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Cellular and Molecular Biology > Lecture 9: Enzymology of BacterialDNA replication Rewatch > Flashcards

Lecture 9: Enzymology of BacterialDNA replication Rewatch Flashcards

Tuesday 15th October 2024

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1
Q

Who established the directionality of DNA synthesis?

A

Reiji and Tsuneko Okazaki in 1968

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2
Q

Away from the replication fork….

A

lagging strand

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3
Q

Towards replication fork…

A

leading strand

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4
Q

What provides the energy for polymerisation during DNA replication?

A

Hydrolysis of the incoming nucleotide

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5
Q

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

A

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

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6
Q

What is interesting about the M13 bacteriophage?

A

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

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7
Q

Describe the life cycle of the M13 bacteriophage

A
  • 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
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8
Q

What did Tsuneko Okazaki want to know?

A

how dna replication begins

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9
Q

Describe Tsuneko Okazaki ‘s experiment

A
  • 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.
  • Proved that the primer for DNA synthesis is made of RNA, not DNA, as the DNA had been degraded.
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10
Q

What did Arthur Kornberg notice in 1971?

A

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

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11
Q

Describe RNA primer synthesis

A

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

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12
Q

Describe the lagging strand synthesis

A

① DNA primase manufactures an RNA primer on the lagging strand template

② The primed duplex is captured by Pol III and clamped. This forces the lagging strand template into a loop

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

⑥ DNA primase primes the lagging strand template…

⑦ DNA Pol I and DNA ligase repair the gap

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

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13
Q

What are Okazaki fragments?

A

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

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14
Q

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

A

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

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15
Q

What does the clamp holder hold?

A

2 molecules of Pol III

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16
Q

Are the polymerases both oriented in the same direction?

A

Yes

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17
Q

What is the clamp loader associated with ?

A

A helicase (6 subunits, each ATPase)

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18
Q

What are the helicase associated with?

A

The DNA primase

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19
Q

What is Pol1?

A

The repair enzyme

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20
Q

What is DNA Pol I in association with?

A

DNA ligase

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21
Q

Describe the leading strand synthesis

A
  • DNA helicase unwinds the DNA helix, separating the strands
  • ② DNA primase manufactures an RNA primer on the leading strand template

③ The primed duplex is captured by Pol III

④ The clamp holder transfers the two halves of the β clamp to Pol III

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

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22
Q

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

A

Yes

23
Q

Why does DNA Pol III have low processivity?

A

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

24
Q

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

A

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

25
Q

How can single stranded DNA be formed?

A

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

26
Q

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

A

In order to prevent inappropriate base pairing in ssDNA during replication

27
Q

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

A

SSB

28
Q

What enzymes deal with DNA supercoiling?

A

Topoisomerases

29
Q

What happens when you overwind DNA?

A

You get a positive supercoil

30
Q

What happens when you underwind DNA?

A

You get a negative supercoil

31
Q

IS most DNA negatively supercoiled?

A

Yes

32
Q

Can positive supercoiling occur upstream of the replication fork ?

A

Yes

33
Q

Does positive supercoiling make strand separation difficult or easy?

A

Difficult

34
Q

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

A

Negatively supercoiled DNA

35
Q

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

A

Yes

36
Q

Name a type II topoisomerase that is found in bacteria

A

Gyrase

37
Q

Describe how a type II topoisomerase in bacteria works

A

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

38
Q

Describe how a type I topoisomerase in bacteria works

A

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.

39
Q

Is bacterial DNA polymerisation bi-directional?

A

Yes

40
Q

Topoisomerase IV (a type II topoisomerase) do?

A

separates the catenated daughter chromosomes by a double stranded break and religation.

41
Q

What is the major problem regarding DNA replication in eukaryotes?

A

Telomeres

42
Q

What happens to a person the older their telomeres get?

A

Their telomeres get shorter

43
Q

What happens when the ends of telomeres get too short?

A

They start eating into genes that are required

44
Q

How many telomeres do we have per haploid chromosome?

A

2 (so 4 for a diploid cell)

45
Q

How do chromosomes get shorter with each replication?

A
  • As the replication complex reaches the telomere, at the lagging strand there is enough space to lay down a new primer.
  • The DNA polymerase complex then falls off
  • The primers are then erased
  • The gap made by the primers is filled by a DNA polymerase and repaired by a ligase.
  • The gap on the lagging strand can’t be filled by a DNA polymerase as there is no primer
46
Q

Why do most chromosomes get shorter with each replication?

A

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

47
Q

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

A

Yes

48
Q

What is telomerase?

A

A reverse transcriptase that extends the telomeres

49
Q
A

Telomerase provides an RNA template to synthesise a DNA copy of the template at the 3’ end of the parental lagging strand template. The result is that telomeres are built of repetitive motifs.

50
Q

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

A
  • some germline cells
  • epithelial cells
  • haematopoietic cells
  • and in > 90% of cancer cell lines.
51
Q

What is responsible for the immortal phenotype of cancer cells?

A

Telomerase

52
Q

Does telomerase only bind to the 3’ end?

A

Yes

53
Q
A

Cellular and Molecular Biology (25 decks)

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