Lecture 3 - DNA Replication Flashcards
What is the mutation rate during DNA replication? What does this mean for the frequency of protein alteration?
1 per 10^9 (billion) nucleotides
Protein: a 400 AA protein suffers 1 alteration every 200,000 years
How often do cells turnover?
Depends on the cell type:
- Epithelial cells of the gut: every few days
- Most every 7 to 15 years (eg: skeletal muscle)
- Frontal cortex: never
How does the mutation rate of DNA replication limit the size of our genome?
Because we want a genome that is large enough to allow complexity, but not so large that the odds of gaining a mutation is high => limits the genome to 50,000 essential proteins, and our genome is 2/3 of that limit
What is the genetic measurement minute?
1 minute = ~40,000 bp along the DNA molecule
Function of gene mut?
Mutagenesis
Function of gene dna?
DNA replication
Function of gene pol?
DNA polymerase
Function of gene rpo?
RNA polymerase
Function of gene uvr?
UV resistance
Function of gene rec?
Recombination
Function of gene dam?
DNA adenine methylation
Function of gene lig?
DNA ligase
Function of gene Ter?
Termination of replication
Function of gene ori?
Origin of replication
What is the Meselson-Stahl experiment?
- Cells were grown for many generations in a medium containing only heavy nitrogen, 15N, so that all the nitrogen in their DNA was 15N, as shown by a single band when centrifuged in a CsCl density gradient.
- Once the cells had been transferred to a medium containing only light nitrogen, 14N, cellular DNA isolated after one generation equilibrated at a higher position in the density gradient because it had replicated into hybrid helices with both 15N and 14N
- A second cycle of replication yielded a hybrid DNA band and another band, containing only [14N]DNA, confirming semiconservative replication
What is a theta, θ, structure?
Structure resulting from the replication of a circular chromosome because both strands are replicated simultaneously and bidirectionally
How many replication forks per origin of replication?
2
In what direction are nucleic acids synthesized?
5’ => 3’
In what direction is the DNA template being read?
3’ => 5’
What are the 2 types of eukaryotic replication and which one is more common?
- Unidirectional
2. Bidirectional (more common)
How to distinguish between unidirectional and bidirectional replication?
Autoradiography: when tritium (3H) is added for a short period just before replication is stopped, the label would be found at one or both replication forks, respectively
How long are Okazaki fragments?
150 to 200 nucleotides long
What enzyme catalyzes DNA elongation during replication?
DNA polymerase
Describe the 2 steps of DNA elongation and write out the equation.
- Mg2+ on DNA polymerase between 2 Asp residues facilitates the attack of the free 3’ (-OH) on primer strand to the alpha phosphate on an dNTP
- Another Mg2+ on DNA polymerase between 3 Asp residues facilitates the release of PPi
(dNMP)n + dNTP => (dNMP)n+1 + PPi
How do the Mg2+ of DNA polymerase catalyze DNA elongation during replication?
Both ions stabilize the structure of the pentacovalent transition state by interacting with negative charges on Os of the 3 phosphate groups of the incoming dNTP
Describe the mechanism of RNA replication.
RNA polymerases use a similar mechanism than DNA polymerase I
What are the 2 elements that DNA polymerases require?
- DNA template
2. RNA primer
What are 2 elements that help ensure the high level of accuracy of DNA replication?
- The standard A=T and G≡C base pairs have very similar geometries, and an active site sized to fit one will accommodate the other, so the active site of DNA polymerase which will be very specific to proper base pair
- DNA polymerase contains 3’ => 5’ exonuclease activity to chew back DNA to proofread its own work
How is it possible for an improper base pair to fit in the active site of DNA polymerase?
Chemical or randomly modified base that can fit
Describe the position of the 3’ -> 5’ exonuclease activity on DNA polymerase and how it works.
The exonuclease activity is located ahead of the polymerase activity as the enzyme is oriented in its movement along the DNA. A mismatched base has different H bonds with its base pair and impedes translocation of DNA polymerase I to the next site. Sliding backward, the enzyme corrects the mistake with its 3′→5′ exonuclease activity, then resumes its polymerase activity in the 5′→3′ direction.
What is a common base pair mismatch?
A rare tautomeric form of cytosine (C*) resembles T and can pair with A
What are tautomers? Eg?
2 molecules with the same molecular formula but different connectivity (chair vs boat conformations)
What are the 3 types of DNA polymerases in prokaryotes and eukaryotes? What are the 2 main differences between the 3?
I, II, and III in prokaryotes
Alpha, epsilon, and delta in eukaryotes
Main differences:
- Processivity = number of nucleotides added before the polymerase dissociates:
- I: 3-200
- II: 1,500
- III: more than 500,000 - Number of subunits:
- I: 1
- II: 7
- III: over 10
How are prokaryotic and eukaryotic DNA polymerases I different from the 2 others?
They contain a 5’ => 3’ exonuclease
What is a core of DNA polymerase III made of?
- Alpha subunit = polymerase activity
- Epsilon subunit = 3’ =>5 ‘ exonuclease activity
- Theta subunit = stabilization of epsilon subunit
How many cores does each DNA polymerase III have? What is the purpose of this?
2 so that both DNA strands can be simultaneously replicated
What is the role of the DNA helicase subunit of DNA polymerase?
Unwinding the DNA
Where are the beta clamps found on DNA polymerase? What is their role?
At the base of the cores and constitute a ring of proteins that surround the DNA guiding the DNA polymerase along the strand
Describe the composition of the beta clamps of DNA polymerase.
2 beta subunits with a core of alpha helices surrounded by a ring of beta pleated sheets
What are the 3 phases of DNA replication and transcription? Which is the most critical part?
- Initiation (most critical)
- Elongation
- Termination
What is another name for DNA helicase in prokaryotes?
DnaB protein
What enzyme is responsible for relieving the torsional strain generated by DNA unwinding during replication? 2 names
DNA gyrase = DNA topoisomerase II
What enzyme catalyses the synthesis of the RNA primer needed by DNA polymerase? 2 names
What kind of activity does it have?
Primase = DnaG protein
Has RNA polymerase activity
What is the role of the origin of replication?
Strand-separate the DNA to generate replication forks
What are the DUE sequences of the origin of replication? How many are there? How many base pairs in each? What is particular about them?
DNA Unwinding Element (3 of them): 13-bp sequences
Very AT rich
Where does the energy required for DNA replication initiation come from?
ATP
Describe the steps of DNA replication initiation.
- DNA a protein and ATP bind at the replication origin and twist the DNA to cause these AT rich sequences to pop open
- 2 DNA b proteins/helicases bind to DNA at each replication fork at DUE sequences, which requires both DNA c protein and ATP
At what 2 sites of the origin of replication does DNA a protein bind?
- R sites
2. I sites bound by Dna A only when the protein is complexed with ATP
What can we assume with PPi is a product of a reaction?
The reaction is irreversible because pyrophosphatase will degrade it
Do all enzymes have a processivity property?
YUP
What do a lot of the subunits of DNA polymerase III have in common?
Single stranded binding proteins that project single stranded nucleic acids from endonucleases
How many DNA polymerase cores at each replication fork?
2
What secondary structure interacts the most with DNA?
Alpha helix
What is found behind the primase on the DNA strand? Role?
Single stranded binding proteins protecting the single stranded DNA
What is the order of the enzymes on the DNA during elongation?
- DNA gyrase
- DnaB helicase of DNA polymerase
- Primase
- Single stranded binding proteins
- DNA polymerase
Describe elongation of the lagging strand. 5 steps
- Dna B/helicase unwinds DNA
- Primase binds Dna B and synthesizes the RNA primer and then dissociates
- DNA polymerase III adds nucleic acids until the fragment extends as far as the primer of the previously added Okazaki fragment and then discards the beta clamp it was using
- Clamp loader adds a new beta clamp to the DNA polymerase III core
- DNA polymerase I will use its 5’ to 3’ exonuclease activity to chew through the primer and right behind it its 5’ to 3’ polymerase activity will replace the primer
- DNA ligase uses ATP or NAD+ to make a phosphodiester bond between the 5’ end and 3’ end of the fragments (releasing AMP + PPi or NMN)
What happens to the discarded beta clamps during the DNA replication of the lagging strand?
Recycled: broken down and resynthesized
Describe the mechanism of the loading of the beta clamp from the core during the DNA replication of the lagging strand.
3 ATPs on the clamp loader are hydrolyzed to ADP which dissociates the clamp loader releasing 3 Pis and leaving the beta clamp on the DNA to close
Describe the DNA replication termination in prokaryotes.
The counterclockwise and clockwise replication forks encounter Tus proteins bound to Ter sequences positioned in 2 clusters with opposite orientations and you end up with concatenated chromosomes which are unwound by DNA topoisomerase IV (type 2)
What does Tus stand for?
Terminus utilization substance
How many times does replication happen per cell cycle? Why?
Once because it’s regulated by components of the cell cycle and is part of it
What happens at the origins of replication prior to it starting in eukaryotes? What is this called?
Assembly of a pre-replicative complex:
Origin is already bound by ORC at the origin of replication and Ccd6 binds using ATP which promotes the loading of the replicative helicase bound to Cdt1 along with ATP hydrolysis
What does MCM stand for? What is its role?
Minichromosome maintenance protein = bacterial DnaB helicase
What does ORC stand for?
Origin recognition complex
What does the commitment to replication require in eukaryotes? What is this called?
Synthesis and activity of S-phase cyclin-CDK complexes which interact with the proteins involved in the pre-replicative complex = licensing
During what phase of the cell cycle does DNA replication occur?
S phase
How does the eukaryotic cell cycle differ from the prokaryotic one?
It’s coordinated with extracellular events
How does the presence of multiple origins of replication affect DNA replication in eukaryotes?
Different regions of a chromosome are replicated at different times (highly condensed chromatin replicates later)
How long does it take for a whole eukaryotic human chromosome to be replicated?
~8 hrs
What is the last step of DNA replication of a chromosome? Explain the mechanism.
Telomere synthesis by telomerases using telomerase RNA as a template which then loops to “hide” the end of a chromosome
What is the active site of the telomerase protein called?
Palm
What are the MCM and ORC part of?
The cell cycle
What are the 4 parts of DNA polymerase III?
- 2 cores
- 2 beta clamps (1 on each core)
- Helicase
- 1 clamp loader
Describe elongation of the leading strand. 3 steps
- Dna B/helicase unwinds DNA
- Primase binds Dna B and synthesizes the RNA primer and then dissociates
- DNA polymerase III adds nucleic acids until termination is signaled
What is the equivalent of DNA b protein in eukaryotes?
Helicase MCM2-7
What 5 steps in DNA replication require ATP hydrolysis?
- DNa a protein binding to origin of replication
- DNa b and c binding to origin of replication
- Pre-replicative complex formation in eukaryotes
- Clamp loader detachment from beta clamp left on DNA
- DNA ligase activity (can also use NAD+)
What are AAA+ ATPases? Examples?
ATPases Associated with diverse cellular Activities
Eg: DNa, DNa B, DNa C proteins
What does NMN stand for?
Nicotinamide mononucleotide
