7.1 (DNA Structure)

Question 1

Outline Hershey and Chase’s experiment.

Answer 1

• In the mid-twentieth century scientists were unsure as to whether proteins or chromosomes were the genetic material of cells.
• Viruses infect cells and transform them into virus-producing factories:
• Viruses inject their genetic material into cells.
• The non-genetic part of the virus remains outside the cell.
  Infected cells produce large numbers of the virus
• The cell bursts releasing the copied virus
• Hershey and Chase chose to study the T2 bacteriophage, which infects the E. Coli bacterium (image of both above), because of its very simple structure consisting of just:
• Protein coat (capsid)
• DNA inside the coat
• Amino acids containing Radioactive isotopes were used to label the virus:
• Sulfur (35S) for the protein coat (capsid)
• Phosphorus (32P) for the DNA
• The experiments combined T2 bacteriophage with E. Coli bacteria. At the end of the experiment a centrifuge was used to separate them:
  8 The smaller virus remained in the supernatant (liquid)
• The bacteria formed a pellet
• Separate experiments with the two isotopes found that:
• Sulfur (35S) remained in supernatant
• Phosphorus (32P) was found in the pellet
• Hershey and Chase deduced that DNA therefore was the genetic material used by viruses because DNA (labelled by 32P) was being transferred into the bacteria

Question 2

What is x-ray diffraction?

Answer 2

When X-rays are directed at a material some is scattered by the material. This scattering is known as diffraction. For X-ray diffraction to work well the material ideally should be crystallised so that the repeating pattern causes diffraction to occur in a regular way. DNA cannot be crystallised but the molecules were arranged regularly enough for the technique to work.

Question 3

Outline the evidence which supports complementary base pairing.

Answer 3

• (X-ray diffraction showed that) the DNA helix is both tightly packed and regular* therefore pyrimidines need to be paired with purines
• The electrical charges of adenine and thymine are compatible (and opposite) allowing two hydrogen bonds to form between them
• The pairing of cytosine with guanine allows for three hydrogen bonds to form between them

Question 4

How is is eukaryotic DNA supercoiled?

Answer 4

• Nucleosomes both protect DNA and allow it to be packaged, this in turn allows DNA to be supercoiled.
• Nucleosomes are formed by wrapping DNA around histone proteins

Question 5

Why does eurkaryotic need to be supercoiled?

Answer 5

• Essential to pack genetic material into the nucleus
• To organise DNA to allow cell division to occur (most DNA supercoiling occurs at this time)
• To control DNA expression - supercoiled DNA cannot be transcribed
• Allow cells to specialise by permanently supercoiling DNA (heterochromatin)
• Transcription of active chromatin (Euchromatin) can be promoted or inhibited by the associated histones

Question 6

Outline the role of helicase.

Answer 6

• Unwinds the DNA Helix
• Separates the two polynucleotide strands by breaking the hydrogen bonds between complementary base pairs
• ATP is needed by helicase to both move along the DNA molecule and to break the hydrogen bonds
• The two separated strands become parent/template strands for the replication process

Question 7

Outline the role of DNA polymerase.

Answer 7

• Creates complementary strands
• Free nucleotides are deoxynucleoside triphosphates
• The extra phosphate groups carry energy which is used for formation of covalent bonds
• DNA polymerase always moves in a 5’ to 3’ direction
• DNA polymerase catalyses the covalent phosphodiester bonds between sugars and phosphate groups
• DNA Polymerase proof reads the complementary base pairing. Consequently mistakes are very infrequent occurring approx. once in every billion bases pairs

Question 8

Define RNA primers and their role.

Answer 8

• RNA primers consists of a short sequence (generally about 10 base pairs) of RNA nucleotides
• RNA primers provide an attachment and initiation point for DNA polymerase III

Question 9

What is DNA replication said to be and why?

Answer 9

Each new strand contains one original and one new strand, therefore DNA Replication is said to be a semi-conservative process

Question 10

Summarise the enzymes involved in DNA replication (6).

Answer 10

• DNA Helicase: unwinds and separates the double stranded DNA by breaking the hydrogen bonds between base pairs
• DNA Gyrase (aka topoisomerase): moves in advance of helicase and relieves strain and prevents supercoiling on the separated strands
• DNA Ligase joins the Okazaki fragments together to create a continuous strand
• DNA Polymerase I removes the RNA primers and replaces them with DNA
• DNA Polymerase III adds deoxynucleoside triphosphates (dNTPs) to the 3’ end of the polynucleotide chain, synthesising in a 5’ - 3’ direction
• RNA Primase synthesises a short RNA primer on each template strand to provide an attachment and initiation point for DNA polymerase III

Question 11

Outline DNA replication.

Answer 11

• DNA replication occurs during (S phase of ) interphase, in preparation for cell division
• Helicase unwinds the double helix separating the strands of DNA
• It breaks the hydrogen bonds between the two strands
• Single stranded binding proteins keep the separated strands apart so that nucleotides can bind
• DNA gyrase moves in advance of helicase and relieves strain and prevents the DNA supercoiling again.
• Each strand of parent DNA is used as template for the synthesis of the new strands
• Synthesis always occurs in 5´ → 3´ direction on each new strand
• Therefore synthesis is continuous on leading strand (in the same direction as helicase) and dis-continuous on lagging strand (away from from helicase)
• This leads to the formation of Okazaki fragments on the lagging strand
  To synthesise a new strand first an RNA primer is synthesized on the parent DNA using RNA primase
• Next DNA polymerase III adds the nucleotides (to the 3´ end) added according to the complementary base pairing rules; adenine pairs with thymine and cytosine pairs with guanine; (names needed, letters alone not accepted)
• Nucleotides added are in the form of as deoxynucleoside triphosphate. Two phosphate groups are released from each nucleotide and the energy is used to join the nucleotides in to a growing DNA chain
• DNA polymerase I then removes the RNA primers and replaces them with DNA
• DNA ligase next joins Okazaki fragments on the lagging strand
• Because each new DNA molecule contains both a parent and newly synthesised strand DNA replication is said to be semi-conservative