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Genetics PMY-3 > L2. DNA replication In Prokaryotes > Flashcards

L2. DNA replication In Prokaryotes Flashcards

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1
Q

What is Conservative DNA replication?

A

Conservative Replication- the parental double strand is entirely duplicated to create a new double strand

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2
Q

What is Semiconservative DNA replication?

A

Parental strands are separated and used as a template to synthesize a new strand

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3
Q

What is Dispersive DNA Replication?

A

Parental strands are cleaved into segments, replicated and the resulting DNA strands are a “patchwork” of original DNA and new DNA

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4
Q

What are the thought to be the 3 possible ways of DNA replication?

A

Conservative replication

Semiconservative replication-thought to make the most sense but had to be proven

Dispersive replication

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5
Q

What technique did researchers studying DNA replication rely on?

A

Centrifugation

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6
Q

How is centrifugation done?

A

Bacteria are harvested from the culture

  • Bacteria in media are placed in a centrifuge tube and centrifuged in a fixed angle centrifuge to separate bacteria from media
  • bacteria is heavier than the media and pellets(collects) at the bottom of the centrifuge tube
  • Bacterial pellet is then collected (harvested) and lysed(opened up) to extract the genomic DNA
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7
Q

What is Sedimentation Equilibrium Centrifugation?

A
  • A solution of 6M CsCl is placed in a centrifuge tube
  • DNA sample is added(more than one molecule may be added)
  • While the sample is being centrifuged, the CsCl forms a density gradient where the molecules are more concentrated near the bottom of the tube and less at the top of the tube
  • The DNA settles in the tube where its density matches the density of the CsCl
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8
Q

After sedimentation equilibrium Centrifugation, as one descends the tube….

A

Increases the density of CsCl

Molecules at the top are lighter/less heavy molecules

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9
Q

What results from sedimentation equilibrium Centrifugation for 50-60 hrs?

A

After 50-60 hr at 100,000 x g results in the generation of a gradient of CsCl and banding of DNA

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10
Q

When did the Meselson-Stahl experiment occur?

A

1958

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11
Q

Give a brief overview of the Meselson -Stahl experiment

A

E. Coli was grown in a growth medium that had heavy isotope of nitrogen to incorporate in DNA duplication 15N

They then harvested the bacteria, isolated the DNA and used Sedimentation Equilibrium Centrifugation to determine the density of the DNA

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12
Q

Give in detail, the Meselson-Stahl experiment methodology

A
  • E. Coli DNA becomes uniformly labeled with N-15 in nitrogenous bases
  • used sedimentation equilibrium Centrifugation to determine the density of the DNA
  • N-15 labeled E. Coli transferred to N-14 only medium and allowed to replicate once so all newly formed DNA would contain (generation 1)
  • they then harvested the bacteria, isolated the DNA and used Sedimentation Equilibrium Centrifugation to determine the density of the DNA

They isolated a band of medium density

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13
Q

What was realized by isolating a band of medium density in the Meselson-Stahl experiment ?

A
  • A hybrid of N-15 and N-14
  • Not consistent with a Conservative Replication
  • May be either a Semi-conservative or Dispersive
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14
Q

How could of Meselson and Stahl discover whether DNA replication was semi conservative or Dispersive?

A

If they grew the bacteria for another generation

Dispersive: you would continue to see one hybrid band containing equal amount of N-15 and N-14

Semi-conservative: you would see one hybrid band containing N-15 and N-14 and a new band of only. N-14 appear

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15
Q

Who and when discoveredSemi-conservative replication in eukaryotes?

A

J. Herbert Taylor, Phillip Woods and Walter Hughes in 1957

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16
Q

What cells were used for Taylor, Woods and Hughes Experiment?

A

From root tips of broad beans Vicia faba

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17
Q

How was DNA replication observed in Taylor, Hughes and woods Experiment?

A

DNA was labelled using radioactive 3H-thymidine

Autoradiography was used to follow the incorporation of radioactive isotope into replicating DNA
-Photographic X-ray film is placed onto sample, radioactivity exposes the film

DNA is monitored for two cell cycles and radioactive isotope is followed at metaphase

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18
Q

Give an overview of the methodology of of the Taylor, Phillips and Hughes experiment

A
  • 3H-thymidine added to cellsjust before replication
  • After replication, the DNA is isolated at metaphase and exposed to X-ray film
  • film show evidence that both sister chromatids are labeled at metaphase
  • cells monitored for a second cycle, replication of DNA occurs without adding more 3H-thymidine
  • after replication, DNA isolated and exposed to X-ray film which shows that only one sister chromatid continues to be labelled with 3H-thymidine (occasional evidence of sister chromatid exchange )
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19
Q

What are the 5 components needed for DNA synthesis?

A
  1. All four dNTPs (deoxynucleoside triphosphosphate)
    • building blocks of the DNA molecule
  2. A fragment of DNA to act as a template
  3. DNA polymerase
  4. Magnesium ions (Mg2+)
    - required for DNA polymerase activity
  5. A primer providing a free 3’ -OH group
20
Q

How are DNA strands separated in DNA replication?

A
  • Several proteins are involved in first separating the DNA strands at the “ Origin of Replication”
  • Enzymes called Helicases and Topoisomerases will unwind the DNA
  • These create a Replication Bubble
  • Each side of the replication bubble is called a replication fork
21
Q

What is a DNA primase in DNA replication?

A
  • An enzyme that synthesizes short RNA sequences called Primers
  • These primers then serve as a starting point for DNA synthesis providing the 3’-OH group for synthesis of a DNA strand
  • uses DNA as a template
22
Q

How many types of DNA polymerase 3xist in bacteria?

A

3 types

23
Q

What are the properties of DNA polymerase 1?

A

Erases primer and fills in gaps on lagging strand

  • 5’ to 3’ polymerization
  • 3’ to 5’ exonuclease activity
  • 5’ to 3’ exonuclease activity
24
Q

What are the properties of DNA polymerase 2?

A

DNA repair

  • 5’ to 3’ polymerization
  • 3’ to 5’ exonuclease activity
25
Q

What are the properties of DNA polymerase 3?

A

Performs the majority of DNA synthesis

  • 5’ to 3’ polymerization
  • 3’ to 5’ exonuclease activity
26
Q

What is the catalytic activity of DNA polymerase 3?

A

DNA polymerase catalyze the formation of a phosphodiester bond between the 3’ OH group of the deoxyribose on the last nucleotide and the 5’-phosphate of the dNTP precursor

27
Q

What is the polymerase activity of DNA polymerase?

A
  • DNA polymerase finds the correct precursor dNTP that can form a complementary base pair with the nucleotide on the template strand of the DNA
  • Pyrophosphate is liberated as a result of this incorporating a single base
28
Q

What is the mis-incorporation activity of DNA polymerase 3?

A

Occasionally DNA polymerase will mis-incorporate a nucleotide into a growing DNA chain.

These misincorporations need to be repaired prior to the next DNA replication or a mutation will occur

29
Q

Give an example of DNA polymerase 3 mis-incorporation activity

A
  • Rare tautomeric form of C ( C*) happens to base-pair withA and is thereby incorporated by DNA polymerase into the primer strand
  • rapid tautomeric shift of C* to normal cytosine (C) destroys its base-pairing with A
  • unpaired 3’ -OH end of the primer blocks further elongation of primer strand by DNA polymerase
30
Q

What is the exonuclease activity of DNA polymerase 3?

A

3’ to 5’ activity/proofreading exonuclease activity of DNA polymerase clips off any unpaired residues at the primer terminus. It continues this activity until a base paired 3’ -OH Terminus is encountered

31
Q

Describe the beginning of DNA replication

A
  • Primase synthesizes a primer on the top and bottom strand
  • DNA polymerase 3 elongates (synthesizes) the complimentary strand on the top and bottom strand
  • Replication fork opens up more
  • DNA polymerase 3 continues to synthesize the bottom strand (leading strand)
  • Top strand(leading strand ) needs another primer added
  • Now DNA polymerase 3 can continue to synthesize the top strand
  • the replication fork opens up more
  • primase has to add a new primer for DNA polymerase 3 to continue synthesize the top strand
32
Q

What are the functions of topoisomerase 1 and 2

A

Relaxes over-wound DNA:

Severs over-wound DNA strands then reattaches it

33
Q

What is the function of ligase?

A

Joins DNA strands together by catalyzing the formation of a phosphodiester bond

34
Q

What is the function of helicase?

A

Unwinds helix

35
Q

What is the function of: Single strand binding proteins?

A

Prevent reattachment of original DNA strands

36
Q

What is the function of DNA polymerase 1?

A

Replacement if RNA primer with DNA

37
Q

What is the function of DNA polymerase delta (d)?

A

Eukaryotic enzyme responsible for lagging strand synthesis

38
Q

What is DNA polymerase epsilon(e)?

A

DNA synthesis of leading strand in eukaryotic cells

39
Q

What is DNA polymerase gamma(y)?

A

Mitochondrial DNA replication in eukaryotic cells

40
Q

What is DNA polymerase alpha-primase complex(Pol a-DNA primate complex)

A

RNA/DNA primers in eukaryotes to initiate DNA synthesis (about 10 ribonucleotides then adds about 20 deoxynucleotudes)

41
Q

What is the function of DNA polymerase 3 in prokaryotic replication?

A

Elongation of new strand

42
Q

Describe helicase in eukaryotes

A

Helicase- Mcm, isoforms 2-7

Function- replication helicase, unwinds helix

43
Q

What is DNA Gyrase?

A

Topoisomerase 2

44
Q

What is the function of topoisomerase 1?

A

Breaks one of the double strand and relegates it to relieve Supercoiling

45
Q

What is the function of gyrase(topoisomerase 2)?

A

Needs 2 molecules of ATP to religase 2 DNA strands together and relieve tension/Supercoiling

46
Q

What happens ahead of helicase?

A

Ahead of the helicase, DNA becomes supercoiled and topoisomerase is needed

Genetics PMY-3 (27 decks)

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