthesis during chromosome replication occurs in a 5’ to 3’ direction on both strands.
True or False:
DNA synthesis during chromosome replication is continuous on one strand and discontinuous on the other.
True
True or False:
DNA synthesis during chromosome replication is continuous on both strands.
False:
DNA synthesis during chromosome replication is continuous on one strand and discontinuous on the other.
True or False:
DNA synthesis during chromosome replication is discontinuous on both strands.
False:
DNA synthesis during chromosome replication is continuous on one strand and discontinuous on the other.
True or False:
DNA synthesis during chromosome replication is continuous on the leading strand.
True
True or False:
DNA synthesis during chromosome replication is continuous on the lagging strand.
False:
DNA synthesis during chromosome replication is continuous on the leading strand and discontinuous on the lagging strand.
True or False:
DNA synthesis during chromosome replication is discontinuous on the lagging strand.
True
True or False:
DNA synthesis during chromosome replication is discontinuous on the leading strand.
False:
DNA synthesis during chromosome replication is continuous on the leading strand and discontinuous on the lagging strand.
What are the three phases of chromosome replication?
Initiation
Elongation
Termination
Fill in the gaps: Replication Stage One - Initiation:
The origin of replication is recognised by _______ proteins that _____ ___ the double helix and recruit _______.
____ _______ unwind the helix to expose single-stranded DNA. DNA synthesis needs a ______, because ____ ___________ can only add nucleotides to an existing ___ ___. The ______ is a short _____ _______ synthesised by ________.
The origin of replication is recognised by initiator proteins that open up the double helix and recruit helicases.
DNA helicases unwind the helix to expose single-stranded DNA. DNA synthesis needs a primer, because DNA polymerase can only add nucleotides to an existing 3’ end. The primer is a short RNA strand synthesised by primase.
Fill in the gaps: Replication Stage Two - Elongation:
After the ______ is synthesised, the ______ _____ is recruited. ____ __________ is associated with DNA via the _______ ______.
Each base in the parental DNA is read by ____ __________, and complementary bases are added to the growing strand in a ___ __ ___ direction.
DNA synthesis on the _______ strand is ________ but on the ______ strand is ________.
After the primer is synthesised, the sliding clamp is recruited. DNA polymerase is associated with DNA via the sliding clamp.
Each base in the parental DNA is read by DNA polymerase, and complementary bases are added to the growing strand in a 5’ to 3’ direction.
DNA synthesis on the leading strand is continuous but on the lagging strand is discontinuous.
When does termination of replication occur?
Termination of replication occurs when:
- Two different forks meet
- DNA polymerase meets the previously replicated strand
- The fork reaches the end of a linear chromosome
Fill in the gaps: Replication Stage Three - Termination:
Once one of three scenarios occur to cause termination of replication, the ________ _________ are __________. The ____ ______ are removed and replaced with _____. ____ _______ connects _______ strands.
Once one of three scenarios occur to cause termination of replication, the replication complexes are disassembled. The RNA primers are removed and replaced with DNA. DNA ligase connects adjacent strands.
True or False:
The three domains of DNA polymerase are often referred to as bar, body and shackle, since together they resemble a padlock.
False:
The three domains of DNA polymerase are often described as thumb, fingers and palm as together they resemble a right hand.
True or False:
The three domains of DNA polymerase are often described as thumb, fingers and palm as together they resemble a right hand.
True
What is the role of the palm domain of DNA polymerase?
It contains the catalytic site for nucleotide addition and forms a cleft in which elongating dsDNA fits.
What is the role of the finger domain of DNA polymerase?
The single stranded DNA template wraps through the finger domain (like being threaded through multiple fingers) to help position the incoming nucleotide.
What is the role of the thumb domain of DNA polymerase?
The thumb domain holds the elongating double stranded DNA and maintains contact with the single strand template necessary for processive synthesis.
Name the three domains of DNA polymerase
The thumb
The fingers
The palm
What is the role of DNA polymerase in DNA replication?
It catalyses the addition of a new nucleotide to the 3’ OH of the last nucleotide of the growing strand.
Why does the template strand have opposite orientation to the newly synthesised strand during DNA replication?
DNA strands are antiparallel
Fill in the gaps:
New DNA synthesis is template directed, meaning it involves ___________ of a ____ in the template strand and the addition of a nucleotide with the _____________ _____ to the new _________ strand.
New DNA synthesis is template directed, meaning it involves recognition of a base in the template strand and the addition of a nucleotide with the complementary base to the new daughter strand.
What is the main replicative polymerase in bacteria for the leading strand and for the lagging strand?
DNA polymerase III , for both leading and lagging strand synthesis.
What is the main replicative polymerase in eukaryotes for the leading strand and for the lagging strand?
DNA polymerase ε for the leading strand
DNA polymerase δ for the lagging stand
True or False:
Replicative DNA polymerases are highly conserved
True
True or False:
Replicative DNA polymerases are weakly conserved
False:
Replicative DNA polymerases are highly conserved
True or False:
All DNA polymerases synthesise only in the 5’ to 3’ direction
True
True or False:
All DNA polymerases synthesise only in the 3’ to 5’ direction
False:
All DNA polymerases synthesise only in the 5’ to 3’ direction
Define processivity
The ability of the enzyme to continuously polymerize without releasing the template strand
Fill in the gaps:
DNA polymerases remain attached to DNA for _____ _______ before _________ - they are ________.
DNA polymerases remain attached to DNA for long stretches before dissociation - they are processive.
What are the three subunits of DNA polymerase III?
Alpha , epsilon and theta
α, ε, θ
What is the function of the alpha subunit of DNA polymerase III?
Polymerisation activity
What is the function of the ε subunit of DNA polymerase III?
Exonuclease activity
(removes successive nucleotides from the end of a polynucleotide molecule)
What is the function of the θ subunit of DNA polymerase III?
Stabilisation of subunit ε
What is the function of DNA polymerase III?
It is the main replicative polymerase in bacteria for both the leading strand and for the lagging strand
What is the function of DNA polymerase ε
It is the main replicative polymerase in eukaryotes for the leading strand
What is the function of DNA polymerase δ
It is the main replicative polymerase in eukaryotes for the lagging stand
Fill in the gaps: Mechanism of catalysis by DNA polymerase:
The active site catalyses a ________ ______ reaction. This links the ___ _________ of the incoming nucleotide to the ___ ____ of the growing DNA, forming a ____________ bond.
There is a __________ ______ by the ___ ____ on the __ ___________ of the incoming dNTP, releasing two phosphates as ___________.
The ________ of the released __________ provides energy.
The active site catalyses a phosphoryl transfer reaction. This links the 5’ phosphate of the incoming nucleotide to the 3’ OH of the growing DNA, forming a phosphodiester bond.
There is a nucleophilic attack by the 3’ OH on the α-phosphate of the incoming dNTP, releasing two phosphates as pyrophosphate.
The hydrolysis of the released pyrophosphate provides energy.
What is a nucleophile
An electron donor
True or False:
Bacteria have one origin (of replication) per chromosome
True
True or False:
Bacteria have many origins (of replication) per chromosome
False:
Bacteria have one origin (of replication) per chromosome
True or False:
Eukaryotes have many origins (of replication) per chromosome
True
True or False:
Eukaryotes have one origin (of replication) per chromosome
False:
Eukaryotes have many origins (of replication) per chromosome
True or False:
Bacteria do not have specific DNA sequences to define origins of replication
False:
Bacteria have specific DNA sequences to define origins of replication, which bind to the initiator protein with high affinity
True or False:
Bacteria have specific DNA sequences to define origins of replication, which bind to the initiator protein with high affinity
True
True or False:
Eukaryotes have specific DNA sequences to define origins of replication
False:
Only a few eukaryotes have specific DNA sequences to define origins of replication
True or False:
A few eukaryotes have specific DNA sequences to define origins of replication
True
What is required to initiate replication in bacteria and eukaryotes?
Origins of replication (ori)
What are the regions called where double stranded DNA is unwound and separated ready for replication proteins to attach?
Origins of replication (ori)
What are origins of replication (ori)?
Regions where double stranded DNA is unwound and separated, ready for replication proteins to attach
Fill in the gaps:
All initiator proteins are ____ _________.
All initiator proteins are AAA+ ATPases.
True or False:
All initiator proteins are AAA+ ATPases
True
True or False:
All initiator proteins are AA+ ATPases
False:
All initiator proteins are AAA+ ATPases
What is an AAA+ ATPase?
ATPases associated with various cellular activities
(extra info for reference: like DNA replication, protein degradation, membrane fusion, microtubule serving, peroxisome biogenesis, signal transduction, and regulation of gene expression)
True or False:
Bacteria have specific DNA sequences to define origins of replication, which bind to the initiator protein with low affinity
False:
Bacteria have specific DNA sequences to define origins of replication which bind to the initiator protein with high affinity
Fill in the gaps:
When an ______ protein binds to an origin of replication, this facilitates the unwinding of the adjacent ___ ____ region.
When an initiator protein (AAA+ ATPases) binds to an origin of replication, this facilitates the unwinding of the adjacent AT rich region.
In E.coli, what is the name of the initiator protein?
DnaA
Where does DnaA bind?
At oriC , a 245 bp sequence with multiple DnaA boxes and adjacent AT rich region, a DNA unwinding element
What are the DnaA boxes?
TTAT[C/A]
CA[C/A]A
want more explanation on what these are, bookmark it
What do DnaA proteins bind to?
DnaA boxes on oriC
What happens when DnaA is bound to ATP?
It self-associates into a helical multisubunit complex
When happens when DNA wraps around the spiral DnaA complex?
The DNA is bent and the adjacent AT rich sequence is unwound locally
Fill in the gaps: DnaA binding at oriC:
DNA wrapping around the DnaA complex induces the _____ unwinding of the _______ _______. A _____ ________ ______ assembles onto the _________ ______ helicase.
The _____ _______ ______ then binds to ______, bound at the origin. The ______ ________ _______ places the _____ helicase around the _______ _______ DNA at the origin.
The ______ ________ dissociates from the _____ helicase. The origin is ready for the recruitment of _______ and other replication proteins.
DNA wrapping around the DnaA complex induces the local unwinding of the AT-rich region. A DnaC helicase loader assembles onto the hexameric DnaB helicase.
The DnaC helicase loader then binds to DnaA, bound at the origin. The DnaC helicase loader places the DnaB helicase around the single stranded DNA at the origin.
The DnaC loader dissociates from the DnaB helicase. The origin is ready for the recruitment of primase and other replication proteins.
True or False:
DnaC helicase loader assembles onto the hexameric DnaB helicase
True
True or False:
DnaB helicase loader assembles onto the hexameric DnaC helicase
True or False:
DnaC helicase loader assembles onto the hexameric DnaB helicase
True or False:
DNA wrapping around DnaA complex induces unwinding of the CG-rich region
False:
DNA wrapping around DnaA complex induces unwinding of the AT-rich region
True or False:
DNA wrapping around DnaA complex induces unwinding of the AT-rich region
True
True or False:
DnaB helicase binds to DnaA bound at the origin
False:
DnaC helicase loader binds to DnaA bound at the origin
True or False:
DnaC helicase loader binds to DnaA bound at the origin
True
True or False:
DnaC helicase loader places DnaB helicase around single stranded DNA at origin
True
True or False:
DnaB helicase places DnaA around single stranded DNA at origin
False:
DnaC helicase loader places DnaB helicase around single stranded DNA at origin
What initiator protein binds origins in eukaryotic organisms?
Origin Recognition Complex (ORC)
Give an example of a eukaryote with origins defined by specific sequences
S cerevisiae
True or False:
Yeast origins have two core sequences, A and B1.
True
True or False:
Yeast origins have two core sequences, A1 and B1.
False:
Yeast origins have two core sequences, A and B1.
True or False:
Yeast origins have two core sequences, B1 and B2.
False:
Yeast origins have two core sequences, A and B1.
Fill in the gaps:
In _________, replication can be initiated at multiple different sites, determined by the __________ that they are ______ ___ _____.
________ may be influenced by __________ _________ and ___________ and other __________ specific DNA binding proteins.
In eukaryotes, replication can be initiated at multiple different sites, determined by the probability that they are bound by ORC.
Binding may be influenced by nucleosome positioning and modification and other sequence specific DNA binding proteins.
How many subunits do Origin Recognition Complexes (ORC) consist of?
6
How many stages are there for unwinding the origin in eukaryotes?
2
Fill in the gaps:
Origins are selected (licensed) in ____ ____ - the ___________ _______ is established.
The ____ binds to DNA to _______ ______.
______ and ______ cooperate with ____ as ____ ________ _______.
Two ring-shaped _______ _______ are loaded sequentially in a ___________ orientation.
The ____ dissociates once the _______ pair is loaded.
In G1 the ________ ____ ________ is _______, remains ________ both DNA strands.
_________ _______ _______ is referred to as ___________ ______.
Origins are selected (licensed) in late M/G1 - the prereplication complex is established.
The ORC binds to DNA to establish origins.
Cdc6 and Cdt1 cooperate with ORC as DNA helicase loaders.
Two ring-shaped MCM2-7 hexamers are loaded sequentially in a head-to-head orientation.
The ORC dissociates once the MCM2-7 pair is loaded.
In G1 the loaded MCM complex is inactive, remains encircling both DNA strands.
Loaded MCM2-7 helicase is referred to as the prereplicative complex.
When are origins selected / licensed and the prereplication complex established?
In late M/G1
What cooperate with the ORC, as DNA helicase loaders?
Cdc6 and Cdt1
What hexamers are loaded sequentially in a head-to head orientation?
Two ring-shaped MCM2-7
When does the ORC dissociate?
Once the MCM2-7 pair is loaded
When is the loaded MCM complex inactive, still encircling both DNA strands?
In G1
What is loaded MCM2-7 helicase referred to as?
the prereplicative complex.
What is the prereplicative complex?
Loaded MCM2-7 helicase
Fill in the gaps: PLEASE BOOKMARK WANT TO CLARIFY THE STEPS BY WATCHING A VIDEO
DNA helicases that are loaded around double stranded DNA at origins during the G1 phase of the cell cycle are activated in S-phase.
In S-phase the MCM2-7 complex is phosphorylated by DDK which allows recruitment of Cdc45 and Sld3.
S-phase CDK phosphorylates Sld2 and Sld3 allowing recruitment of GINS complex.
Replicative helicase complex - CMG complex (Cdc45-MCM-GINS) (guessing this is formed????)
Activated CMG helicase opens DNA. It transitions from binding double stranded DNA to encircling single stranded DNA at the origin. The mechanism for this is unknown. It also moves 3’ to 5’ on the leading strand template.
DNA helicases that are loaded around double stranded DNA at origins during the G1 phase of the cell cycle are activated in S-phase.
In S-phase the MCM2-7 complex is phosphorylated by DDK which allows recruitment of Cdc45 and Sld3.
S-phase CDK phosphorylates Sld2 and Sld3 allowing recruitment of GINS complex.
Replicative helicase complex - CMG complex (Cdc45-MCM-GINS) (guessing this is formed????)
Activated CMG helicase opens DNA. It transitions from binding double stranded DNA to encircling single stranded DNA at the origin. The mechanism for this is unknown. It also moves 3’ to 5’ on the leading strand template.
When are the DNA helicases that are loaded around dsDNA at origins during G1 activated?
In S-phase
What happens to the the MCM2-7 complex in S-phase? What does this allow?
The MCM2-7 complex is phosphorylated by DDK, which allows recruitment of Cdc45 and Sld3.
What does CDK phosphorylate in S-phase? Why?
CDK phosphorylates Sld2 and Sld3. This allows the recruitment of GINS complex.
What does activated CMG helicase do?
Activated CMG helicase opens DNA. It transitions from binding double stranded DNA to encircling single stranded DNA at the origin. It moves 3’ to 5’ on the leading strand template.
What does MCM stand for in the hexameric MCM complex?
Mini Chromosome Maintenance
Compare/contrast DNA helicases for prokaryotes and eukaryotes.
Prokaryotes:
DnaB helicase (E. coli replicative DNA helicase) has 6 identical subunits.
It is loaded onto ssDNA by the DnaC helicase loader complex.
It moves 5’ to 3’ on the lagging strand template.
Eukaryotes:
The core of the replicative helicase is the hexameric MCM complex.
It is formed of different subunits, MCM2-7.
It is loaded onto dsDNA in G1.
The full eukaryotic CMG (Cdc45, MCM, GINS) DNA helicase is assembled and activated in S phase.
It transitions to encircle ssDNA.
It moves 3’ to 5’ on the leading strand template
Compare subunits for DNA helicases for prokaryotes and eukaryotes.
Prokaryotes: DnaB helicase (E. coli replicative DNA helicase) has 6 identical subunits.
Eukaryotes: The core of the replicative helicase is the hexameric MCM complex, formed from different subunits, MCM2-7.
Compare loading of DNA helicases for prokaryotes and eukaryotes.
Prokaryotes: DnaB helicase (E. coli replicative DNA helicase) is loaded onto ssDNA by the DnaC helicase loader complex.
Eukaryotes: The hexameric MCM complex is loaded onto dsDNA in G1.
Compare movement of DNA helicases for prokaryotes and eukaryotes along the strand templates.
Prokaryotes: DnaB helicase (E. coli replicative DNA helicase) moves 5’ to 3’ on the lagging strand template.
Eukaryotes: The full eukaryotic CMG moves 3’ to 5’ on the leading strand template.
Fill in the gaps: Compare DNA helicases for prokaryotes and eukaryotes:
Prokaryotes:
____ ______ (E. coli replicative DNA helicase) has __ _______ subunits.
It is loaded onto _______ by the ______ ______ ______ complex.
It moves ___ to ___ on the _______ strand template.
Eukaryotes:
The core of the replicative helicase is the _______ ____ _______.
It is formed of different subunits, ______.
It is loaded onto ______ in ___.
The full eukaryotic _____ (_____, ____, ______) DNA helicase is assembled and activated in ___ phase.
It transitions to encircle ______.
It moves ___ to ___ on the _______ strand template.
Prokaryotes:
DnaB helicase (E. coli replicative DNA helicase) has 6 identical subunits.
It is loaded onto ssDNA by the DnaC helicase loader complex.
It moves 5’ to 3’ on the lagging strand template.
Eukaryotes:
The core of the replicative helicase is the hexameric MCM complex.
It is formed of different subunits, MCM2-7.
It is loaded onto dsDNA in G1.
The full eukaryotic CMG (Cdc45, MCM, GINS) DNA helicase is assembled and activated in S phase.
It transitions to encircle ssDNA.
It moves 3’ to 5’ on the leading strand template
True or False:
Replication machinery can access double-stranded DNA
False:
Double-stranded DNA is inaccessible to replication machinery.
True or False:
Double-stranded DNA is inaccessible to replication machinery.
True
True or False:
DNA helicases travel along the DNA, continuously unwinding it at the replication fork.
True
True or False:
DNA helicases travel along the DNA, continuously rewinding it at the replication fork.
False:
DNA helicases travel along the DNA, continuously unwinding it at the replication fork.
True or False:
Helicases bind to and move directionally along ssDNA, displacing the complementary strand
True
True or False:
Helicases bind to and move directionally along dsDNA, displacing the template strand
False:
Helicases bind to and move directionally along ssDNA, displacing the complementary strand
True or False:
DNA helicases are pentameric ring proteins
False:
DNA helicases are hexameric ring proteins
True or False:
DNA helicases are hexameric ring proteins
True
What shape are DNA helicase proteins?
Hexameric rings
What defines helicase polarity?
The direction the helicase moves on the strand that is bound
What does the difference in helicase polarity between prokaryotes and eukaryotes suggest?
Independent evolution
True or False:
Single stranded DNA exposed by helicase activity can form secondary structures.
True
True or False:
Double stranded DNA helixes can form secondary structures.
False:
Single stranded DNA exposed by helicase activity can form secondary structures.
In bacteria, what single-stranded binding protein binds to the single-stranded DNA?
Single-stranded DNA-Binding protein (SSB)
In eukaryotes, what single-stranded binding protein binds to the single-stranded DNA?
Replication protein A (RPA)
True or False:
Single-stranded DNA-Binding protein (SSB) is the eukaryote single-stranded binding protein that binds to the single-stranded DNA
False:
Replication protein A (RPA) is the eukaryote single-stranded binding protein that binds to the single-stranded DNA
True or False:
Replication protein A (RPA) is the eukaryote single-stranded binding protein that binds to the single-stranded DNA?
True
What is the function of single-stranded DNA-binding proteins?
Keep unwound DNA strands open
Protects single-strand DNA from nucleases
What releases overwound DNA by transiently breaking DNA and allowing supercoils to relax?
Topoisomerases
True or False:
Positive supercoils impede the progress of DNA
True
True or False:
Positive supercoils escalate the progress of DNA
False:
Positive supercoils impede the progress of DNA
Fill in the gaps:
As DNA is unwound by helicases, _______ _____ is introduced, which results in _________ ahead of the fork. This is _______ supercoiling.
As DNA is unwound by helicases, torsional stress is introduced, which results in over-winding ahead of the fork. This is positive supercoiling.
For every turn of the DNA helix that is unwound, how many supercoils are introduced ahead of the fork?
Is it positive or negative supercoiling?
One supercoil is introduced ahead of the fork for each turn of the DNA helix that is unwound.
It is a positive supercoil.
What was the Meselson-Stahl experiment?
E.coli cultures first grown in N15 medium (heavy).
DNA composition is 15N / 15N for the two strands respectively.
Continue growing in N14 medium (light).
DNA composition becomes 15N / 14N hybrid.
Continue growing in N14 medium (light).
DNA composition becomes either 14N / 14N or 15N / 14N hybrid.
Different compositions identified based on their density and therefore position in tests.
What does DNA primase synthesise?
RNA primers, which start the synthesis of new DNA strands
Why are primers required for DNA synthesis?
DNA polymerases cannot initiate DNA synthesis de novo (from scratch), they require an existing 3’OH for synthesis (primers provide this)
What is a primer?
A short piece of RNA
True or False:
DNA primase is a type of RNA polymerase
True
True or False:
DNA primase is a type of DNA polymerase
False:
DNA primase is a type of RNA polymerase
True or False:
RNA polymerases can synthesis de novo (from scratch) opposite a complementary base.
True
True or False:
DNA polymerases can synthesise de novo (from scratch) opposite a complementary base.
False:
DNA polymerases cannot synthesise de novo, they require an existing 3’OH, provided by a primer.
RNA polymerases can synthesis de novo (from scratch) opposite a complementary base.
True or False:
RNA polymerases cannot synthesis de novo (from scratch) opposite a complementary base.
False:
DNA polymerases cannot synthesise de novo, they require an existing 3’OH, provided by a primer.
RNA polymerases can synthesis de novo (from scratch) opposite a complementary base.
True or False:
DNA polymerases cannot synthesis de novo (from scratch) opposite a complementary base.
True
Where does priming occur on the leading strand?
Only at the origins
Where does priming occur on the lagging strand?
For every new Okazaki fragment (a new primer is synthesised for every section)
Describe bacterial primase DnaG
(subunits, type of polymerase, what it synthesises, what it hands over to)
Bacterial primase DnaG is a single subunit RNA polymerase.
It synthesises an RNA primer of 10-30 bases, then hands over to DNA pol III
Describe eukaryotic polymerase α-primase complex
Eukaryotic polymerase α-primase complex has 4 subunits (2 primase, 2 polymerase).
The 2 subunit primase makes an RNA of 10-30 bases. The polymerase α subunit then adds a short piece of DNA called initiator DNA (iDNA).
What is the name of the short piece of DNA that polymerase α-primase adds?
What size is it?
Initiator DNA (iDNA)
100 to 200 nucleotides
Why is polymerase α-primase not very accurate?
It has no proof reading ability
What direction does DNA polymerase move in and synthesise in?
It travels from the 3’ to 5’ of the template strand, adding nucleotides in a 5’ to 3’ direction.
True or False:
The sliding clamp structure is conserved
True
(Ring structure)
(though the subunits it is made of is not)
True or False:
The sliding clamp structure is not conserved
False:
The sliding clamp ring structure is conserved (though the subunits it is made of is not)
What is the name of the bacterial sliding clamp?
Beta protein
What does the bacterial sliding clamp Beta-protein structure consist of?
It is a ring consisting of two Beta subunits (homodimer) each with three similar domains
What is the name of the eukaryotes sliding clamp?
Proliferating Cell Nuclear Antigen (PCNA)
What does the Proliferating Cell Nuclear Antigen (PCNA) structure consist of?
It is a ring consisting of three PCNA polypeptides (homotrimer) each with two similar domains
What is the structure of a sliding clamp?
Ring shape, with 35 Å (3.5 nm) hole that encloses the double stranded DNA
What does processivity mean?
The ability of DNA polymerase to carry out continuous DNA synthesis on a template DNA without frequent dissociation
Why are sliding clamps important?
They enable high processivity of replicative DNA polymerase.
They increase the speed of nucleotide additions.
Fill in the gaps:
The sliding clamp binds ____ ________, sliding along the DNA with the _________ keeping it ________ to the ____. If the ________ releases the _____ of the nascent strand it cannot ________ away, so it ______ the _____ and continues DNA synthesis.
The sliding clamp binds DNA polymerase, sliding 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 away, so it rebinds the 3’OH and continues DNA synthesis.
True or False:
PCNA interacts with eukaryotic DNA polymerase δ through a motif of 8 amino acids in each PCNA subunit
True
True or False:
PCNA interacts with eukaryotic DNA polymerase θ through a motif of 6 amino acids in each PCNA subunit
False:
PCNA interacts with eukaryotic DNA polymerase δ through a motif of 8 amino acids in each PCNA subunit
True or False:
The Beta-protein has a short peptide motif that interacts with bacterial DNA polymerase III
True
True or False:
The Beta-protein has a short peptide motif that interacts with DNA polymerase ε
False:
The Beta-protein has a short peptide motif that interacts with bacterial DNA polymerase III
How long does it take for polymerase to reattach without the sliding clamp?
1 second
What is the job of a clamp loader?
To open and load the sliding clamps onto DNA at the prime template junction
What structure is the clamp loader in bacteria and eukaryotes?
It is made up of 5 subunits, and is a ring structure usually. It is a spiral structure when opened up.
What is the clamp loader in bacteria?
γ-complex
There are 3 copies of γ (or τ), one each δ and δ’)
{bookmark / look up this, not sure how it works}
What is the clamp loader in eukaryotes?
Replication Factor C (RFC)
There are 5 different but related polypeptides
What causes the conformational change that drives clamp loading?
Some of the clamp loader subunits are AAA+ ATPases. When ATP binds, the conformational change drives clamp loading
Fill in the gaps: Clamp loader
The clamp loader has low ______ for the sliding clamp until the loader is ________ __ ____.
On ________ ____, the clamp loader binds to the sliding clamp, forming a _____ shape and _________ the clamp.
The clamp loader-sliding clamp complex has a high ______ with the ______-________ _______.
When they bind, this stimulates _______ activity, which _______ the sliding clamp and ________ the clamp loader.
The sliding clamp remains associated with DNA.
The sliding clamp recruits ____ __________ to begin _________. The same region that was bound to the ______ ________ now binds to the ____ _________.
The clamp loader has low affinity for the sliding clamp until the loader is bound to ATP.
On binding ATP, the clamp loader binds to the sliding clamp, forming a spiral shape and opening the clamp.
The clamp loader-sliding clamp complex has a high affinity with the primer-template junction.
When they bind, this stimulates ATPase activity, which closes the sliding clamp and releases the clamp loader.
The sliding clamp remains associated with DNA.
The sliding clamp recruits DNA polymerase to begin elongation. The same region that was bound to the clamp loader now binds to the DNA polymerase.
When does the clamp loader have low affinity for the sliding clamp?
Before the clamp loader binds to ATP
What shape is formed when the clamp loader binds to the sliding clamp?
Spiral
What does the clamp loader-sliding clamp complex have a high affinity with?
The primer-template junction
When the clamp loader-sliding clamp complex binds with the primer-template junction, what does this do?
Stimulates ATPase activity, which closes the sliding clamp and releases the clamp loader.
What region of the sliding clamp binds to the DNA polymerase that it recruits to begin elongation?
The same region that was bound to the clamp loader
(should find out what this actually is?)
Fill in the gaps: Polymerase switching
The __________ ________ ______ synthesises RNA and ______ DNA. The complex dissociates and is replaced by one of the _________ eukaryotic DNA polymerases, __ __ __.
This handover is called _________ _______.
A _________ polymerase, __ __ __, is recruited by the _______ ______. It acts as a _____ for where the polymerase needs to come.
This mechanism ensures the replicative DNA polymerases are loaded onto DNA at the right _____ and in the right _____ to begin __________.
The polymerase α-primase complex synthesises RNA and initiator DNA. The complex dissociates and is replaced by one of the processive eukaryotic DNA polymerases, δ or ε.
This handover is called polymerase switching.
A replicative polymerase, δ or ε, is recruited by the sliding clamp. It acts as a label for where the polymerase needs to come.
This mechanism ensures the replicative DNA polymerases are loaded onto DNA at the right time and in the right place to begin elongation.
What is polymerase α-primase complex replaced by?
A processive eukaryotic DNA polymerase, either δ or ε depending on if it is the leading or lagging strand.
What is polymerase switching?
When the polymerase α-primase dissociates and is replaced by one of the processive eukaryotic DNA polymerases, δ or ε.
What recruits the replicative polymerases to begin elongation?
The sliding clamp recruits the replicative polymerases, δ or ε, acting as a label for where they need to go to.
True or False:
All organisms have multiple DNA polymerases with specialised functions
True
True or False:
Not all organisms have multiple DNA polymerases with specialised functions
False:
All organisms have multiple DNA polymerases with specialised functions
True or False:
DNA polymerase active sites (palm) are highly conserved.
True
True or False:
DNA polymerase active sites (palm) are not conserved.
False:
DNA polymerase active sites (palm) are highly conserved.
How are DNA polymerases grouped?
DNA polymerases are grouped into families according to the evolutionary lineage of the rest of the protein.
True or False:
DNA polymerases are more similar within family groups than within organisms.
True
True or False:
DNA polymerases are more similar within organisms than within family groups.
False:
DNA polymerases are more similar within family groups than within organisms.
DNA polymerases are more similar within family groups than within organisms. What does this indicate?
Early divergence of DNA polymerase groups.
Fill in the gaps:
Specificity of the DNA polymerase active site promotes ________ ____ _________. The active site is selective for correct ____ ______. The correct nucleotide fits precisely in the active site only when base paired with the ________ _____.
Mismatches have a ________ _____ to correctly matched bases and _____ ____ in the active site well.
___ ______ is required for this level of “__________”.
Base selectivity ensures an error rate of less than __________.
Specificity of the DNA polymerase active site promotes faithful DNA replication. The active site is selective for correct base pairing. The correct nucleotide fits precisely in the active site only when base paired with the template strand.
Mismatches have a different shape to correctly matched bases and don’t fit in the active site well.
No energy is required for this level of “proofreading”.
Base selectivity ensures an error rate of less than 1/100,000.
What about DNA polymerases promotes faithful DNA replication?
The specificity of their active sites. The correct nucleotide fits precisely in the active site only when the base pairs with the template strand.
What is the error rate from base selectivity?
Less than 1/100,000
Fill in the gaps:
Replicative DNA polymerases have __ __ __ proofreading ________ activity. The DNA __________ active site has ______ affinity for _____ when the incorrect nucleotide is present.
The proofreading _________ active site has an ________ affinity for ______ when the incorrect nucleotide is present, so the _________ active site removes the incorrect nucleotide. _______ is required for this process.
DNA synthesis is then resumed.
Replicative DNA polymerases have 3’ to 5’ proofreading exonuclease activity. The DNA polymerisation active site has reduced affinity for 3’OH when the incorrect nucleotide is present.
The proofreading exonuclease active site has an increased affinity for 3’OH when the incorrect nucleotide is present, so the exonuclease active site removes the incorrect nucleotide. Energy is required for this process.
DNA synthesis is then resumed.
True or False:
Replicative DNA polymerases have 3’ to 5’ proofreading exonuclease activity.
True
True or False:
Replicative DNA polymerases have 5’ to 3’ proofreading exonuclease activity.
False:
Replicative DNA polymerases have 3’ to 5’ proofreading exonuclease activity.
True or False:
The DNA polymerisation active site has reduced affinity for 3’OH when the incorrect nucleotide is present.
True
True or False:
The DNA polymerisation active site has increased affinity for 3’OH when the incorrect nucleotide is present.
False:
The DNA polymerisation active site has reduced affinity for 3’OH when the incorrect nucleotide is present.
True or False:
The proofreading exonuclease active site has an increased affinity for 3’OH when the incorrect nucleotide is present
True
True or False:
The proofreading exonuclease active site has reduced affinity for 3’OH when the incorrect nucleotide is present
False:
The proofreading exonuclease active site has an increased affinity for 3’OH when the incorrect nucleotide is present
How is the incorrect nucleotide removed by the proofreading exonuclease active site?
Proofreading exonuclease active site has an increased affinity for 3’OH when the incorrect nucleotide is present, so the exonuclease active site removes the incorrect nucleotide
Is energy is required for the incorrect nucleotide to be removed by the proofreading exonuclease active site?
Yes
True or False:
The proofreading exonuclease activity is part of the replicative DNA polymerases - they have two functions
True
True or False:
The proofreading exonuclease activity is performed by a separate protein than the replicative DNA polymerases
False:
The proofreading exonuclease activity is part of the replicative DNA polymerases - they have two functions
What is the difference between processive and distributive / non-processive synthesis polymerisation?
Processive: DNA polymerases stay attached for many 1000s of nucleotides. Association with the sliding clamp processivity factor enhances this.
Distributive: DNA polymerases add a few nucleotides before falling off.
What rate does DNA polymerase III work at with and without association with the sliding clamp?
Without: 10 base pairs per second
With: 1000 base pairs per second
How is movement of the leading and lagging strand DNA polymerases coordinated at the replication fork?
By looping round of the lagging strand template.
Relative to the direction of movement of the replication fork, what direction is the leading strand synthesised?
The same direction (this is why it can be continuous synthesis)
Relative to the direction of movement of the replication fork, what direction is the lagging strand synthesised?
The opposite direction (this is why the synthesis is discontinuous)
Define replisome
A complex of proteins acting at the replication fork that coordinates rate of synthesis between the leading and lagging strand during DNA replication
What does the bacterial replisome consist of?
DnaB helicase, DnaG primase, DNA polymerases, and the clamp loader complex.
What is the function of the bacterial replisome? How does it achieve this?
The replisome ensures the lagging strand DNA polymerase remains associated at the fork even though it is released at the end of each Okazaki fragment.
The lagging strand and leading strand bacterial DNA polymerases are tethered together via binding to the flexible linker protein Tau, an alternative subunit of the clamp loader complex.
If the leading and lagging strand synthesis was not coordinated in the bacterial replisome what would happen?
More and more single stranded DNA would be exposed on the lagging strand, making it unprotected and vulnerable to attack from nucleases.
What coordinates leading and lagging strand synthesis is eukaryotes and in bacteria?
Bacteria: replisome
Eukaryotes: Ctf4
True or False:
The leading and lagging strand synthesis in eukaryotes is coordinated by the replisome
False:
The leading and lagging strand synthesis in eukaryotes is coordinated by Ctf4
True or False:
The leading and lagging strand synthesis in eukaryotes is coordinated by Ctf4
True
What is the equivalent of the bacterial Tau protein linking the lagging and leading strand polymerases for eukaryotes?
There is no equivalent, the DNA polymerase ε
and DNA polymerase δ are not directly tethered.
What is the function of multisubunit Ctf4?
It acts as a hub to couple CMG helicase, DNA polymerase ε and polymerase α-primase at the fork
Fill in the gaps:
Okazaki fragments are _______ after synthesis.
In bacteria when the DNA polymerase III encounters the _________________________ it disassociates from DNA.
The sliding clamp _____________ and recruits ____ ________ ___, which binds to the 3’ __ at the end of the RNA primer and removes the RNA (with __’ to __’ exonuclease activity) and fills the gap with DNA.
A _____ in the phosphodiester backbone (a “____”) is left in the DNA, which is then ______ by ____ ______ ___.
Okazaki fragments are joined after synthesis.
In bacteria when the DNA polymerase III encounters the previously synthesised Okazaki fragment it disassociates from DNA (when it reaches the next RNA primer).
The sliding clamp remains attached and recruits DNA polymerase I, which binds to the 3’ OH at the end of the RNA primer and removes the RNA (with 5’ to 3’ exonuclease activity, removing it ahead of itself) and fills the gap with DNA.
A break in the phosphodiester backbone (a “nick”) is left in the DNA, which is then sealed by DNA ligase I.
What are the three activities of DNA polymerase I?
5’ to 3’ DNA synthesis activity (like with all DNA polymerases)
3’ to 5’ Proofreading exonuclease activity (taking off last base if incorrectly added)
5’ to 3’ exonuclease activity (can remove RNA or DNA ahead of where it is synthesising - called nick translation, it is like removing what is there and replacing it)
Which DNA polymerase has 5’ 3’ exonuclease activity?
DNA polymerase I
(It still has 3’ to 5’ proofreading exonuclease activity as well like other DNA polymerases)
What happens in bacteria when DNA polymerase III encounters a previously synthesised Okazaki fragment / another RNA primer?
It disassociates from DNA.
The sliding clamp remains attached and recruits DNA polymerase I to carry out “nick translation”.
What is nick translation?
When DNA polymerase I removes the RNA primer with 5’ to 3’ exonuclease activity and fills the gap with DNA.
Fill in the gaps:
DNA polymerase _ in eukaryotes__________________ when it meets the RNA primer / another Okazaki fragment. This ______ the _’ end of the existing Okazaki fragment / RNA primer + some ______ DNA previously synthesised (which may not have been very accurate) and DNA from the template strand, making a _____.
___________________ (enzyme) then ______ the _______.
DNA _______ seals the DNA “nick”. This joins the Okazaki fragments.
DNA polymerase δ in eukaryotes continues to synthesise DNA when it meets the RNA primer / another Okazaki fragment. This displaces the 5’ end of the existing Okazaki fragment / RNA primer + some initiator DNA previously synthesised (which may not have been very accurate) and DNA from the template strand, making a flap.
Flap endonuclease (Fen1) then cleaves the flap DNA. DNA ligase I seals the DNA “nick”. This joins the Okazaki fragments.
True or False:
DNA polymerase δ in eukaryotes continues to synthesise DNA when it meets the RNA primer / another Okazaki fragment.
True
True or False:
DNA polymerase δ in eukaryotes dissociates from DNA when it meets the RNA primer / another Okazaki fragment.
False:
DNA polymerase δ in eukaryotes continues to synthesise DNA when it meets the RNA primer / another Okazaki fragment.
(This displaces some existing Okazaki fragment, making a flap that is then cleaved)
Why is it a problem as more positive supercoils are formed ahead of the replication fork?
The more positive supercoils there are ahead of the replication fork the harder it is for the helicase to separate the strands.
For every turn of DNA that is unwound, how many supercoils are formed? Is this positive or negative supercoiling?
DNA ahead of the fork gains one positive supercoil for each DNA turn that is unwound.
Give two examples of when increased supercoiling might occur and make strand separation more difficult
When bacterial DNA has been replicated all the way around the chromosome and is nearing the end.
When 2 replication bubbles are nearing each other in eukaryotes.
Torsional stress generated ahead of convergent replication forks is resolved by what?
Topoisomerases
How do topoisomerases resolve supercoils to allow replication to be continued?
They transiently break DNA to remove the supercoils.
What is the function of topoisomerases?
To change supercoiled DNA to relaxed DNA
What are the names of the different types of topoisomerases?
Type IA
Type IB
Type II
What is DNA gyrase II?
Found in bacteria, it takes positive supercoils, turns them into relaxed DNA and then negatively supercoils them.
Why can introducing negative supercoils be useful?
When done ahead of a replication fork, some of the unwinding that will be done by the helicase can be ‘absorbed’ / offset by these negative supercoils.
True or False:
All organisms have topoisomerases
True
True or False:
Not all organisms have topoisomerases
False:
All organisms have topoisomerases
What organisms have type IA topoisomerases?
Eukaryotes and bacteria
What organisms have type IB topoisomerases?
Eukaryotes
What organisms have type II topoisomerases?
Eukaryotes and bacteria
Where does termination of replication occur for bacteria?
At ter sites within the termination zone
What is each ter site bound by in bacteria?
Each ter site is bound by a single molecule of Tus (“Termination utilisation substance”)
What does Tus stand for?
Termination utilisation substance
True or False:
ter sites bound by Tus stall the fork only in one direction
True
True or False:
ter sites bound by Tus stall the fork in all directions
False:
ter sites bound by Tus stall the fork only in one direction
What happens if an anticlockwise fork encounters a stalled clockwise fork at a Tus bound ter site?
Replication terminates as the replisomes move past each other.
Fill in the gaps:
In bacteria, termination of replication occurs at ___ _____ within the _____________. Each ____ is bound by a single molecule of ___. They _____ the fork only in one direction.
______ is used most frequently - it is the first to be encountered by the ________ fork. If the _________ fork encounters a stalled __________ fork at a ___ _______ __ site, replication terminates as the _________ move past each other.
DNA polymerase I and DNA ligase complete replication as termination is completed.
In bacteria, termination of replication occurs at ter sites within the termination zone. Each ter is bound by a single molecule of Tus. ter sites bound by Tus stall the fork only in one direction.
terC is used most frequently - it is the first to be encountered by the clockwise fork. If the anticlockwise fork encounters a stalled clockwise fork at a Tus bound ter site, replication terminates as the replisomes move past each other.
DNA polymerase I and DNA ligase complete replication as termination is completed.
Which ter sites are part of the clockwise fork trap?
(Not sure if this is just in the diagram or if actually is named) C B F G J
Which ter sites are part of the anticlockwise fork trap?
(Not sure if this is just in the diagram or if actually is named) A D E I H
Which ter is used most frequently?
terC
Which topoisomerase is used when all of the parental DNA has been copied?
Type II (cuts through the 2 strands)
Which topoisomerase is used when the parental DNA has only been partially copied?
Type IA (cuts through the part that is still only 1 strand)
Fill in the gaps:
In eukaryotes there are _______ origins of replication. Termination occurs ________ on the chromosome where __________________. They _______ at many _________ _____ due to their stochastic nature.
After replication, forks and replication machinery disassemble and the remaining DNA is filled in.
In eukaryotes there are multiple origins of replication. Termination occurs anywhere on the chromosome where two replication forks meet. Replication forks meet at many different sites due to their stochastic nature.
After replication, forks and replication machinery disassemble and the remaining DNA is filled in.
True or False:
In eukaryotes, termination of replication can occur anywhere on the chromosome, when two replication forks meet.
True
True or False:
In bacteria, termination of replication can occur anywhere on the chromosome, when two replication forks meet.
False:
In eukaryotes, termination of replication can occur anywhere on the chromosome, when two replication forks meet.
In bacteria, termination of replication occurs at ter sites within the termination zone.
True or False:
In bacteria, termination of replication occurs at ter sites within the termination zone.
True
True or False:
In eukaryotes, termination of replication occurs at ter sites within the termination zone.
False:
In eukaryotes, termination of replication can occur anywhere on the chromosome, when two replication forks meet.
In bacteria, termination of replication occurs at ter sites within the termination zone.
