Lecture 9: Enzymology of Bacterial DNA replication [G]

Question 1

Who established the directionality of DNA synthesis?

Answer 1

Reiji and Tsuneko Okazaki in 1968

Question 2

What makes DNA replication semi-continous?

Answer 2

This combination of continuous and discontinuous synthesis is why DNA replication is called semi-continuous.

Question 3

Away from the replication fork….

Answer 3

lagging strand

Question 4

Towards replication fork…

Answer 4

leading strand

Question 5

How many nucleotides are Ozaki fragments in eukaryotes?

Answer 5

~100-200 nucleotides in eukaryotes

Question 6

How many nucleotides are Ozaki fragments in E. coli.?

Answer 6

~1000-2000 nucleotides in E. coli.

Question 7

What provides the energy for polymerisation during DNA replication?

Answer 7

Hydrolysis of the incoming nucleotide. If synthesis occured in the 3’ → 5’ direction, there would be no 5’ triphosphate available for hydrolysis: no energy for polymerisation.

Question 8

Tsuneko Okazaki. 5’ → 3’ synthesis permits EDITING: 3’ → 5’ does not

Answer 8

Tsuneko Okazaki. 5’ → 3’ synthesis permits EDITING: 3’ → 5’ does not

Question 9

What is interesting about the M13 bacteriophage?

Answer 9

it has a single-stranded DNA genome, not a double-stranded one

Question 10

Describe the life cycle of the M13 bacteriophage

Answer 10

• M13 binds to the F pilus of E. coli and squirts its single stranded genome into E coli.
• This single-stranded genome is then replicated into a double stranded genome that’s called the replicative form.
• Replication by ‘rolling circle’, and a long strand is spooled off and cut into pieces to make single stranded genomes.
• These single-stranded genomes are then packaged and the new virus is extruded out through the E coli.
• You then get the progeny phage
• Track by monitoring molecular weight of dna during replication

Question 11

What did Tsuneko Okazaki want to know?

Answer 11

how dna replication begins/ the specifics

Question 12

Describe Tsuneko Okazaki ‘s experiment

Answer 12

• Tsuneko Okazaki took the DNA and digested it with DNase and looked to see what was left.
• She got DNA nucleotides and little stretches of RNA. (she got no long DNA polypeptides)
• Proved that the primer for DNA synthesis is made of RNA, not DNA, as the DNA had been degraded.

Question 13

What did Arthur Kornberg notice in 1971?

Answer 13

The replication of M13 phage DNA from single-stranded (ss) infective form to double-stranded (ds) replicative form (RF) by an E. coli extract is prevented by rifampicin.

Rifampicin is an inhibitor of E. coli RNA polymerase

Question 14

Describe RNA primer synthesis

Answer 14

DNA primase synthesizes an RNA primer to initiate DNA synthesis on the lagging strand.

Question 15

Describe the lagging strand synthesis

Answer 15

① DNA primase manufactures an RNA primer on the lagging strand template. These primers provide free 3’-OH groups for DNA polymerase to begin DNA synthesis.

② The primed duplex(template and primer) is captured by Pol III and clamped. This forces the lagging strand template into a loop to align it with the replication machinery.

③ The helicase continues to unwind, Pol III replicates the leading strand continuously and extends the new primer on the lagging strand …

④ … until the old Okazaki fragment has been pulled back to Pol III.

⑤ The lagging strand and template are unclamped, releasing Pol III from the DNA.

⑥ DNA primase primes the lagging strand template with a new RNA primer.

⑦ DNA polymerase I replaces the RNA primer with DNA by removing ribonucleotides and adding deoxyribonucleotides. DNA ligase then seals the remaining nick, creating a continuous DNA strand.

⑧ … and the process restarts by clamping the new lagging strand primer

Question 16

Why is DNA only synthesises in the 5’ to 3’ direction?

Answer 16

• Incorporation of nucleotides involves hydrolysis of a triphosphate group, releasing energy.
• If synthesis were 3′ to 5′, incorrect nucleotide removal would leave a monophosphate at the primer, halting the process.
• The 5′ to 3′ direction allows editing and correction of errors.

Question 17

Describe the enzyme helicase

Answer 17

• Unwinds DNA strands, creating replication forks.
• Consumes large amounts of ATP due to its six ATPase subunits.

Question 18

What does DNA ligase do?

Answer 18

• It seals nicks between Okazaki fragments, requiring ATP.
• Uses ATP to form a phosphodiester bond between 3′-OH and 5′-phosphate groups.

Question 19

What are Okazaki fragments?

Answer 19

short DNA sequences that are created during DNA replication when the lagging strand is synthesized

Question 20

What are the steps of the Ozaki fragment joining by DNA ligase?

Answer 20

Step ①: DNA ligase uses ATP as an energy source, releasing pyrophosphate and attaching AMP(adenosine monophosphate) to the 5’ phosphate of the downstream fragment

Step ②: AMP is released and a phosphodiester bond is formed between the 3’-OH of the upstream Okazaki fragment and the 5’ phosphate of the downstream fragment

Question 21

What does the clamp holder hold?

Answer 21

2 molecules of Pol III

Question 22

Are the polymerases both oriented in the same direction?

Answer 22

Yes

Question 23

What is the clamp loader associated with ?

Answer 23

A helicase (6 subunits, each ATPase)

Question 24

What are the helicase associated with?

Answer 24

The DNA primase

Question 25

What is Pol1?

Answer 25

The repair enzyme

Question 26

What is DNA Pol I in association with?

Answer 26

DNA ligase

Question 27

Describe the leading strand synthesis

Answer 27

• DNA helicase unwinds the DNA helix, separating the strands
• ② DNA primase manufactures an RNA primer on the leading strand template. The primer provides a free 3′-OH group for the DNA polymerase to start synthesis.

③ The RNA-DNA hybrid is recognized and bound by DNA polymerase III. Pol III begins extending the primer by adding deoxynucleotides in the 5′ to 3′ direction.

④ The clamp holder transfers the two halves of the β clamp to Pol III, enabling high processivity.

⑤ New clamp halves maintain the clamp holder in a state of readiness

⑥ Helicase continues to unwind, and Pol III replicates the leading strand continuously in the 5′ to 3′ direction.

Question 28

Is it true that Pol III has very low processivity until it’s clamped?

Answer 28

Yes

Question 29

Why does DNA Pol III have low processivity?

Answer 29

If it was a highly processive enzyme, it could not release the new Okazaki fragment easily.

Question 30

Is DNA replication in eukaryotes similar to DNA replication in prokaryotes?

Answer 30

Yes, except for the fact that DNA replication in eukaryotes is slower and uses different proteins/enzymes

Question 31

How can single stranded DNA be formed?

Answer 31

if DNA is denatured and fails to re-anneal properly.

Question 32

Why does DNA replication require a supporting cast of SSB – single stranded DNA binding protein?

Answer 32

to stabilize the unwound DNA and prevent it from reannealing or forming secondary structures during replication.

Question 33

What is constantly being displaced by Pol III and being replaced as the helix is unwound?

Answer 33

SSB

Question 34

What enzymes deal with DNA supercoiling?

Answer 34

Topoisomerases

Question 35

What happens when you overwind DNA?

Answer 35

You get a positive supercoil

Question 36

What happens when you underwind DNA?

Answer 36

You get a negative supercoil

Question 37

Is most DNA negatively supercoiled?

Answer 37

Yes

Question 38

Can positive supercoiling occur upstream of the replication fork ?

Answer 38

Yes

Question 39

Does positive supercoiling make strand separation difficult or easy?

Answer 39

Difficult

Question 40

Is negatively supercoiled DNA or positively supercoiled DNA easier to replicate?

Answer 40

Negatively supercoiled DNA

Question 41

Is it true that topoisomerases convert very tight positive supercoils into negative supercoils?

Answer 41

Yes

Question 42

Name a type II topoisomerase that is found in bacteria

Answer 42

Gyrase

Question 43

Describe how a type II topoisomerase in bacteria works

Answer 43

① Topo II binds the positive supercoil …

② … makes a nick in both DNA strands …

③ … passes the DNA loop through the break and re-ligates it, making a negtaive supercoil

Question 44

Describe how a type I topoisomerase in bacteria works

Answer 44

① Topo I binds the negative supercoil …

② … makes a nick in one DNA strand …

③ … unwinds the DNA and re-ligates it, therefore allowing the replication complex to carry on moving forward.

Question 45

Is bacterial DNA polymerisation bi-directional?

Answer 45

Yes

Question 46

What does topoisomerase IV (a type II topoisomerase) do?

Answer 46

separates the catenated daughter chromosomes(two linked copies of the circular DNA) by a double stranded break and religation.

Question 47

What is the major problem regarding DNA replication in eukaryotes?

Answer 47

Telomeres

Question 48

What happens to a person the older their telomeres get?

Answer 48

Their telomeres (ends of chromosomes) get shorter

Question 49

What happens when the ends of telomeres get too short?

Answer 49

They start eating into genes that are required

Question 50

How many telomeres do we have per haploid chromosome?

Answer 50

2 (so 4 for a diploid cell)

Question 51

How do chromosomes get shorter with each replication?

Answer 51

• On the lagging strand, small pieces of DNA (called Okazaki fragments) are made using RNA primers.
• After replication, the RNA primers are removed.
• The gaps left behind by these primers are usually filled by DNA polymerase and the fragments are joined by DNA ligase.
• However, there is a problem with the very end of the lagging strand. At the very end of the chromosome, there’s no place to put the last primer.
• Without a primer, DNA polymerase can’t fill the gap.
• This leaves a small section of the DNA at the end of the lagging strand unreplicated, which is why telomeres (protective caps on chromosomes) get a little shorter after each cell division.

Question 52

Why do most chromosomes get shorter with each replication?

Answer 52

Because it’s an inbuilt ageing mechanism that stops cells dividing forever.

Question 53

Is it true that some cells express telomerase that extends telomeres?

Answer 53

Yes

Question 54

What is telomerase?

Answer 54

A reverse transcriptase that extends the telomeres

Question 55

How does telomerase prevent telomere shortening?

Answer 55

During DNA replication, the ends of chromosomes (telomeres) cannot be fully copied by regular DNA polymerase. Telomerase compensates for this by lengthening the telomeres, ensuring that important genetic information isn’t lost.

Question 56

In which cells is telomerase active in for extending the telomeres?

Answer 56

• some germline cells
• epithelial cells
• haematopoietic cells
• and in > 90% of cancer cell lines.

Question 57

What is responsible for the immortal phenotype of cancer cells?

Answer 57

• Telomerase
• In cancer cells, telomerase is often continously reactivated, allowing for unlimited growth.

Question 58

Does telomerase only bind to the 3’ end?

Answer 58

Yes

Question 59

Somatic cells = normal cells

Answer 59

Somatic cells = normal cells

Question 60

Which cells is telomerase usually inactive in?

Answer 60

In most normal (somatic) cells, telomerase is inactive, leading to gradual telomere shortening. This contributes to aging and limits the number of times a cell can divide.