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BIO111 Cell Biology > Lecture 17 & 18 DNA replication and repair > Flashcards

Lecture 17 & 18 DNA replication and repair Flashcards

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1
Q

Two strands of the DNA helix are ____.

A

Two strands of the DNA helix are antiparallel

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2
Q

the nucleotide sequence of one DNA strand is _____ to the nucleotide sequence of its partner strand

A

the nucleotide sequence of one DNA strand is complimentary to the nucleotide sequence of its partner strand

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3
Q

What are the models of DNA replication?

A

A) watson and crick theorised this model

B) others agreed on this model

B) others agreed on this model

An experiment was needed to show which model was correct?

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4
Q

[1] What is the experiment which proved that DNA is semi conservative?

A

1598 (meselson stahl)

  • worked out a method to differentiate the new DNA from the old DNA, using a heavy isotope of Nitrogen.
  • grew medium with the heavy and normal N isotope for many generations (to incorportate N into DNA)
  • burst open bacterial cells and loaded contents into tubes with cesium chloride salt which created a gradient
  • DNA will float or sink at the same density of the surrounding salt solution
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5
Q

[2] What is the experiment which proved that DNA is semi conservative? 1st Generation

A
  • differentiate parental DNA from new DNA
  • took bactieria from heavy medium and transfered to light medium (N-14)
  • let it replication one round, then extracted DNA, spun in density gradient

- DNA was in an intermediate position in the tube

(now we ruled out conservative model)

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6
Q

[3] What is the experiment which proved that DNA is semi conservative? 2nd generation

A

- ruled out dispersive model

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7
Q

DNA acts as a ____ for its own duplication. How?

A

DNA acts as a template for its own duplication.

  • due to complimentary nucleotide base pairing
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8
Q

DNA replication catalysed by DNA ____. How?

A

DNA replication catalysed by DNA polymerase.

  • DNA polymerase catalyses the addition of nucleotides to the 3′ end
  • The energy is provided by the incoming nucleotide
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9
Q

How does DNA replication start?

A

• To be used as a template:

  • the double helix must be opened
  • two strands separated to expose unpaired bases
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10
Q

What is the replication fork?

A
  • A localised replication region moves along parental DNA helix - has a Y shape – called a replication fork
  • DNA replication is bidirectional
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11
Q

DNA replication comes form the study of what group of organisms?

A

Bacteria

  • the mechanisms are conserved however differ from eukaryotes
  • circular chromosomes
  • replication fork moves relatively fast
  • Copy entire genome in around 30min
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12
Q

Eukaryotic chromosomes contain multiple origins of replication. What does this mean?

A
  • reduced time for DNA replication
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13
Q

The DNA replication fork is ____. How?

A

The DNA replication fork is asymmetrical.

At each replication fork:

  • one template runs in 3′ to 5′ direction
  • the other template runs in the opposite 5′ to 3′direction
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14
Q

DNA synthesis can only occur 5′-to-3′. What does this mean for the lagging strand (going away from fork)?

A
  • Lagging strands has the formation of Okazaki fragments made in correct 5’-3’ direction
  • Leading strand – grows continuously
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15
Q

What are the proteins that help to open up the DNA double helix?

A
  • For DNA synthesis to proceed: the double helix must be unzipped ahead of the replication fork
  • Two types of replication proteins are needed:

- DNA helicases

- Single-strand DNA-binding (SSB) proteins

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16
Q

What is DNA helicase?

A

Uses the energy of ATP hydrolysis to propel itself forward, prying apart the double helix

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17
Q

What are Single-strand DNA-binding (SSB) proteins?

A
  • the lagging strand fragemnts would natural become in an unorganised shape, making it very hard for the free nucelotides to bind
  • SSB coats the strand keeping it at straight composture for free nucelotides to bind.
  • bind tightly and cooperatively to ss DNA
  • also called helixdestabilizing proteins
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18
Q

What is the accuracy of DNA replication?

A
  • Fidelity of 5’-3’ polymerization = 1 error per 105 nucleotides
  • Incorrect pairs (mispairs) can be formed
  • Yet DNA polymerase only makes 1 mistake for every 107 nucleotides copied

DNA polymerase is self-correcting

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19
Q

Why DNA polymerase is selfcorrecting?

A

If DNA polymerase did nothing:

  • Wrong nucleotide would be incorporated (1 in 105 )
  • Frequent mutations (~30,000) would be produced
  • By self-correcting mutation rate reduced to 1 in 107 (1 error for every 10 million nucleotides copied)
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20
Q

What are the proofreading mechanisms?

A
  1. Monitoring: DNA polymerase ‘doublechecks’ the exact base pairing
  2. Exonucleolytic proofreading: Error correcting reaction - DNA polymerase can correct the mismatched nucleotides
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21
Q

Proofreading mechanisms: how does monitoring work?

A

DNA polymerase undergoes conformational change more readily with correctly base-paired nucleotide.

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22
Q

Proofreading mechanisms: how does Exonucleolytic proofreading work?

A

3′-to-5’ exonuclease

  • nuclease - cleaves the DNA strand*
  • exo - cleaving the end*
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23
Q

Why does DNA replication only occur in the 5’-to-3’ direction?

A

only occur in the 5’-to-3’ direction allows for efficient error correction, if it was in the other direction it would block further chain elongation.

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24
Q

[1] How can the polymerase start replication?

A

The mechanism involves a different enzyme called DNA primase

  • Doesn’t need base-paired end
  • Uses ribonucleoside triphosphates to synthesize short RNA primers
  • RNA polymerase
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25
Q

[2] How can the polymerase start replication, in reference to primers?

A
  • On the leading strand : Primer is only needed at the start of replication
  • On the lagging strand : New primers are needed continually

In eukaryotes: – RNA primers are about 10 nucleotides long (made at intervals of 100–200 nucleotides in eukaryotes)

26
Q

Why is an RNA primer preferred to a DNA primer?

A
  • Primase can begin new polynucleotide chains, but it does not proofread
  • Primers frequently contain mistakes (1 in 105 )
  • RNA primers are automatically marked as: - ‘suspect copy’ and are anyways removed and replaced by DNA
27
Q

DNA ligase joins ____ .

A

DNA ligase joins new Okazaki fragments.

28
Q

What are the 3 additional enzymes needed for lagging strand synthesis?

A
  • nuclease – degrades the RNA primer
  • repair DNA polymerase – replaces the RNA with DNA
  • DNA ligase – joins the 3′ end of the new DNA fragment to the 5′ end of the previous one
29
Q

What is the summary DNA replication?

A

The proteins at a replication fork cooperate to form a replication machine

30
Q

How is the lagging strand shaped?

A

The lagging strand is actually folded

  • allows for the lagging strand to have access to the complex of proteins when replication occurs
31
Q

What is a sliding ring?

A

holds a moving DNA polymerase onto the DNA

An accessory protein (PCNA in eukaryotes):

  • Functions as a regulated sliding clamp
  • Forms a large ring around DNA double helix
  • Keeps the polymerase firmly on the DNA
  • Increases processivity of the DNA polymerase
32
Q

What is a clamp holder?

A
  • Assembly of the clamp around DNA requires ATP hydrolysis by the clamp loader
  • Leading strand - loading needs to occur once
  • Lagging strand – the clamp is removed and reattached at the start of each new Okazaki fragment
33
Q

What is the “winding problem” refering to?

A

The “winding problem” arises during DNA replication

If the tension is not relieved the DNA will become overwound and supercoiled.

34
Q

[1] What is function of DNA topoisomerases?

A

to relieve the tension and prevent supercoiling

.

35
Q

[2] What is DNA topoisomerase I?

A

produces a transient singlestrand break

36
Q

[3] What is DNA topoisomerase II?

A

makes a transient double-strand break - Prevents tangling

37
Q

What is an end-replication problem?

A
  • DNA replication proceeds only in the 5′-to-3′ direction.
  • Lagging strand is synthesized in discontinuous fragments.

• A special problem arises: as the replication fork reaches the end of a chromosome

lagging strand would get shorter with each round of DNA replication

38
Q

What is the end-replication problem in bacteria?

A

This is not a problem beause DNA is circular and there are no ends

39
Q

What is Telomerase?

A
  • Eukaryotes “end-replication” problem:*
  • have special repetitive nucleotide sequences at ends of chromosomes (GGGTTA in humans)
  • incorporated into structures called telomeres
  • attract an enzyme called telomerase
  • elongates telomere DNA in the 5′-to-3′ direction
40
Q

How does Telomerase function?

A

Telomerase replicates the ends of chromosomes

41
Q

Telomerase is a _____.

A

Telomerase is a reverse transcriptase.

making DNA from RNA template

42
Q

What is present at the end of a mammalian chromosome?

A

A t-loop at the end of mammalian chromosomes to product ends of chromosomes → Cells can distinguish between ends of chromosomes and double-strand DNA breaks

prevents single transded dna from degrading, it is folded on itself and tucked inside

43
Q

What is the importance of DNA repair?

A
  • Genetic stability requires accurate replication, but also repair mechanisms
  • Most spontaneous changes in DNA are immediately corrected by a set of processes called DNA repair.
  • Importance of DNA repair is demonstrated by:

– Large investment in DNA repair enzymes

– Increased mutation rate following inactivation of a DNA repair gene

44
Q

A mismatch repair system removes ____.

A

A mismatch repair system removes replication errors.

Mismatch repair corrects 99% of replication errors

45
Q

How does them mismatch repair system work?

A

detects the potential for distortion in the DNA helix

• Must be able to distinguish between newly synthesized strand and original DNA strand

  • The two proteins involved in mismatch repair (MutS and MutL) are present in both bacteria and eukaryotic cells
  • e.g. MSH2 (MutS) mutations predispose to colon cancer*
46
Q

What are spontaneous DNA changes?

A

Sources of DNA damage:

  • Endogenous e.g. reactive metabolites (ROS, H20), replication errors.
  • Exogenous e.g. UV radiation, x-rays, mutagenic chemicals
47
Q

Without DNA repair, what would happen to spontaneous DNA damage?

What are the two examples of the most frequent cause of damage?

A

spontaneous DNA damage would rapidly change DNA sequences

  1. Depurination
  2. Deamination
  3. Thymine dimers (UV radiation)
48
Q

What is Depurination?

A

Depurination removes a purine base from a nucleotide

49
Q

Depurination - What happens when there is a baseless DNA region?

A
50
Q

What is deamination?

A

results in spontaneous loss of an amine group e.g. converts a cytosine base to a uracil

Most common example is with cytosine

51
Q

Deamination - what happened if this error was not corrected?

A
52
Q

Deamination - what happenes if the error goes undetected by the body?

A

Mutation “hotspots”

can affect how and when the gene is expressed

53
Q

What are Thymine dimers?

A

relates to UV radiation

Thymine dimers covalent linkage between adjacent pyrimidine bases.

two nucleotides fuse together

54
Q

What are the 2 pathways to remove DNA damage?

A
  1. Base excision repair (BER)
  2. Nucleotide excision repair (NER)

in both:

  • the damage is excised (nuclease)
  • the original DNA sequence is restored (repair DNA polymerase)
  • the double helix is sealed (DNA ligase)
55
Q

Base excision repair (BER) - How is the damage recognized?

A
  • recognize specific, non-bulky lesions in DNA.
  • damaged bases removed by specific glycosylases.
  • creates AP sites, with a baseless sugar. e.g. U is restored to a C
56
Q

Nucleotide excision repair (NER) - How is the damage recognized?

A
  • repairs bulky, helixdistorting DNA damage
  • bacteria - 12 nucleotide gap
  • human - ~30 nucleotide gap
  • Global vs transcriptioncoupled
    e. g. individuals with xeroderma pigmentosum, Cockayne syndrome
57
Q

What happens when a double strand breaks and what are 2 mechanisms for repair?

A
  • Double-strand breaks can lead to fragmentation of chromosomes and loss of genes
  • Especially difficult to repair as each chromosome contains unique information
  • 2 distinct mechanisms have evolved to deal with this type of damage:

- non-homologous end-joining

- homologous recombination

58
Q

How do the mechanisms if a double strand breaks work?

A
59
Q

What is Homologous recombination?

A
  • Genetic exchange takes place between a pair of homologous DNA sequences
  • Can only occur after DNA has been replicated and before the cell divides.
  • e.g. breast cancer caused by mutations in BRCA1 or BRCA2
60
Q

Examples of severe consequences due to failure to repair DNA damage?

A

Mutations in germ cells can lead to inherited diseases

  1. Sickle-cell anaemia A permanent change in a single nucleotide in the haemoglobin gene –> Haemoglobin with an incorrect sequence of amino acids
  2. Palmoplantar keratoderma​ (skin damage)
  3. Cancer mutations in somatic cells (The chance that a cell will become cancerous greatly increases with age)

BIO111 Cell Biology (11 decks)

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