Week 1:
1. What is DNA synthesis?
Molecular events that allow assembly of new DNA strands.
2. What is DNA replication and what does it include?
The copying of double stranded DNA which is used for cell reproduction before cell division.
Include:
– DNA chain synthesis
– Initiation, elongation and termination
– Accuracy
– Daughter strand separation
– Distribution of chromosomes
3. Who proposed that DNA replicated by semi-conservatively?
Watson and Cricket.
4. How many phases are there in DNA replication?
3
G1: Growth: phase-1
G0 Growth arrest
S : Synthesis phase; DNA
replication
G2: Growth: phase-2
M: Mitosis, cell division
5. Explain the semiconservative model?
Parental strands separate making a copy of itself. Two strands
separate into daughter cells each with one new and one old strand.
6. Explain the conservative model?
, The parental molecule creates an entirely new double stranded
molecule, this helps conserve one the molecules in to two old
strands.
7. Explain the dispersive model?
The two parental strands are distributed randomly between the
two daughter molecules.
8. What is the Meselson-stahl experiment and what does
it propose?
This was an experiment devised to determine which
model DNA replicated through.
The experiment includes:
- E. coli were grown for several generations in a medium with
the heavier 15N isotope.
- E. coli cells with only 15N in their DNA were transferred to a 14N
medium and were allowed to divide;
-DNA was extracted periodically and was centrifuged on a salt
density gradient.
Results
• After one generation, DNA is “Half-heavy”. This is consistent with both semi-conservative
and dispersive models
• After ~ two generations, DNA is of two types: “Light” and “Half-heavy”. This is consistent
with only the semi-conservative model
9. Where does replication start?
• The point at which replication occurs is called the replication fork.
• The replication fork is the site of the progressive unwinding of the double-stranded
DNA and the duplication of the template DNA, and it travels along the length of the
DNA molecule, away from the ORI.
10. What is the origin of replication for prokaryotes?
The ORI in E.coli is called OriC and is a well-defined structure.
• The origin of E.coli is 245bp in length and contains the following elements.
, – AT-rich region (3 copies of 13 bp repeats)
– DNA boxes (5 copies of 9 bp repeats)
– GATC methylation sites (methylation of the Bacterial Origin regulates Initiation)
11. What does the model proposed by Okazaki explain about DNA replication?
When DNA replicated one strand is synthesised as a continuous molecule and the other is
made in pieces. Leading (continuous) lagging (discontinuous pieces).
– A polymerase reads the leading strand and adds complementary nucleotides to the
lower leading strand.
– The lagging strands direction of synthesis is in the opposite direction of the replication
fork, this makes it more complicated for the strand to replicate.
– The lagging strand is synthesised in short segments, the primase reads the template of
DNA and starts a synthesis of a short complementary RNA primer.
– A DNA polymerase then extends the pried segments forming Okazaki fragments.
12. What are the termination sequences in E. coli called?
They are called ter sites and they serve as binding sites for the Tus terminator protein.
Tus can block the progression of replication forks in specific directions
13. When does replication terminate?
Replications terminate when two growing forks meet in the terminus region (located 180
around the circular chromosome from the ORI).
Week2: Proteins and enzymes of DNA replication
Summary of the functions of proteins involved in DNA replication?
➢ DNA Polymerase: makes the new DNA strands, removes RNA primers, and fills in
gaps
➢ Helicase: separates double-stranded DNA into single strands
➢ Single-strand binding proteins (SSB): prevent the strands of parental DNA from re-
forming a double helix
➢ DNA Primase: synthesizes short RNA primers
➢ Topoisomerases/gyrases: removes coils ahead of replication fork
➢ Ligase: covalently attaches Okazaki fragments in the lagging strand after synthesis
1. How do DNA polymerases catalyse the formation of new poly-nucleotide chains?
Through the addition of nucleotides to the growing strand of new DNA.
All prokaryotic and eukaryotic DNA polymerases add nucleotides to the 3′-OH end of a
polynucleotide chain, so that the new chain grows in the 5’ ➔3’ direction.
2. How many different DNA polymerases have been found for E. coli and eukaryotic?
Prokaryotic DNA polymerases:
1) DNA Polymerase I: involved in DNA replication
2) DNA Polymerase II: replication restart
3) DNA Polymerase III: principal DNA replication enzyme (Replicase)
4) DNA Polymerase IV: functions in repair
5) DNA Polymerase V: functions in repair
, Eukaryotic DNA polymerases:
➢ DNA polymerase , and : main polymerases involved in nuclear DNA replication
(Replicases)
➢ DNA polymerase : DNA repair
➢ DNA polymerase : mitochondrial DNA replication
3. What is exonuclease activity?
Some polymerases have the ability to destroy newly synthesised strands right after the new
strand has been made.
• The 5’ -3’ exonuclease activity is used to excise RNA primers in a reaction called
“nick translation”
• The 3’-5’ exonuclease activity acts as a proof-reading mechanism: correcting
polymerase mistakes.
• It removes incorrectly matched bases, so that the polymerase can try again
4. How do we correct polymerase mistakes?
DNA polymerase moves along a single strand of DNA, building the complementary strand as
it goes. The two stranded molecule passes through the DNA polymerase molecule after
synthesis is complete. If the wrong base is inserted then the bond is unstable. Because the
double strand is passing through the DNA polymerase the missing base can be detected and
replaced.
Normal extension proof reading extension
5. Explain the structure of DNA polymerase?
The Klenow fragment of DNA polymerase I from E. coli comprises two main parts of the full
enzyme, including the polymerase unit. This unit approximates the shape of a right hand
with domains that are referred to as the fingers, the thumb, and the palm.
1. What is DNA synthesis?
Molecular events that allow assembly of new DNA strands.
2. What is DNA replication and what does it include?
The copying of double stranded DNA which is used for cell reproduction before cell division.
Include:
– DNA chain synthesis
– Initiation, elongation and termination
– Accuracy
– Daughter strand separation
– Distribution of chromosomes
3. Who proposed that DNA replicated by semi-conservatively?
Watson and Cricket.
4. How many phases are there in DNA replication?
3
G1: Growth: phase-1
G0 Growth arrest
S : Synthesis phase; DNA
replication
G2: Growth: phase-2
M: Mitosis, cell division
5. Explain the semiconservative model?
Parental strands separate making a copy of itself. Two strands
separate into daughter cells each with one new and one old strand.
6. Explain the conservative model?
, The parental molecule creates an entirely new double stranded
molecule, this helps conserve one the molecules in to two old
strands.
7. Explain the dispersive model?
The two parental strands are distributed randomly between the
two daughter molecules.
8. What is the Meselson-stahl experiment and what does
it propose?
This was an experiment devised to determine which
model DNA replicated through.
The experiment includes:
- E. coli were grown for several generations in a medium with
the heavier 15N isotope.
- E. coli cells with only 15N in their DNA were transferred to a 14N
medium and were allowed to divide;
-DNA was extracted periodically and was centrifuged on a salt
density gradient.
Results
• After one generation, DNA is “Half-heavy”. This is consistent with both semi-conservative
and dispersive models
• After ~ two generations, DNA is of two types: “Light” and “Half-heavy”. This is consistent
with only the semi-conservative model
9. Where does replication start?
• The point at which replication occurs is called the replication fork.
• The replication fork is the site of the progressive unwinding of the double-stranded
DNA and the duplication of the template DNA, and it travels along the length of the
DNA molecule, away from the ORI.
10. What is the origin of replication for prokaryotes?
The ORI in E.coli is called OriC and is a well-defined structure.
• The origin of E.coli is 245bp in length and contains the following elements.
, – AT-rich region (3 copies of 13 bp repeats)
– DNA boxes (5 copies of 9 bp repeats)
– GATC methylation sites (methylation of the Bacterial Origin regulates Initiation)
11. What does the model proposed by Okazaki explain about DNA replication?
When DNA replicated one strand is synthesised as a continuous molecule and the other is
made in pieces. Leading (continuous) lagging (discontinuous pieces).
– A polymerase reads the leading strand and adds complementary nucleotides to the
lower leading strand.
– The lagging strands direction of synthesis is in the opposite direction of the replication
fork, this makes it more complicated for the strand to replicate.
– The lagging strand is synthesised in short segments, the primase reads the template of
DNA and starts a synthesis of a short complementary RNA primer.
– A DNA polymerase then extends the pried segments forming Okazaki fragments.
12. What are the termination sequences in E. coli called?
They are called ter sites and they serve as binding sites for the Tus terminator protein.
Tus can block the progression of replication forks in specific directions
13. When does replication terminate?
Replications terminate when two growing forks meet in the terminus region (located 180
around the circular chromosome from the ORI).
Week2: Proteins and enzymes of DNA replication
Summary of the functions of proteins involved in DNA replication?
➢ DNA Polymerase: makes the new DNA strands, removes RNA primers, and fills in
gaps
➢ Helicase: separates double-stranded DNA into single strands
➢ Single-strand binding proteins (SSB): prevent the strands of parental DNA from re-
forming a double helix
➢ DNA Primase: synthesizes short RNA primers
➢ Topoisomerases/gyrases: removes coils ahead of replication fork
➢ Ligase: covalently attaches Okazaki fragments in the lagging strand after synthesis
1. How do DNA polymerases catalyse the formation of new poly-nucleotide chains?
Through the addition of nucleotides to the growing strand of new DNA.
All prokaryotic and eukaryotic DNA polymerases add nucleotides to the 3′-OH end of a
polynucleotide chain, so that the new chain grows in the 5’ ➔3’ direction.
2. How many different DNA polymerases have been found for E. coli and eukaryotic?
Prokaryotic DNA polymerases:
1) DNA Polymerase I: involved in DNA replication
2) DNA Polymerase II: replication restart
3) DNA Polymerase III: principal DNA replication enzyme (Replicase)
4) DNA Polymerase IV: functions in repair
5) DNA Polymerase V: functions in repair
, Eukaryotic DNA polymerases:
➢ DNA polymerase , and : main polymerases involved in nuclear DNA replication
(Replicases)
➢ DNA polymerase : DNA repair
➢ DNA polymerase : mitochondrial DNA replication
3. What is exonuclease activity?
Some polymerases have the ability to destroy newly synthesised strands right after the new
strand has been made.
• The 5’ -3’ exonuclease activity is used to excise RNA primers in a reaction called
“nick translation”
• The 3’-5’ exonuclease activity acts as a proof-reading mechanism: correcting
polymerase mistakes.
• It removes incorrectly matched bases, so that the polymerase can try again
4. How do we correct polymerase mistakes?
DNA polymerase moves along a single strand of DNA, building the complementary strand as
it goes. The two stranded molecule passes through the DNA polymerase molecule after
synthesis is complete. If the wrong base is inserted then the bond is unstable. Because the
double strand is passing through the DNA polymerase the missing base can be detected and
replaced.
Normal extension proof reading extension
5. Explain the structure of DNA polymerase?
The Klenow fragment of DNA polymerase I from E. coli comprises two main parts of the full
enzyme, including the polymerase unit. This unit approximates the shape of a right hand
with domains that are referred to as the fingers, the thumb, and the palm.
