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BIOL253 Genetics > DNA Structure and Supercoiling > Flashcards

DNA Structure and Supercoiling Flashcards

Lecture 1 (Complete) (Video notes included)

1
Q

Why is Fred Griffith important?

A

In 1928 he demonstrated the Transforming Principle: material isolated from heat-killed virulent bacteria can transform non-virulent bacteria into a virulent form. He used Streptococcus pneumoniae.

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2
Q

Why were Oswald Avery, Colin MacLeod and Maclyn McCarty important?

A

In a follow-up experiment to Griffith’s Transforming Principle, in 1944 they demonstrated nucleic acids were the material that carried the information, by fractionating material isolated from heat-killed bacteria.

Transforming activity destroyed when nucleic acids treated with deoxyribonuclease but not with ribonuclease, so transforming principle was DNA.

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3
Q

Why were Alfred Hershey and Martha Chase important?

A

In 1952 they confirmed DNA as the genetic material by labelling bacteriophage T2 with either 35S [labels proteins] or 32P [labels nucleic acids].

Only 32P detected in infected bacteria and in phage progeny.

35S isolated in phage ghosts which fail to enter bacteria.

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4
Q

What is a polynucleotide?
Give two examples

A

A polymer of a nucleotide

DNA and RNA

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5
Q

When writing nucleotide sequences, which direction is convention?

A

5’ to 3’

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6
Q

How are nucleotides joined together in DNA?

A

A phosphodiester bond between the 3’ OH of one sugar and the phosphate attached to the 5’ hydroxyl of the next sugar.

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7
Q

What is the repeating unit of DNA?

A

The sugar phosphate backbone

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8
Q

What is a dAMP?

A

A nucleotide

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9
Q

What is the similarity / difference between a DNA sugar and RNA sugar?

A

Both have pentose sugars (5 carbon) but for RNA there is an extra oxygen at the 2’ carbon (OH) whereas for DNA it is only hydrogen (H) .

Therefore ribose is more reactive.

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10
Q

True or false:
Nitrogenous bases / nucleobases are planar rings, typically uncharged under physiological conditions.

A

True

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11
Q

True or false:
Nitrogenous bases / nucleobases are planar rings, typically charged under physiological conditions.

A

False:
Nitrogenous bases / nucleobases are planar rings, typically uncharged under physiological conditions.

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12
Q

What does it mean if a base is a pyrimidine?

A

It is made up of a single ring

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13
Q

What does it mean if a base is a purine?

A

It is made up of a double ring

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14
Q

What are single-ringed bases called?

A

Pyrimidines

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15
Q

What are double-ringed bases called?

A

Purines

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16
Q

Which bases are pyrimidines?

A

Cytosine, Thymine, Uracil

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17
Q

Which bases are purines?

A

Adenine, Guanine

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18
Q

True or False:
Positions in sugars are referred to using the prime symbol

A

True

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19
Q

True or False:
Positions in sugars are referred to without the prime symbol

A

False:
Positions in sugars are referred to using the prime symbol

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20
Q

True or False:
Positions in bases are referred to using the prime symbol

A

False:
Positions in bases are referred to without the prime symbol

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21
Q

True or False:
Positions in bases are referred to without the prime symbol

A

True

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22
Q

What is a tautomer?

A

A molecule (eg; a base) where a proton has migrated to a different place

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23
Q

What percentage of bases are in the tautomeric form at any one time?

A

<0.01%

(But still have a significant effect, as human genome is 3Gbp = 3000000000 base pairs. 0.0033% of this number is = 100,000 bases)

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24
Q

True or False:
Bases flip back and forth between the common form and rare tautomeric form.

A

True

25
Q

True or False:
If a base transforms to the tautomeric form it cannot return to its original form.

A

False:
Bases flip back and forth between the common form and rare tautomeric form.

26
Q

When does it matter if a base is in the common or tautomeric form?

A

During replication (can result in damage and mutations)

27
Q

What is a nucleoside?

A

A base and a sugar, without the phosphate

28
Q

How are nucleosides named?

A

They are named for their base and their sugar, eg; adenosine (ribose), deoxyadenosine (deoxyribose)

29
Q

How are bases and sugars joined?

A

A glycosidic bond between the C1’ of the sugar and the:
N1 of a pyrimidine
or
N9 of a purine

30
Q

Where do phosphates bind to nucleosides to create nucleotides?

A

On the C5’ of the sugar

31
Q

What additional biological functions are nucleotide monomers involved in?

A

ATP , energy carrier and phosphoryl donor

CoA , acyl group activation and transfer

S-adenosyl methionine , methyl group donor

NAD+ /NADH NADP+ / NADPH , oxygen-reduction

FAD/FADH2 , oxygen-reduction

32
Q

What are Chargaff’s rules?

A

Amount of purine bases = Amount of pyrimidine bases
so [A]+[G]=[C]+[T]

Amount of guanine = amount of cytosine
so [G]=[C]

Amount of adenine = amount of thymine
so [A]=[T]

33
Q

What did Franklin and Wilkins do?

A

X-ray diffraction analysis of DNA showed:
Helical structure characteristic of two intertwined helices
Spacing of lines suggested dimensions of helix

34
Q

What dimensions of the DNA helix were inferred from Franklin and Wilkins’ experiment?

A

3.4 nm, approximately 10 bp / turn [34 Å]
0.34 nm rise per bp [3.4 Å]
2 nm helix diameter [20 Å]

35
Q

What are Watson-Crick base pairs?

A

A pairs with T with 2 H-bonds
C pairs with G with 3 H-bonds

A-T and C-G base pairs have similar widths

Two DNA strands associate via weak hydrogen bonds to form double-stranded DNA

36
Q

What is the structure of B-DNA?
(helix diameter, bp/turn, distance between bases, morphology, major and minor grooves, helix location)

A

2 nm helix diameter
10.5 bp / turn
0.34 nm between bases
One full turn of helix is 3.57 nm
Long, thin morphology
Major groove: Wide, intermediate depth
Minor groove: Narrow, intermediate depth
Helix axis location: Through base pairs

37
Q

Describe the properties of B-DNA

A

Two complementary, antiparallel, polynucleotide DNA strands wind around each other, forming a right-handed (clockwise) double helix.

Hydrophilic sugar phosphate backbone on outside of molecule, hydrophobic bases form a stack on the inside.

Van der Waals interactions between bases stabilise DNA.

Base stacking can contribute significantly to stability but depends on neighbouring bases.

38
Q

What is the importance of the major and minor groove?

A

In the major groove:
-Each base pair provides different chemical information
- Sequence specific binding proteins can recognise the different sequence combinations of hydrogen bond acceptors, donors and methyl groups available

In the minor groove:
- A-T & T-A are indistinguishable from each other, as are G-C & C-G

39
Q

Fill in the gaps:
Proteins that bind to DNA but are not sequence specific bind to the ______ groove.

Proteins that bind to sequence specific DNA bind the ______ groove.

A

Proteins that bind to DNA but are not sequence specific bind to the minor groove.

Proteins that bind to sequence specific DNA bind the major groove.

40
Q

What conformation is DNA predominantly in in cells?
Why?

A

B conformation
High humidity

41
Q

What conformation is DNA in when there is low humidity?

A

A DNA

42
Q

What conformation is DNA in when there is high humidity?

A

B DNA

Diameter = 2 nm
Right-handed helix (clockwise)
10.5 bp / turn

43
Q

What conformation is DNA in when exposed to lots of methylation of cytosine, torsional stress, high salt concentrations?

A

Z DNA

44
Q

What are some non-B DNA structures formed in genomic repetitive sequences with their sequence requirements?

A

Cruciform conformations, require inverted repeats

Slipped (hairpin) structure conformations, require direct repeats

Quadruplex conformations, require oligo (G)n tracts
[AG3(T2AG3)3 sequence, Single Strand] [Hoogsteen base paring] Seen in telomeres

45
Q

What is supercoiled DNA?

A

DNA under tension that twists in on itself

46
Q

What is relaxed DNA?

A

Open, uncoiled circular DNA

47
Q

Where can supercoils form?

A

In constrained linear DNA, during replication for example

48
Q

How are supercoils generated?

A

If parts of the DNA within the helix are untwisted, the whole molecule will wrap around itself to compensate for the amount of twists missing

49
Q

What is Lk?

A

Linking number:
Number of times one strand wraps around the other (fixed for circular DNA and constrained linear molecules)

50
Q

What is Tw?

A

Twist
Number of turns in a DNA fragment (+1 per 360 twist)

51
Q

What is Wr?

A

Writhe
Number of supercoils (can be positive or negative)

52
Q

What equation involves linking number, twist and writhe?

A

Lk = Tw + Wr

53
Q

How can you tell if something is positively or negatively supercoiled?

A

If you unwind the supercoil and this opens up / loosens the DNA strand (separates a the strands of a small section) this is a negative supercoil

54
Q

What enzymes use energy to introduce and remove supercoils from DNA, by temporarily breaking DNA and twisting it?

A

Topoisomerases

55
Q

What are topoisomerases?

A

They are enzymes that use energy to introduce and remove supercoils from DNA by temporarily breaking DNA and twisting it

56
Q

Why is negative supercoiling useful?

A

It facilitates DNA strand separation, as unwinding negative supercoils opens up the DNA strand

57
Q

Why is there a major and minor groove formed?

A

Because of hydrogen bonding, the opposite sugar-phosphate backbones are not equally spaced

58
Q

Describe the structure of A DNA
(helix diameter, bp/turn, distance between bases, morphology, major and minor grooves, helix location and direction of turn)

A

Diameter = 2.6 nm
Right-handed helix (clockwise)
11 bp / turn
Even sized grooves
Short and fat morphology
Major groove: extremely narrow, deep
Minor groove: very wide and shallow
Helix axis: major groove

Can be induced by DNA binding proteins

59
Q

Describe the structure of Z DNA
(helix diameter, bp/turn, distance between bases, morphology, major and minor grooves, helix location and direction of turn)

A

Diameter = 1.8 nm
Left-handed helix (anticlockwise)
12 bp / turn
Elongated and thin morphology
Major groove: flattened out on helix surface
Minor groove: extremely narrow, very deep
Alternating pyrimidine / purines

BIOL253 Genetics (15 decks)

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