Topic 5 (DNA Replication) Flashcards
Why is eukaryotic chromosome replication challenging?
Multiple origins of replication, which may cause breakage
What were the findings of the Meselson and Stahl experiment?
Semi-conservative replication
True/False? DNA can be synthesized de novo
False
True/False? RNA can be synthesized de novo
True
On which end are nucleotides added to in replication?
3’
What kind of exonuclease activity does eukaryotic DNA Pol have?
3’ to 5’
Which two phosphates in the form of pyrophosphate are cleaved once a nucleotide is added to the growing backbone?
Beta and gamma
Define processivity. Example?
An enzyme’s ability to catalyze “consecutive reactions without releasing its substrate”; the average number of nucleotides added each time when an enzyme binds a primer:template junction
A give a course-relevant processive enzyme
DNA Pol
How many active sites does DNA Pol have for the 4 nucleotides?
1 used for all of them
What are the 3 functions of DNA Pol?
- Sterically distinguish between dNTPs and rNTPs (RNA)
- Show kinetic selectivity for adding the correct base-paired dNTP (A-T, G-C)
- Initiate synthesis using either an RNA or DNA primer
Why can’t DNA Pol add bases onto the 3’ end of ddNTPs?
ddNTPs lack a 3’ OH, so it cannot attack the 5’ phosphate of the incoming base
Explain how DNA Pol sterically distinguishes between DNA and RNA bases
Discriminator amino acids in its active site interact with the 2’ H. If an rNTP is in the active site (2’ OH), this causes steric hindrance and cannot be added (slide 10 for diagram)
Explain how DNA Pol distinguishes between base pairs
If the incoming base does not hydrogen bond correctly with the template base (incorrect orientation), it will not fit properly into the active site and cannot be added to the growing strand (slide 11 for diagram)
What does the structure of DNA Pol resemble?
A hand
What is the function of the “fingers” in DNA Pol?
Binds and encloses the dNTP
What is the function of the “thumb” in DNA Pol?
Maintains the correct position of the primer in the active site and a strong association between DNA Pol and its substrate
What is the function of the “palm” in DNA Pol? What cation is used?
Catalytic site and monitors base pairing of the most recently added bases (3’ to 5’ exonuclease activity); Mg2+ used for catalysis
What is the function of DNA helicase?
Unwinds DNA at the replication fork to create ssDNA template for primase
What is the function of primase?
RNA polymerase that makes short RNA primers used to anneal to an ssDNA template to serve as a starting point during replication
True/False? Primase can start de novo
True
In which direction does DNA Pol place bases during replication?
5’-3’
In which direction does DNA Pol read the template strand during replication?
3’-5’
What is the function of RNAse H?
Removes RNA primers from RNA:DNA hybrid to complete DNA synthesis
Which 3 subunits of eukaryotic DNA Pol are essential for function?
Alpha, delta, and epsilon
What is the function of DNA Pol alpha?
Primer synthesis
What is the function of the delta subunit?
Lagging strand synthesis
What is the function of the epsilon subunit?
Leading strand synthesis
True/False? All three subunits of eukaryotic DNA Pol have about the same processivity
False. Alpha has a relatively low processivity
Why is the alpha subunit’s processivity lower than the others?
Allows for polymerase switching
Explain polymerase switching
Replacement of DNA Pol alpha with epsilon and delta at the leading and lagging strands, respectively
What is the function of the sliding clamp? Where is it situated relative to the polymerase?
Increases DNA Pol processivity by keeping DNA Pol in close proximity to the template; 5’ of the polymerase
Define the origin of replication
Sites at which DNA unwinds to initiate synthesis
Define the replicator
The DNA seq that directs the initiation of DNA replication. It includes binding sites for the initiator protein and AT-rich seq that unwind easily
What is the initiator protein?
DNA binding proteins that sequentially bind to DNA involved in the initiation of replication
What is the replicon?
The DNA replicated from a particular origin of replication
What is the origin recognition complex (ORC)?
6-protein complex that recognizes the replicators and is required to recruit other replication proteins such as helicase
True/False? ORC interactions require ATP hydrolysis
True
True/False? ORC binding leads to strand separation
False. Strands do not separate until S phase begins (ORC must be activated first)
Describe the order of activation of replicators
3 and 5 activate
2 is passively activated by extension of 3
4 is passively activated by extension of 5
1 activates independently
When does each replicator become inactive? Why?
After it is replicated to prevent multiple replications
What does origin activation trigger?
Triggers the replicator associated complex to initiate DNA unwinding and DNA Pol recruitment
What is replicator selection?
The process to identify seq that will direct the initiation of replication
When does helicase loading begin?
Late G1
When does replicator activation begin?
S phase
What are the steps in helicase loading?
- Recognition of the replicator by the ORC
- ORC recruits helicase loading proteins (Cdc6, Cdt1) in the expense of ATP
- Cdc6 hydrolyses ATP and dissociates with 2 Cdt1’s while Mcm2-7 dimer wraps around DNA
- ORC hydrolyzes ATP
- Mcm2-7 dimer is ready to be activated
Which protein does Cdt1 recruit before binding to the ORC?
Mcm2-7
How many Cdt1’s and Mcm2-7’s are recruited per ORC?
2 of each
How are the Mcm2-7’s oriented?
Head to head
When is Cdc6 released?
After Mcm2-7 binds, the ATP is hydrolyzed and both Cdt1 and Cdc6 are released
What happens once the ORC hydrolyzes its ATP?
Activation of the Mcm2-7 dimer
When does the loaded helicase start unwinding the DNA?
S phase
What are the steps in helicase activation?
- DDK phosphorylates Mcm2-7 dimer and CDK phosphorylates Sld2/3
- Cdc45/Mcm2-7 (phosphorylated)/GINS/Pol epsilon/Dbp11/Sld2/3 (phosphorylated) associate together
- Formation of the CMG complex (Cdc45/Mcm2-7 (phosphorylated)/GINS/Pol epsilon), (Dbp11/Sld2/3 (phosphorylated) dissociate)
- Unwinding begins
- Pol alpha/primase and Pol delta are recruited
Is Pol epsilon recruited before or after DNA unwinding?
Before
Is Pol alpha recruited before or after DNA unwinding?
After
Is Pol delta recruited before or after DNA unwinding?
After
True/False? The sliding clamp can only associate with Pol delta
False. Can associate with Pol epsilon too
What happens when Mcm2-7 associates with Cdc45 and GINS?
Double hexamer into two single hexamer, one going in each direction of the DNA strand
True/False? After helicase activation, helicase encircles double stranded DNA as a single hexamer
False. It encircles single stranded DNA
Why is it important that DNA replicates only once during S phase?
Multiple replication events may have detrimental effects on the phenotype of the cell (may be cancerous, cytotoxic, etc)
How can eukaryotic cells limit thousands of origins of replication to become activated only once per cell cycle?
Temporal regulation of helicase loading and activation
What is low CDK activity required for?
Helicase loading in eukaryotes
What is high CDK activity required for?
Initiation of DNA replication
What happens to the Mcm2-7 complexes after S phase?
They are all removed from the DNA
In what phase(s) is CDK activity low? Why?
G1; helicase loading requires low levels
In what phase(s) is CDK activity high? Why?
S, G2, and M; helicase activation requires high levels (S) and prevents loading of new helicase (G2 and M) to restrict one replication per cycle
True/False? Circular DNA can be completely replicated
True
Which enzyme separates the two daughter DNA molecules after replication in prokaryotes?
Topo II (gyrase)
Why do prokaryotes lack telomeres?
Not needed because entire genome can be replicated
What is the end replication problem?
A small amount of DNA is not synthesized at the 5’ end of the new strand, so upon another round of replication, one strand will be shorter than the other
Which strand is shortened following the end replication problem?
Lagging
How many rounds of replication does it take to shorten the genome by one equivalent of a primer?
2 rounds
What is the ratio of complete:partially complete:shortened DNA following 2 cycles?
1:2:1
What are the possible solutions to the end replication problem?
- Use a protein primer instead of RNA
- Use telomeric sequence
Which organisms use protein primers?
Some linear bacterial chromosomes and viruses
What about the structure of a protein primer allows for full replication of the genome?
Uses the OH group of an amino acid residue (Ser, Tyr, Thr) to substitute as the 3’ end of an RNA primer
Which location should the protein primer associate with to ensure replication of the entire genome?
Very end of the 3’ end of the template strand
What is the human telomeric sequence? Richness?
5’-TTAGGG-3’; TG rich
What does the telomeric sequence serve as?
Origin of replication for the 3’ end of each chromosome
What is telomerase?
A ribonucleoprotein complex that consists of an RNA subunit and a reverse transcriptase subunit that polymerizes telomeres
What is the function of the RNA subunit in telomerase?
Serves as a template so no exogenous DNA template is used
What is the function of the reverse transcriptase subunit in telomerase?
DNA polymerase that uses the RNA template subunit
Where does telomerase associate with the DNA?
Past the 3’ end of the template
What are the similarities between telomerase and DNA Pol?
Require a template
Extend at the 3’-OH end
Use the same nucleotide precursors
Processive
What are the differences between telomerase and DNA Pol?
Telomerase:
RNA component
Does not require exogenous template
Able to use ssRNA substrate to make ssDNA (able to reverse transcribe)
RNA:DNA helicase activity displaces its RNA template from the DNA for repeated rounds of synthesis
True/False? Newly synthesized telomeric DNA is double stranded
False. Single stranded
How many copies of the complement telomeric sequence does telomerase have?
1.5x
What are the steps to telomere elongation?
- Telomerase associates past the 3’ end of ssDNA
- Reverse transcription of RNA subunit
- RNA template dissociates and reanneals to the last 4bp of the telomere (moves downstream)
- Repeat step 2-3
Why doesn’t the inclusion of the telomeres effect cellular function?
They are non-protein coding
True/False? After telomerase adds a telomere and the DNA is replicated, there is a 5’ overhang left of the telomere expansion
False. 3’ overhang
What inhibits telomerase activity in yeast?
Rap1 and Rif1/2
What recruits telomerase in yeast?
Cdc13
What inhibits telomerase activity in humans?
POT1 protein complex
What length of telomeres requires telomerase inhibition?
Long telomeres
What is the function of Shelterin? How?
Protects telomeres from recombination and chromosome fusion; creates a t-loop structure with the DNA that protects the telomeres (end creates a loop with itself)
