DNA replication Flashcards
What direction do the DNA strands run to one another?
The DNA strands run anti-parallel to one another
In the double helix, where are the base pairs located?
The base pairs are stacked on the inside
What 2 grooves are there on DNA?
There is a major and a minor groove in DNA
What charge is the phosphodiester backbone in DNA?
The backbone has a negative charge
Is the DNA a right or left handed double helix?
DNA runs in a right handed double helix
What are the building blocks of DNA called?
Deoxynucleotide triphosphates
What does DNA synthesis require and what are the different types of this?
It requires deoxynucleotide triphosphates
dNTP- dCTP, dGTP, dATP, dTTP
On the following diagram label the phosphate groups either alpha, beta or gamma
On DNA there is a 5’, 4’, 3’, 2’ and 1’ carbon, why are these referred to as prime carbons?
A prime carbon is a carbon that is located on a sugar
What is the driving force of DNA synthesis?
The hydrolysis of pyrophosphate
What functional group is located at both the 5’ and 3’ end of ssDNA?
An alcohol group
When new nucleotides attach to DNA, what end do they join onto?
They attach onto the 3’ end of DNA
In the Watson-Crick (W-C) model of DNA, what are the base pairs and the number of H bonds between each one?
The base pairs are;
AT- 2 H bonds
CG- 3 H bonds
Whats some of the issues with DNA replication?
Everytime a cell divides its entire DNA content must be exactly replicated so that a comple copy is given to each daughter cell
Whats the genome size for E. coli and humans?
E. Coli (4.6x106 bp)
Humans (6x109 bp)
What type of replication is DNA replication?
Semi-conservative replication (as demonstrated by Meselson and Stahl)
What happened in the experiment that Meselson and Stahl undertook in order to support their theory for semi-conservative replication?
- They grew bacteria on heavy, non-radioactive isotope of nitrgoen (15N)
- Cells containing the labelled DNA were then transferred to a medium containing the normal isotope of Nitrogen (14N)
- The DNA was then isolated after each generation
- The DNA was seperated by a density gradient centrifugation
- At zero: All DNA has 15N
- After 1 generation: All DNA has a density between 15N and 14N
- After 2 generations: 50% has a density of 14N and 50% is an intermediate
- After 3 generations: 75% has a density of 14N and 25% is an intermediate
Is there an origin of replication of DNA on the E. coli chromosome?
Yes
In E. Coli does DNA replicate bidirectionally or unidirectional ?
What does this mean?
From the origin of replication in E.Coli, the DNA replicates bidirectionally
This means it starts from its origin and replicates round the circle
The following image shows bidirectional replication, what experiment was done to prove this was the case and why does this show bidirectional as opposed to unidirectional?
Firstly the genome was exposed to a pulse of low radioactivity before then being chased with high radioactivity
Bidirectional replication showed to start of strong, go weak in the middle then strong at the ends (like seen in the image with the thin and thicker bands)
Unidirectional replication tends to starts week before being strong at the end
What 4 main issues make DNA replication a much more complex idea then whats originally thought and why are these make it difficult?
- The antiparallel nature of the DNA strands:
when DNA is opened up it is exposing in different directions and as DNA syntheses only works in 5’ to 3’ then the 3’ to 5’ is a problem
- The coiling of the two strands around each other: could get twisted up as moving so fast per second
- The circular nature of (bacterial) genomes: start at one end and have to go around in a circle and also possible multiple origins of replication
- Stacking of bases within the helix: everything you need is in the middle of the helix
What was the first enzyme that was isolated and by who?
The first enzyme to be isolated what DNA Polymerase I by Kornberg in 1958
Tell me about the structure of DNA polymerase I (number of chains, mw, molecules per cell)
Its a single polypeptide chain (mw 109,000) about 400 molecules per cell
What does DNA polymerase I require?
It requires all 4 dNTPs, template strands and primers
Why can DNA polymerase I only add onto existing 3’-OH terminus
As its not self-priming
Whats the strand thats used to produce the DNA in the 5’ to 3’ direction?
The 3’ which is the template strand
Polymerisation is processive what does this mean?
This means that it moves along the DNA before randomly falling off after about 10-100 nucleotides added per binding event. This is roughly 10 nucleotides per second
What does Pol I bind to ?
Nicked or gapped (not to intact ds DNA or ss DNA)
Whats Nick translation?
Its when there is a breakage in the phosphodiester backbone and then pol I can attach to the 3’ end of the DNA and synthesis and produce more DNA whilst removing further DNA bases in front as it movves along the DNA chain
DNA polymerase I is an enzme with multiple activities, what are they?
5’-3’ polymerase
3’-5’ exonuclease (proof-reading)
5’-3’ exonuclease (nick translation)
When DNA polymerase I is treated with protease, what does it produce?
Small N-terminal fragment (5’- exonuclease) and large C-terminal fragment (Klenow fragment) which contains polymerase and 3’-exonuclease
The crystal structure of the Klenow fragment looks as shown in the image, label each of the parts and its direction?
What is at each site?
Where does DNA sit?
Palm has an active site
Fingers is the position template
Thumb binds the DNA as it leaves and is important for processivity
DNA sits in the palm of the enzyme
What does Klenow fragments require and what are they held in place by?
They require 2 bound metal ions of Mg2+ and these are held in place by aspartate residues
What is the role of the Klenow fragment?
It positions the 3’- end of the primer and incoming dNTP in order to enhance the catalytic reaction
Tell me about the Klenow fragments shape and why it is like this?
The shape is complementary to the correctly formed base pair in order to lead to specificity e.g. GC and AT pairs are the same size and shape
incorrect pairings are wrong in size and shape in order to fit into the active site of the enzyme
How does the Klenow fragment achieve the complimentary shape?
Its achieved by specific hydrogen bonding (‘molecular ruler’) and the conformational change of the polymerase upon binding to its correct substrates
What can’t the Klenow fragments incorporate and why?
They cannot incorporate ribonucleotide triphosphates due to a steric clash with the 2’-OH on the sugar
How does the enzyme “decide” between polymerase and exonuclease activity?
The exonuclease pocket is seperate from the polymerase site on the enzyme
Addition of a new base is fast compared to the exonuclease activity
However, addition to a mismatched base is slow and allows time for the strand to contact the exonuclease site
Is Pol I the main replicative enzyme?
No, its importany but not the main
Tell me about the rate of polymerisation in Pol I, why is this the case?
Polymerisation is slow (it would take >100 hours to replicate the E. Coli genome)
Its only moderatly processive (20-100 nts per binding event but needs to replicate the whole genome)
Theres too many copies per cell (>400; which is wasted energy)
Whats the role for DNA pol I in vivo ?
in vivo: In vivo refers to when research or work is done with or within an entire, living organism
PolA is the gene for the protein DNA pol I
PolA mutants are viable and can replicate (1% normal activity) but accumulated small DNA fragments and cells were UV sensitive with a high rate of mutation
Suggests that Pol I has a role in replication (and repair) but is NOT the ‘real’ replicative enzyme (will revisit Pol I later)
PolA is a protein involved in the origin of replication
What is pol II and pol III only detectable in?
Whats their activity and why is this the case?
polA mutants , the combined activity of pol II and pol III is less than 5%
This is because they are usuaully masked by DNA pol I
What are the 5 polymerases that have now been isolated in E. coli?
Whats each of their functions?
Pol I: Functions in repair and replication
Pol II: Functions in DNA repair
Pol III: Principle DNA replication enzyme (the main replicative enzyme)
Pol IV: Functions in DNA repair
Pol V: Functions in DNA repair
How many molecules does DNA pol III have per cell?
10 molecules per cell
Tell me about the rate of polymerisation in pol III
High rate of polymerisation (1600 nucleotides per second)
Whats DNA pol III essential for?
Viability, cells can’t divide without it
Tell me about the processivity of pol III
Its higly processive, it doesn’t fall off once it starts replicating DNA as the strand is clamped by another protein
What protein is pol III clamped by?
>50,000 nucleotides are added per binding event because it is clamped onto the DNA by the beta subunit (dnaN)
Whats pol III structure described as?
Complex multimeric enzyme
Tell me about pol III polypeptide components?
It has 22 polypeptide components of at least 10 types
DNA pol III is a Holoenzyme, with 2 core enzyme pol III which each consist of three subunits, what are these subunits?
α, ɛ and θ
What does the alpha subunit of pol III undertake?
polymerisation of 3’-OH additions
Whats does the e subunit of DNA pol III undertake?
3’ exonuclease- proof reading
What does DNA pol III not contain?
a 5’-3’ exonuclease
How can we determine which enzymes (proteins) are important?
Since any mutation will be lethal this is studied using conditionally lethal mutants - temperature-sensitive mutants
Powerful tools for studying loss-of-function phenotypes, in particular essential genes, since the mutation is only lethal (conditionally) at certain temperatures, e.g. cells survive at 20º C but not 37º C
Mutation usually effects protein structure
Then by seeing which proteins are effecting and what can’t occur if they don’t work, we can see their importance
Mutants are divided into two broad categories, what are these and what do these mutants do?
Give examples for each
- Quick stop mutants: These immediately halt DNA replication (e.g. dnaE, dnaG, lig…)
- Slow stop mutants: These allow replication to finish but stop further replication from happening after (e.g. dnaA)
Dont need to know but for interest, here are some enzymes involved in dna replication
So give some general facts as to why so many enzymes are needed for DNA replication
- DNA strands are antiparallel
- All DNA polymerases are 5’-3’, extending at the 3’-OH of a pre-existing strand. Not self-priming – need a primer
- DNA strands are plectonemically coiled and need to be physically separated for semi-conservative replication
- Pol III can’t do anything if there is something ahead of it, it has to stop, at this case then another pol like Pol I can take over
Since the two DNA strands are antiparallel to each other, and all polymerases work from 5’-3’, how is the lagging strand synthesized?
Overall synthesis of this strand must be in the 3’-5’ direction
What was Okazaki’s experiment?
E. Coli culture —► Infect cells with phage T4 —► Add 3H-thymdine
—► Take samples at intervals —► lyse with alkali (pH 13)- ss DNA
—► alkaline sucrose density gradient
What did Okazaki’s experiment show?
- The short fragments are always present and there is more of the longer DNA fragments
- Short fragments are the precursors of the long one’s and can be “chased” into long ones
- Can do a pulse-chase experiment (as shown in image)
This image shows what Okazaki’s experiment proved, what’s wrong in it?
The image shows that the leading strand is made in bits, where actually it is formed as a continuous part so this is where the image is incorrect
Tell me about lagging strand synthesis, the enzyme used and what the enzyme requires
The lagging strand is synthesised in short pieces, which are subsequently joined together by DNA ligase.
DNA ligase requires a 3’-OH and 5’-P at adjacent complementary base pairs. It also need ATP (or NAD) and joins the nick
some organisms may use the NAD instead of ATP for this process
DNA replication is descirbed as being semi-discontinuous, why is this the case?
The leading strand is synthesised continuously and the lagging strand is synthesised discontinuously, and this is why it is called semi-discontinuous replication
Okazaki’s experiment suggested that both strands are synthesised discontinually, but the quantitative interpretation of Okazaki’s experiment was wrong, why?
The reason is that U is being removed from the newly synthesised DNA which results in transient breaks which results in short fragments
What is an Okazaki’s fragment?
Okazaki fragments are pieces of DNA that are transient components of lagging strand DNA synthesis at the replication fork
They are only found in the lagging strand as this is discontinuous. The leading strand doesn’t have them as its produced continuously
What are the steps to the synthesis of thymidylate and whats used at each stage?
- UMP kinase (pyrH)
- ribonucleoside diphosphate reductase (nrdAB)
- nucleoside diphosphate kinase (ndk)
- dUTPase (dut)
- thymidylate synthase (thyA)
- dTMP kinase (tmk)
- DNA pol III (dnaE)
What are the 2 ways that uracil (U) appear in DNA?
are they offensive?
- U is incorporated in place of T roughly once in every 1200 bases opposite A
Pol III uses UTP in place of TTP
This incorporation is non-offensive as U has the same base pairing properties as T
- U can arise in situ from the spontaneous deamination of C
This is offensive because it generates a GU base pair in place of GC, which will cause mutation to AT at the next round of replication.
GC –> GU –> AU
How is uracil removed from DNA?
In the majority of species, uracil residues are removed from DNA by Uracil-N-glycosylase (ung)
However, in certain archaeal organisms uracil can be eliminated by AP(apyrimidinic) endonuclease (xthA)
When uracil is removed from DNA, how is the gap in the chain filled?
The gap is then filled in by DNA pol I (nick translation) and sealed by DNA ligase
Whats the main purpose of uracil-N-glycosylase ?
To remove U opposite G, but it doesnt discriminate U opposite A
During replication temporary nicks are created on the leading strand, what are these known as?
What two mutations can cause this?
pseudo-Okazaki fragments
ung and dut mutants can cause this
When is true semi-discontinuous replication evident?
If the removal of U is prevented
ung- mutant (no uracil-N-glycolsylase)
Whats dut-mutant?
dut- mutant – defective dUTPase, causes increased levels of dUTP –> more incorporation into DNA –> v. short fragments as more Us are removed at the replication fork
What are the two methods for proof reading ?
- Strand extension at the 3’-end only proceeds if the correct base has been added. If not then the 3’-exonuclease activity of pol III (dnaQ) removes it.
- dnaQ- mutants accumulate errors
What do all known DNA polymerases require?
a primer and extended onto the 3’-OH
What are primers synthesises by?
An enzyme called primase (dnaG)
What type of polymerase is primase?
An RNA polymerase (but not the one involved in transcription) its a specialist RNA that makes primers involved in DNA replication and as the primers are RNA its called RNA polymerase
Tell me some features/ characteristics of primase?
- self-priming (adding nucleotides in the 5’-3’ direction)
- no editing functions (proof reading)
- primers are 5-10 nucleotides in length
When is primases activity increased?
In the presence of helicase (this enzyme opens up the replication fork)
How is the theory of primase tested?
What happens with the RNA primers are removed and the gaps need to be sealed?
- Pol I takes over on the lagging strand when the newly synthesised strand meets the previous RNA primer and removes the RNA primer by nick translation- using its 5’-3’ exonuclease activity
- The gap is then sealed by DNA ligase
- This seals the “nick” in the phosphodiester backbond, between 3’-OH and a 5’-P
- NO bases are added
Pol I is processive but not very, what does this mean?
It doesn’t know when to stop but will just fall off at some point, sometimes it goes past the RNA prikmer but this doesnt matter
Even though DNA replication on the leading strand is continuous, where are primers required?
Where are primers required on the lagging strand as well?
Leading: Primers at the beginning
Lagging: Primers at beginning and for each Okazaki fragment
For continual opening, the DNA strand must be unwound, what is this done by?
Enzymes called Helicases
The main helicase enzyme is a product of what?
The dnaB gene
Whats meant by cooperativity?
Where the binding of one protein enhances the binding of an adjacent protein
During replication does the dimer dissociate from the DNA template? If it doesn’t then what does it do?
The lagging strand is “looped out” before synthesis in the 5’ to 3’ directions
Label the subunits in the pol III holoenzyme
How does the ß-clamp slide along the DNA chain?
The clamo uses water as a lubricant when sliding along DNA
Label this cryo-EM structure
σ = ΔLk/N (typically -0.06)
What is this accomodated by?
What can it result in?
This could be accomodated by changing the value of h, the helical repeat
But it can also result in coiling the DNA molecule
What leads to positive and negative supercoiling?
Extra helical twists: lead to positive supercoiling
Subtractive twisting: lead to negative supercoiling
How do topoisomerases work?
by nicking the DNA backbone, forming an enzyme-DNA complex, linked between the nicked phosphate and tyrosine in the active site of the protein.
The complex then ‘swivels’, rotating one strand around the other and the nick is then resealed
What does the topoisomerase II (IV) do and how does this effect the value of L?
They cut both strands of a duplex and passess one strand through the other
This process decreases L (the linking number) by 2
How does DNA gyrase effect the value of L?
It decreases L by 2 at a time; A2B2 structure
DnaA is an AAA+ protein, what is this?
ATPases associated with diverse cellular activities
What does OriC contain and what is the role of this?
What does it lead to?
OriC contains a large number of GATC sequences- substrates for dam (DNA adenosine methylase)
These methylates N6 of adenine. immediately after replicated these will be hemi-methylates. Hemi-methylation inhibits initiation
Hemi-methylated DNA at OriC has a strong affinity for what?
The cell lipid membrane-possibly assisting segregation of the two daughter chromosomes
How does the FEN1 exonuclease work?
FEN1 takes junction and cuts it very precisely at the flap overhand
It leaves no spaces
Leaves no nucleotides missing
Instead leaves a nick in the chain which is sealed by DNA ligase
What is the end of replication problem that arises in eukaryotic replication?
Why is the end of replication problem not an issue in organisms with a circular genome?
What can some organisms do to overcome the end of replication problem?
