7.1 DNA Structure And Replication

Question 1

Who found out that DNA was the genetic material of the cell?

Answer 1

Hershey and Chase

Question 2

How did Hershey and Chase discover that DNA was the genetic material of the cell and not proteins? (The experiment) (5)

Answer 2

1. Viruses were grown and radioactively labelled with either:
   Radioactive Sulphur (proteins)
   Radioactive phosphorous (DNA)
2. Virus infect E.coli and they are then separated via centrifugation
3. Bacteria formed pellet and virus remained in supernatant
4. Bacteria was found to be radioactive when infected with P(DNA) but not S(protein)
5. Showed DNA was genetically material because DNA was transferred to bacteria

Question 3

Who used X-ray diffraction to investigate the structure of DNA?

Answer 3

Franklin and Wilkins

Question 4

How does X-ray diffraction work? (3 steps)

Answer 4

1. DNA purified and fibres stretched in thin glass tube (to make parallel)
2. DNA targeted by X-ray beam, which diffracted when contacted with atom
3. Scattering astern of the X-ray was recorded on film and used to analyse molecular structure

Question 5

What inferences could be made about the DNA model after crystallography? (3 things)

Answer 5

= Composition: DNA is a double stranded molecule
= Orientation: nitrogenous bases are closely packed together on the inside and phosphate from an outer backbone
= Shape: DNA molecule twists at regular intervals to form a helix

Question 6

What number of hydrogen bonds do adenine and thymine have?

Answer 6

2

Question 7

What number of hydrogen bonds does guanine and cytosine have?

Answer 7

3

Question 8

What two things does the DNA structures suggest about two mechanisms for DNA replication?

Answer 8

Replication occurs via complementary base pairings
Replication is bi-directional (due to antiparallel nature of strands)

Question 9

What is the process of DNA replication? (11 steps)

Answer 9

1. P1: DNA helicase unwinds & separated the double helix by breaking H bonds
2. Gyrase prevents it from supercooling by reducing torsional strain
3. Strands now expose, act as templates (semi-conservative)
4. Single Stranded Binding proteins bind to DNA strand after separation to prevent re-annealing
5. As synthesis occurs in 5’->3’ direction synthesis continuos on the leading strand & discontinuous on lagging strand
6. P2: RNA primer is synthesised on parent DNA using RNA primase
7. DNA polymerase III adds nucleotide to 3’ end according to complementary base pairing (A-T & C-G)
8. Nucleotides added are in the form as deoxynucleoside triphosphate (2 phosphate groups are released from each nucleotide & energy is used to join nucleotides
9. DNA polymerase I removes RNA primers & replaces them with DNA nucleotides
10. DNA ligand joins Okazaki fragments on lagging strand
11. Each new DNA molecule contains both parent & newly synthesised DNA strand -> semi conservative

Question 10

What does helicase do?

Answer 10

• Unwinds and separates double stranded DNA by breaking hydrogen bonds between base pairs
• Occurs at specific regions, creating a replication fork of 2 strands in anti parallel directions

Question 11

What does DNA gyrase do?

Answer 11

Reduces torsional strain created by the unwinding of DNA helicase
By relaxing positive supercojls

Question 12

What do Single Stranded Binding Proteins do?

Answer 12

• SSB proteins bind to DNA strand after their separation and prevent re-annealing
• Help prevent DNA from being digested by nucleases
• Dislodges from strand when new complementary strand is synthesised by DNA polymerase III

Question 13

What does DNA primase do?

Answer 13

• Generates a short RNA primer on each template strand (-10-15 nucleotides)
• RNA primer provides an initiation pointer for DNA polymerase which can extend nucleotide chain (but doesn’t start one)

Question 14

What does DNA polymerase III do?

Answer 14

Free nucleotides align opposite their complementary base pairings
- Attaches to 3’ end of primer and covalently join free nucleotides in 5’->3’
Moves in opposite directions on 2 strands
- leading towards replication fork and continuously
- lagging move away from fork and synthesis in pieces (Okazaki fragments)

Question 15

What does DNA polymerase I do?

Answer 15

• As the lagging strand is synthesised in a series of short fragments, it has multiple RNA primers along its length
• DNA pol I removes the RNA primers from the lagging strand and replaces them with DNA nucleotides

Question 16

What does DNA lignase do?

Answer 16

Joins Okazaki fragments together to form continuous strand
By covalently joining sugar-phosphate backbones together with phosphodiester bonds

Question 17

what can DNA polymerase not do?

Answer 17

cannot initiate replication
only add new nucleotides to an existing strand

Question 18

what must occur before DNA replication can occur?

Answer 18

RNA primer must first be synthesised to provide an attachment point for DNA polymerase

Question 19

how do free nucleotides exist as? (2)

Answer 19

deoxynucleoside triphosphate (dNTPs)
they have 3 phosphate groups

Question 20

how does DNA polymerase use dNTPs/ deoxynucleoside triphosphates?

Answer 20

by cleaving 2 additional phosphates and uses the energy to form a phosphodiester bond with 3’ end of a nucleotide chain

Question 21

why must DNA polymerase move in opposite directions on 2 strands?

Answer 21

because the double-stranded DNA is antiparallel

Question 22

which direction must the DNA polymerase move on the leading strand?

Answer 22

DNA polymerase must be moving towards te replication fork and can copy continuously

Question 23

what direction must the DNA polymerase move on the lagging strand?

Answer 23

DNA polymerase is moving away from the replication fork and copying is discontinuous

Question 24

what are the steps DNA polymerase moving on the lagging strand? (3 steps)

Answer 24

1. DNA polymerase is moving away from helicase, it must constantly return to copy newly separated stretches of DNA
2. this means that the lagging strand is copied as a series of short fragments (OKAZAKI fragments) each preceded by a primer
3. the primers are replaced with DNA bases and the fragments joined together by combination of DNA polymerase I and DNA ligase

Question 25

what is DNA sequencing?

Answer 25

the process by which base order of a nucleotide sequence is elucidated (made clear)

Question 26

what are the characteristics of dideoxynucleotides and the consequences? (3)

Answer 26

- dideoxynucleotides (ddNTPs) lack 3' hydroxyl group necessary for forming a phosphodiester bond - so, ddNTPs prevent further elongation of a nucleotide chain and effectively terminate replication - the resulting length of DNA sequence will reflect the specific nucleotide position at which the ddNTP was incorporated (eg. is ddGTP terminates a sequence after 8 nucleotides, then the 8th nucleotide in the sequence is a cysosine)

Question 27

describe the way that dideoxynucleotides can be used to determine DNA sequencing using the Sanger method (5 steps)

Answer 27

1. four PCR mixes are set up, each containing stocks of normal nucleotides plus one dideoxynucleotide (ddA, ddT,ddC or ddG) 2. (PCR normally generates over 1 billion copies) so, each PCR mix should generate all the possible terminating fragments for that particular base 3. when fragments are separated using gel electrophoresis, the base sequence can be determined by ordering fragments according to length 4. is a distinct radioactive/ fluorescent labelled primer is included in each mix, the fragments can be detected by automated sequencing machines

Question 28

what should occur id the Sanger method is conducted n the coding strand(non-template strand)?

Answer 28

the resulting sequence found will be identical to the template strand

Question 29

what are five examples of non-coding regions of DNA?

Answer 29

tandemly repeating sequences of DNA (eg. STRs), telomeres, introns, non-coding RNA genes, gene regulatory sequences

Question 30

what are tandemly repeating sequences (eg. STRs) and what do humans used them for? (2)

Answer 30

structural component of heterochormatin and centromeres - used for DNA profiling

Question 31

what are telomeres and what do they do? (2)

Answer 31

regions of repetitive DNA at the end of a chromosome - protects against chromosomal deterioration during replication

Question 32

what are intons and what happens to them? (2)

Answer 32

non-coding sequences within genes - are removed by RNA splicing prior to formation of mRNA

Question 33

what are non-coding RNA genes and an example? (2)

Answer 33

codes for RNA molecules that are not translated into proteins eg, genes for tRNA

Question 34

what are gene regulatory sequences and what is included/examples? (2)

Answer 34

sequences are involved in the process of transcription - includes promoters, enhancers and silencers

Question 35

what is DNA profiling?

Answer 35

technique by which individuals can be identified and compared via DNA profiles

Question 36

what is the process of DNA sequencing? (3 steps)

Answer 36

1. within non-coding regions there will be long stretches of DNA made up of short tandem repeats (STRs) 2. tandem repeats can be excised(cut out) using restriction enzymes and separated suing gel electrophoresis for comparison 3. individuals will have different number of repeats at a given short tandem repeat locus and will generate unique DNA profiles (longer repeats = larger fragments shorter repeats = smaller fragments)

Question 37

in Eukaryotes how is DNA packaged?

Answer 37

with histone proteins to creat compacted nucleosome structure

Question 38

what do nucleosomes do and why? (2)

Answer 38

- help supercoil DNA resulting in compacted structure, allowing for more efficient storage - supercoiling helps protect DNA from damage and allows chromosomes to be mobile during mitosis and meiosis

Question 39

what is the organisation of Eukaryotic DNA? (5)

Answer 39

- DNA is complexed w/ 8 histone proteins (octamer) to form a nucleosome - nucleosomes are linked by an additional histone protein (H1 histone) which is condensed to form a string of chromosomes - these coil around a solenoid structure which is condensed to form a 30nm fibre - the fibres from loops which are compressed and folded around protein scaffold to form chromatin - chromatin will supercoil during cell division to from chromosomes that are visible (when stained) under microscope

Question 40

what does a nucleosome consist of? (4)

Answer 40

a molecule of DNA wrapped around a core of 8 histones - the negatively charged DNA associates with positively charged amino acids on the surface of histone proteins - the histone proteins have N-terminal tails which extrude outwards from the nucleosome - during chromosomal condensation tails from adjacent histone octamer link up and draw nucleosomes closer together