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Genetics > DNA replication > Flashcards

DNA replication Flashcards

History, DNA synthesis and replication, cell cycle, enzymes.

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Biology 101 flashcards
1
Q

In 1953, who suggested a possible copying mechanism for genetic material after finding out pairing rules?

A

Watson and Crick.

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2
Q

In 1958, who found replication to be semiconservative?

A

Meselson and Stahl.

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3
Q

Describe the experiment Meselson and Stahl conducted.

A

They grew bacteria in a 15N - containing medium and 14N - containing medium.

They isolated the DNA, loaded into a centrifuge tube and centrifuged at a high speed for 48 hours to form a caesium chloride density gradient.

Heavy 15N DNA bands had a higher density so closer to bottom of the tube.
Lighter 14N DNA bands = lower density so closer to top of tube.

Introduced 14N bacteria into 15N - containing medium and band settled in middle of tube.

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4
Q

What does the hydroscopic characteristic of caesium chloride mean?

A

This solute isn’t very viscous so it is a good material for equilibrium gradient differential centrifugation where the separation of the particles is size and density dependent.

Hydroscopic = tendency to absorb moisture from the air.

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5
Q

Which direction does DNA synthesis take place in?

A

5’ to 3’ direction.

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6
Q

What is the role of Mg2+ involving DNA polymerase?

A

To stabilise binding between active site and nucleotide.

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7
Q

What is the purpose of a primer with a 3’ hydroxyl end?

A

Allows DNA polymerase a starting point to add deoxynucleotide triphosphates to.

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8
Q

Describe interphase of the cell cycle.

A

G1 phase - cell grows physically larger and copies organelles.
S phase - synthesis of a complete copy of DNA.
G2 - cell grows more and makes proteins/organelles.

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9
Q

What part of the cell cycle is this describing?

The chromosomes are separated and the cell begins to divide.

A

Mitosis.

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10
Q

What are telomeres?

A

Areas of highly repetitive DNA that protect chromosome ends from degradation, recombination and end fusion with other chromosomes.

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11
Q

What are centromeres?

A

Repetitive DNA which forms the spindle attachment site in mitosis.

Repetitive DNA = specific sequence of nucleotides repeated many times.

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12
Q

What are the origins of replication?

A

Special sequence where duplication of the DNA begins (each chromosome will have many origins).

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13
Q

Describe the differences between eukaryotic and bacterial genomes.

A

Eukaryotic genomes = large and arranged as linear chromosomes.

Bacterial genomes = small, compact and usually circular.

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14
Q

Where does the energy for polymerisation come from?

A

The breakage of the phosphoanhydride bond of deoxyribonucleotide triphosphate.

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15
Q

What happens to the released pyrophosphate?

A

Hydrolysed to 2 molecules of inorganic phosphate.

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16
Q

What are replication forks?

A

t’s where the double-stranded DNA helix splits at the origins of replication, creating two single strands that serve as templates for creating new DNA molecules.

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17
Q

How many origins of replication does bacterial DNA have?

A

One.

Forms 2 circular daughter DNA molecules.

18
Q

How is each strand copied at a replication fork?

A

In 5’ to 3’ direction.

19
Q

Describe the synthesis of the leading and lagging strands.

A

Leading strand synthesis = continuous.

Lagging strand synthesis = discontinuous ‘Okazaki fragments’ joined together.

Okazaki fragments = short sequences of DNA nucleotides synthesized discontinuously on the lagging strand during DNA replication.

20
Q

Describe the process of lagging strand synthesis.

A
  1. Primase joins 2 ribonucleotides and synthesises the primer in 5’ to 3’ direction = forms an Okazaki fragment.
  2. Primase synthesises a new primer and DNA polymerase adds nucleotides to the 3’ end of the primer.
  3. Previous RNA primer is removed by nucleases (catalyse cleavage of phosphodiester bonds) and replaced with DNA by repair polymerase.
  4. Nick sealed by DNA ligase.
21
Q

Where do DNA replication enzymes work?

A

At replication forks.

22
Q

What is the role of topoisomerases?

A

‘untwist’ DNA by breaking and reforming phosphodiester bonds.

23
Q

Why are topoisomerases needed?

A

DNA is folded into nucleosomes and chromosomes so helicase unwinding of DNA causes supercoiling (twists) ahead of replication fork (which need to be unwound by topoisomerases).

24
Q

What is the role of nucleases?

A

Hydrolytic enzymes that cleave phosphodiester bonds (between sugars and phosphates of nucleic acids).

25
Q

What is the role of DNA ligase?

A

Seals nick between Okazaki fragments.

26
Q

What is the role of primase?

A

Synthesise RNA primer.

27
Q

What are the roles of helicases?

A

To unwind DNA.

28
Q

What are the roles of DNA polymerases?

A

Synthesise new DNA strands.

29
Q

What is the role of single-stranded DNA binding proteins?

A

Stabilise ssDNA.

30
Q

What is the roles of sliding clamps?

A

To keep DNA polymerase on the DNA.

31
Q

Which direction is DNA polymerisation?

A

5’ to 3’

32
Q

Which direction is exonucleolytic proofreading?

A

3’ to 5’

33
Q

What is exonucleolytic proofreading?

A

Allows accurate replication:

Mismatch triggers proofreading.
Exonuclease activity removes the incorrect nucleotide from the 3’ end.
DNA polymerase repositions itself and adds the correct nucleotide.

34
Q

What is the role of MutS?

A

(Stand-directed) mismatch repair protein:

MutS detects incorrect base pairing in newly-synthesised DNA.

MutS scans along the DNA looking for kinks (due to mismatched base pair) and recruits DNA repair proteins to them.

35
Q

What are mutations in human mismatch repair genes associated with?

A

Predisposition to some cancers.

e.g. mutations in MutS are associated with a type of colon cancer.

36
Q

How many errors per nucleotide added are there for 5’ to 3’ polymerisation?

A

1 in 10^5

37
Q

How many errors are not corrected in 3’ to 5’ exonucleolytic proofreading?

A

1 in 10^2

38
Q

Hoe many errors not corrected are there in strand-directed mismatch repair?

A

1 in 10^3

39
Q

Therefore, how many errors per nucleotide added are not corrected with all replication steps combined?

A

1 in 10^10

40
Q

Describe the process of DNA replication.

A
  1. Helicases unwind parental double helix.
  2. Single-strand binding proteins stabilise unwound parental DNA.
  3. Leading strand synthesised continuously in 5’ to 3’ direction by DNA polymerase.
  4. Lagging strand synthesised discontinuously. Primase synthesises a short RNA primer which is extended by DNA polymerase to form an Okazaki fragment.
  5. After RNA primer is replaced by DNA (by another DNA polymerase) DNA ligase joins Okazaki fragment to the growing strand.

Genetics (10 decks)

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