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Biochem FINAL > Chapter 34: DNA Replication > Flashcards

Chapter 34: DNA Replication Flashcards

1
Q

The process in which the DNA within a cell makes an exact copy of itself.

A

DNA Replication

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2
Q

Eukaryotic cells (self-cycle) replicate in what phase?

A

S phase

Must be done rapidly with control and accuracy

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3
Q

DNA Replication is considered

A

Highly coordinated

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4
Q

The E. coli genome has…

A

4.6 million base pairs

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5
Q

The E. coli is replicated in _____, at a rate of _____!

A

less than 40 minutes

Rate of 2000 nucleotides per second

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6
Q

Old duplex is conserved and new duplex composed of two completely new strands

A

Conservative

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7
Q

Sections of the old duplex dispersed somewhat randomly to the two daughter duplexes

A

Dispersive

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8
Q

Single strand of DNA is _____

A

Unstable

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9
Q

Base pairs face _____

A

Inward - poses a challenge for replication

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10
Q

Conservative replication takes a strand of DNA …

A

opens up the duplex and make a copy and close it back up

Start with original and make an exact copy

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11
Q

Dispersive replication is where you end up with

A

a hybrid where you could break the backbone of the DNA and religate it together

“Patchwork”

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12
Q

Two daughter duplexes with one each of the old and new strands

A

Semiconservative

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13
Q

Semiconservative was proposed by

A

crick and watson start with double stranded, split apart and end up with a copy. Two resulting strands with half of original and after second generation, two new strands

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14
Q

N14 is the _____ of nitrogen

A

Natural isotrope

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15
Q

Meselson Stahl Experiment

A

Developed a method to Get a very fine density gradient established in the tube and match it to the DNA

Can separate DNA that has N14 from N15 based on density

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16
Q

What gets labeled in the Meselson Stahl experiment?

A

Bases get labeled

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17
Q

In the Meselson Stahl experiment, First generation results eliminate _____

Second generation eliminates _____

A

First - conservative model

Second - dispersive model

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18
Q

New DNA is all grown in the presence of..

A

N14

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19
Q

By two generations, you end up with…

A

2 peaks at equal intensity (One peak is 50/50, the other is N14)

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20
Q

By the first generation, we know that the _____ model is not correct.

A

Conservative

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21
Q

By the second generation, progressively enrich…

A

Towards fully N14 but not with the discreet jump with two different peaks

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22
Q

_____ is at the heart of DNA replication

A

DNA Polymerase

Adds nucleotides 1 by 1

Phosphodiester bond formation

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23
Q

What are the 4 important characteristics of DNA Synthesis?

A
  1. Deoxynucleoside triphosphates and Mg2+ are required. dATP, dGTP, dCTP and dTTP
  2. A template strand is used to direct DNA synthesis.
  3. Chain extension is always 5’ to 3’
  4. A primer from which the new strand grows must be present
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24
Q

Binding of the _____ induces a structural change, closes over the nucleotide, forming a pocket into which only the correct nucleotide fits.

A

incoming dNTP

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25
Q

Specificity is determined by two factors:

A
  1. the correct hydrogen bonding between the incoming dNTP and the DNA template
  2. the overall shape of the incoming base
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26
Q

Specificity of replication is dictated by the _____.

A

Complementarity of bases

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27
Q

DNA Replication requires _____.

A

Highly processive polymerases

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28
Q

The holoenzyme responsible for the majority of DNA synthesis in E. coli is _____, a multi-protein complex.

A

DNA polymerase III

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29
Q

DNA polymerase III is highly processive - once it begins catalysis it rarely ____.

A

Releases the DNA substrate.

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30
Q

DNA’s replication process is…

A

Semi-conservative

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31
Q

In the Meselson-Stahl experiment, how many bands will conservative DNA show?

A

2 bands

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32
Q

Separation in the Meselson-Stahl experiment based on…

A

Density

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33
Q

The Meselson-Stahl experiment was used to determine…

A

The correct model for replication

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34
Q

The reaction catalyzed by DNA Polymerase produces…

A

1 extra base and the two phosphate groups drive the reaction forward

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35
Q

The primer will have a _____ and cleaves…

A

Hydroxyl group and cleaves the two phosphates of incoming base

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36
Q

The induced fit is not efficient, but…

A

Cannot afford to have errors in DNA, so it is better to have lost a little energy

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37
Q

dNTP removes _____ correct BP and will fall into the _____ section.

A

1/20

Exonuclease section

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38
Q

The base must match the _____.

A

Active site

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39
Q

1000x more accurate because of…

A

Polymerase I exonuclease activity

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40
Q

A holoenzyme does not require….

A

Any cofactors to activate

“Whole” how it is

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41
Q

DNA Polymerase III almost never…

A

Stops replicating, process keeps chugging

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42
Q

3 distinct stages of DNA replication:

A

Initiation, elongation and termination

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43
Q

Initiation

A

Adding primer

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44
Q

Elongation

A

Adding bases

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45
Q

Termination

A

Connecting fragments and ridding of primer/proofreading

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46
Q

DNA replication is carried out by a large complex containing multiple…

A

Proteins (>20)

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47
Q

The multi-step process of DNA replication:

A

Initiation

Helix must be unwound

Primer synthesis

Primer removal

One fork, two antiparallel strands

Connecting the pieces

Proofreading and error correction

48
Q

Replication begins in E. coli at a unique _____ site called the…

A

245 base pair site called the oriC locus (replication primer)

49
Q

Helicases are _____ and always require…

A

Unwinders and always require ATP

50
Q

Single-stranded binding protein (SSB) bind to _____ to prevent _____.

A

AT rich sites to prevent reattachment of the helix

51
Q

The initiation protein allows…

A

Unwinding of the helix

52
Q

RNA polymerase (primase) enables…

A

DNA synthesis to begin

53
Q

RNA primer adds _____ to serve as primer after DNA is unwound.

A

10 RNA nucleotides

54
Q

DNA template ———->RNA Primer ———->Newly synthesized DNA

What fills in the arrows?

A
  1. Primase
  2. DNA polymerase
55
Q

What prepares the double helix for unwinding?

A

Topisomerases

56
Q

When DNA is unwound for replication, _____ occurs due to overwinding in nearby regions of the helix.

A

Supercoiling

57
Q

Topoisomerases induce OR eliminate…

A

Supercoils

58
Q

Type 1 topoisomerases…

A

Eliminate supercoiling

59
Q

Type 2 topoisomerases…

A

Introduce supercoiling

60
Q

The “negative” supercoiling starts to…

A

Unwind (Areas between supercoiling are where its unwound

61
Q

Bacterial topoisomerase is a…

A

Therapeutic target

62
Q

What are two inhibitors of DNA gyrase that are used to treat bacterial infections?

A

Nalidixic acid and ciprofloxacin

63
Q

What prevents anthrax poisoning?

A

Ciprofloxacin

64
Q

DNA gyrase is what type of topoisomerase?

A

Type 2 in bacteria

65
Q

What two things are needed to separate the DNA strands?

A

Specific helicases and ATP hydrolysis

66
Q

Helicases require _____ and separates _____ to make the DNA available for DNA polymerase.

A

ATP and separates double helix

67
Q

Helicases have how many subunits?

A

6

68
Q

Defects in the helicase can result in…

A

Werner syndrome

69
Q

Werner syndrome causes…

A

Premature aging

70
Q

The site of replication is called the…

A

Replication fork

71
Q

The replication fork moves in one direction, so…

A

Both strands are copied simultaneously

72
Q

All DNA polymerases synthesize DNA only in the…

A

5’ –> 3’ direction

73
Q

Okazaki fragments have…

A

A primer between each fragment and allow 3’ –> 5’

74
Q

What begins each okazaki fragment?

A

RNA Primer

75
Q

What is the complex containing primase enzyme which synthesizes short pieces of RNA at the replication fork (complementary to the lagging-strand template)?

A

Primosome

76
Q

Primosome adds your…

A

Primer

77
Q

DNA polymerase uses the RNA primer to start…

A

The lagging-strand DNA snythesis

78
Q

A _____ is needed to start each Okazaki fragment.

A

Primer

79
Q

The lagging strand is _____ so that it passes through the polymerase active site in _____ direction, allowing synthesis to occur in the _____ direction.

A

Looped

Active site in 3’ –> 5’

Synthesis in 5’ –> 3’

80
Q

DNA polymerase I is the _____ polymerase and deals with _____

A

Proofreading, deals with primer

81
Q

The joining of Okazaki fragments by _____ and _____

A

DNA Polymerase I and DNA ligase

82
Q

Problem: After Okazaki fragment synthesis is complete, there is a _____ between the end of Okazaki fragment and RNA primer.

A

Gap

83
Q

Nick translation

A

DNA Polymerase I extends the Okazaki fragment while its 5–>3 exonuclease activity removes the RNA primer.

84
Q

Nick translation adds _____ where primer was which fixes gaps.

A

Base pair

85
Q

What is required to rejoin the break?

A

DNA Ligase (only needed for lagging strand)

86
Q

DNA Ligase solidifies _____ in DNA and causes DNA to become _____ now.

A

Nicks in DNA

Continuous DNA now

87
Q

What allows for removal of mismatched bases?

A

Exonuclease

88
Q

3’–>5’ exonuclease activity contributes to the remarkably high fidelity of DNA replication - less than ____.

A

10^-8 errors/BP

89
Q

Most uncorrected replication errors are subsequently corrected by….

A

The DNA repair enzymes (NOT proofreading)

90
Q

Mistake rate is approximately…

A

1/100,000 bps, but DNA pol III and repair enzymes fix many

91
Q

Human genome?

A

3.2 x 10^9 billion

92
Q

Fruit fly large chromosomes

A

10x larger than E. coli, more complex (Millions of base pairs)

93
Q

Human genome BPs?

A

6 billion

94
Q

Eukaryotic chromosomes are _____.

A

Large linear, double-stranded DNA molecules

95
Q

Prokaryotes have _____ DNA.

A

Circular

96
Q

_____ of replication are required with each origin of replication representing a unit or replicon.

A

Multiple origins

97
Q

Humans have how many replicons?

A

30,000 replicons

98
Q

Proteins called _____ allow only one replication per replicon per round of DNA synthesis.

A

Licensing factors

99
Q

DNA pol alpha is equivalent to _____ in prokaryotes

A

Pol I

100
Q

Displays primase activity, initiates DNA replication in eukaryotes, generating a DNA molecule approx. 20 dNTP in length

A

DNA Pol alpha

Don’t incorporate RNA

101
Q

DNA poly delta: A more processive enzyme, extends the chains. The switch from alpha to delta is called _____

A

Polymerase switching

102
Q

Unique structures at the ends of linear chromosomes =

A

Telomeres

103
Q

Telomeres are at the _____ of DNA

A

Free end of linear DNA

104
Q

Two problems telomeres have:

A
  1. Susceptible to damage by nucleases
  2. Due to nature of DNA synthesis, one strand will shorten upon each round of DNA synthesis
105
Q

IF telomere is shortened?

A

BAD = cell death

106
Q

What replicates telomeres and fixes the overhang/gap?

A

Telomerase

107
Q

Telomerase is RNA-dependent _____

A

DNA pol

108
Q

Telomerase contains…

A

An RNA template that it uses to extend the leading strand

109
Q

In rapidly dividing cells, including cancer cells, telomeres must be maintained by the telomerase to _____, which would lead to cell death.

A

Prevent shortening

110
Q

_____ activity is characteristic of cancer cells (replicate uncontrollably).

A

High-telomerase activity

111
Q

Which is NOT required for DNA synthesis?

  • deoxynucleotide triphosphates
  • a primer
  • a template strand
  • a DNA polymerase
  • endonucleases
A

Endonucleases

112
Q

The 3’ to 5’ exonuclease activity of DNA polymerase III is critical for:

  • the processivity of polymerization
  • removal of RNA primers
  • the high fidelity of polymerization
  • dimerization of the polymerase subunits
  • DNA polymerase III does not have a 3’ to 5’ exonuclease activity
A

The high fidelity of polymerization

113
Q

Enzymes which use the power of ATP hydrolysis to separate DNA strands are called:

  • Restriction endonucleases
  • Topoisomerases
  • Separases
  • Helicases
  • Primases
A

Helicases

114
Q

You set up a DNA replication reaction in a test tube using enzymes from E. coli. You add all the necessary components EXCEPT that you forget to add DNA ligase. When you then separate the strands to analyze the new DNA, what products would you expect?

  • no DNA product at all
  • normal full length DNA products only
  • a mixture of full length strands, and shorter strands that include both RNA and DNA
  • a mixture of full length strands, and shorter strands made of just DNA
  • only short strands, which are made of just DNA
A

A mixture of full length strands, and shorter strands made of just DNA

115
Q

You try your reaction again, remembering to add DNA ligase. This time, however, you forget to add primase. What products would you expect?

  • no DNA product at all
  • normal full length DNA products only
  • a mixture of full length strands, and shorter strands that include both RNA and DNA
  • a mixture of full length strands, and shorter strands made of just DNA
  • only short strands, which are made of just DNA
A

No DNA product at all

116
Q

Processivity refers to the:

A

Ability to catalyze many reactions without releasing the substrate

117
Q

Which statement about DNA replication in eukaryotes in FALSE? Eukaryotic DNA replication:

  • involves multiple DNA polymerases
  • occurs on linear double-stranded DNA molecules
  • utilizes multiple origins of replication
  • utilizes DNA primers made by primase
  • is highly processive
A

Utilizes DNA primers made by primase

Biochem FINAL (6 decks)

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