slides exam 2 Flashcards
What is DNA replication?
- Duplication of the cellular genome
- Stored genetic information passed to next generation
- Highly regulated
- Requires sufficient resources
- Loss of control leads to cancer—uncontrolled cell division
DNA replication is said to be __________
semiconservative
How do we know about semiconservative replication?
Meselson and Stahl performed an experiment with 2 types of DNA
•DNA contains: C, H, O, N, P
•1 type of DNA contained the abundant isotope: N-14
•1 type of DNA contained a less abundant isotope: N-15
How did this lead to a discovery of semiconservative replication?
Different densities of DNA allows for separation by centrifugation
DNA replication is said to be _______
bidirectional
What is involved in prokaryotic replication?
1 Origin of replication 1 bubble 2 replication forks Semiconservative Bidirectional Sometimes called θ replication
What is involved in eukaryotic replication?
Multiple origins of replication Multiple bubbles 2 replication forks/bubble Bidirectional Semiconservative
What are the exceptions to bidirectionality?
Unidirectional replication occurs in some:
• Prokaryotes
•BacteriophageΦ29
•Plasmids
•Viruses
•Adenovirus
•Respiratory illnesses: common cold, pneumonia, croup, and bronchitis
DNA replication is _______
semi-discontinuous
Does replication have directionality?
Yes 5’ and 3’ ends.
Which direction does DNA polymerase only synthesize?
5’ to 3’ direction.
Can both daughter strands be synthesized in the same direction?
No
Can DNA polymerase initiate synthesize of a daughter strand?
NO, dna polymerase requires an RNA primer than is placed by primate.
What does DNA polymerase require to initiate synthesis of a daughter strand?
RNA primer that is placed by primase
Is DNA replication continuous?
no
What strand is synthesized continuously with the direction of the replication fork opening?
Leading strand
What strand is synthesized discontinuously in the opposite direction the replication fork is opening?
Lagging strand
What is the meaning behind discontinuous replication?
One strand (leading strand) is synthesized continuously whereas one strand (lagging strand) is synthesized discontinuously into fragments that must be glued together with ligase.
What are the fragments of the lagging strand called?
Okazaki fragments
Each Okazaki fragments starts with an RNA primer that is how many nucleotides long?
10-13 nucleotides
What is the fragment length in eukaryotes?
100-200 nucleotides
What is the fragment length in prokaryotes?
1000-2000 nucleotides
Summarize the 3 big takeaways of dna replication:
DNA replication is
- semiconservative
- bidirectional
- discontinuous
Summarize DNA polymerase
- Template: DNA
- Synthesizes: DNA
- Reading direction: 3’ 5’
- Synthesizing direction: 5’ 3’
- CANNOT initiate DNA chains
- Requires an RNA primer placed by primase
In what direction does DNA polymerase read the strand?
3’ to 5’ direction
What process is RNA polymerase involved in?
Transcription
What is RNA polymerases template?
DNA
What does RNA polymerase synthesize?
RNA
What direction does RNA polymerase read?
3’ to 5’
What direction does RNA polymerase synthesize a strand?
5’ to 3’
What process is the ribosome involved in?
Translation
What is a ribosomes template?
RNA
What does a ribosome synthesize?
A polypeptide
What direction does a ribosome read the template?
5’ to 3’
What is the synthesizing direction of a ribosome?
N –> C-terminus
What does DNA polymerase require?
an RNA primer
What is a template strand + a primer strand called?
A primed template
What is an example of an RNA primer?
OH-
What would an example of a primer terminus be?
3’ hydroxyl end
How many DNA polymerases does E. coli have?
5
Name the 5 DNA polymerases of E. coli
Poly I Pol II Pol III Pol IV Pol V
What is the function of E. coli DNA Poly I?
Okazaki fragment processing and DNA repair
What is the function of E. coli DNA Poly II?
Translation Synthesis
What is the function of E. coli DNA Poly III?
Chromosome replication
What is the function of E. coli DNA Poly IV?
Translesion synthesis
What is the function of E. coli DNA Poly V?
Translesion synthesis
What are the 3 DNA polymerases of E. coli that are 3’ –> 5’ exonucleases?
DNA poly I, II, and III
What is the only DNA polymerase of E. coli that functions as a 5’ –> 3’ exonuclease?
DNA polymerase I
What happens while adding nucleotides?
- DNA polymerase incorporates nucleotides 1 at a time
- Mistakes every 105-106 nucleotide additions
- Mistake can be removed
How often do mistakes occur while adding nucleotides? (how many nucleotides before a mistake)
about every 105-106 nucleotide additions there will be a mistake
Can mistakes be removed once they occur?
yes
How many nucleotides are added at a time?
DNA polymerase incorporates 1 nucleotide at a time
Specifics of adding nucleotides:
- 3’ OH— of the RNA primer attacks the α phosphate of the incoming dNTP
- β and γ phosphate leave as a pyrophosphate (PPi)
- dNMP is incorporated to the growing chain
What is the overall reaction of nucleotide addition?
•(dNMP)n + dNTP –> (dNMP)n+1 + PPi
What is the (dNMP)n represent in the reaction of nucleotide addition?
Existing chain
What does dNTP represent in the reaction of nucleotide addition?
incoming nucleotide
What does (dNMP)n+1 represent in the reaction of nucleotide addition?
Chain length +1
What does PPi represent in the reaction of nucleotide addition?
Pyrophosphate
What does magnesium facilitate on DNA polymerase?
The 3’ OH- attack of the α phosphate andthe removal of PPi are facilitated by 2 Mg2+ ions on
What are the 2 polymerase sites?
post insertion and insertion site
What happens at DNA polymerases sites? Which one translocates to the other one?
After a new dNMP is incorporated at the insertion site, the polymerase translocates (moves) until the dNMP is in the postinsertion site
What is pyrophosphorylation?
A nucleotide is release from a polymer by adding PPi
- reverse of an elongated chain
- dNMP is freed from a chain as dNPT when PPi is added
What is the formula for pyrophosphorylation?
(dNMP)n+ PPi (dNMP)n-1+ dNTP
What does pyrophosphatase do?
PREVENT phosphorylation
How does pyrophosphatase do its job?
By hydrolyzing PPi into Pi + Pi
What does the hydrolization of PPi into Pi + Pi explain?
This explains why dNTP is favored over dNDP or dNMP; dNTP + hydrolysis of PPi prevent the chain from undergoing pyrophosphorylation…
In other words, after making the chain longer, you don’t want it to fall apart
T/F Most DNA polymerases have nuclease activity
TRUE
What is a nuclease?
something that cuts phosphodiester bonds in nucleic acids
What are the 2 types of nucleases?
Exonucleases and Endonucleases
What is the difference between the 2 types of nucleases?
- Endonuclease cuts a strand internally
* Exonuclease cuts a nucleotide from the terminus of a strand
Name the 2 types of exonucleases and explain the difference:
- 3’ 5’ nuclease = nuclease that cuts at the 3’ end
- 5’ 3’ nuclease = nuclease that cuts at the 5’ end
- DNA Pol I has both 3’ 5’ and 5’ 3’ exonuclease activity
Which polymerase has activity both exonucleases?
DNA poly I
Define Proofreading:
When the wrong nucleotide is incorporated it can be removed and replaced with a different one
The error rate of mismatch was every 10^4-10^5 bases…
___________ exonuclease activity improves the error rate to ____________
3’ to 5’
10^6 - 10^8
What exonuclease activity is facilitated by magnesium ions?
3’ to 5’ exonuclease activity
Is proofreading the same as pyrophosphorolysis?
NO
Mismatched nucleotides are released as what?
dNMPs
Why does mismatch occur?
Tautomers can “fool” the enzyme into an incorrect base pair..
When tautomers revert back to their normal structure, polymerase can detect the mismatch
FUN FACT:
• Remember DNA pol I has 3’ 5’
and 5’ 3’ exonuclease activity
• 5’ 3’ exonuclease activity can function at the same time as its polymerase activity
What is Nick Translation?
- Allows the degradation of RNA primers at the same time they are replaced with DNA
- RNA primers are degraded from the 5’ end while new DNA is added to the 3’ end
- Nicks are repaired by DNA ligase
Which DNA polymerases intentionally allow mistakes?
DNA Pol IV and V
Which DNA poly is responsible for DNA repair?
II
What does the polymerase crystal structure consist of?
- fingers
- thumb
- palm
- DNA
- polymerase sites
When the correct dNTP is added a ____ degree conformation change is undergone by polymerase
40 degree
What happens when an incorrect base pair tries to attach to polymerase?
- Polymerase cannot fully close with incorrect base pair
- This incorrect base pairing is up to 1000x slower
- Allows time for wrong dNTP to disassociate
- Or allows time for 3’ 5’ exonuclease activity
- Also, a mismatched 3’ terminal nucleotide adds the next nucleotide more slowly more time (6 in the next figure)
What is distributive synthesis?
DNA polymerase adds a nucleotide, fully disassociates then rebinds DNA
What is processive synthesis?
DNA continually adds nucleotides without disassociating
What is the processivity number?
average nucleotides incorporated before polymerase disassociates
• Pol I: 10-100nt
• Pol III: 1000s (more in the next section)
