DNA History, Structure, and Replication

Question 1

Fred Griffith

Answer 1

• Discovered transformation
• Showed that some “active genetic substance” could be transferred from dead bacteria capable of causing disease to live harmless bacteria, making these live bacteria dangerous
• Attempting to develop vaccine for type of bacteria – streptococcus
• Virulence – disease-causing organism (pathogenic)
• S strain
  
  • ​Forms smooth colonies
  • Polysacc. coat protects it from attack by immune system
  • Virulent / pathogenic
• R strain
  
  • Rough colonies
  • No protective coating
  • Avirulent / nonpathogenic
• Something from S strain had been able to transform the R strain into a mouse killer

Question 2

Avery, McLeod + McCarthy

Answer 2

• Proteases (protein-destroying enzymes)​
  
  • ​Added to heat-killed S strain (then mixed with live R strain)
  • Mice still died, factor was not protein
• DNAse (DNA-destroying enzyme)
  
  • Added to heat-killed S strain (then mixed with live R strain)
  • Mice didn’t die, factor was likely DNA

Question 3

Hershey and Chase

Answer 3

• Proved DNA was the genetic material
• Used the T2 bacteriophage (virus that infects and kills bacterial cells)
  
  • Infects living cells and multiplies inside, explode the cell, infect more cells
• Batch of radioactive capsid proteins (³⁵S)
• Batch of radioactive DNA (³²P)
• Allowed each to infect bacteria (E. coli), then remove the viruses on the outside of the bacterial cells by agitating the cells in blender
• Spin tubes in centrifuge – dense bacteria sinks to bottom (forms pellet)
• E. coli bacteria in the pellet contained no ³⁵S
• The offspring of the virus contained lots of ³²P
  
  • Proteins: CHONS
  • DNA: CHNOP

Question 4

Chargaff

Answer 4

• Noticed that in every analysis of DNA that he performed:
  
  • amount of adenine = amount of thymine
  • amount of cytosine = amount of guanine
• Pairs present in equal ratios

Question 5

Watson-Crick-Wilkins-Franklin (and Pauling)

Answer 5

• Wilkins and Franklin – crystallographer, developed X-ray diffraction images of DNA – 2 twisted strands
• Franklin’s data – DNA was an anti-parallel double-stranded molecule
• 2 stranded double helix
• Backbone of molecule composed of the phosphates and deoxyribose sugars of the nucleotides (covalently bonded)
• Rungs composed of nitrogenous base pairs (H bonding)
• Watson and Crick published journal Nature
• Watson, Crick, Wilkins – Nobel Prize
• Watson – published The Double Helix

Question 6

What are the components of a nucleotide?

Answer 6

• Nitrogenous base (A, C, T, G)
• Phosphate group
• Sugar

Question 7

Timeline of discovery

Answer 7

• Griffith discovers that bacteria can change from one form to another (transformation)
• Avery followed up Griffith’s earlier discovery and concluded that the transforming factor is DNA
• Franklin and Wilkins provide evidence that DNA is in the form of a double helix
• Chargaff’s rule
• Hershey and Chase conduct experiments which further prove that DNA was the hereditary material, sufficient to code for growth of a new organism
• Watson and Crick publish 3D structure and composition of DNA

Question 8

What types of bonds hold these molecules together?

Answer 8

• Hydrogen bonds hold the nitrogenous bases together
• Covalent bonds hold the nucleotides together
  
  • Between the sugar of one nucleotide and the phosphate of the next – sugar-phosphate backbone

Question 9

Base pairing rules in DNA?

Answer 9

• There should be a near 1:1 ratio of C to G, and A to T (or U in RNA) in any living organism (Chargaff’s rule)
• Adenine can bond to Thymine or Uracil (A — T / U)
• Guanine can only bond to Cytosine (G — C)

Question 10

Differences between DNA and RNA?

Answer 10

• Both are polymers of nucleotides (sugar + nitrogenous base + phosphate)
• RNA
  
  • sugar is ribose
  • A, G, C, U
  • uracil (U) is substituted for thymine (T)
• DNA
  
  • sugar is deoxyribose
  • A, G, C, T
  • missing an oxygen atom in a hydroxyl group in the sugar,

Question 11

What are 5’ and 3’ ends? (where do these terms come from?)

Answer 11

• Refer to the specific carbon molecules in the deoxyribose backbone of DNA/RNA
• 5’ is the carbon closest to the phosphate group

Question 12

Why is DNA referred to as antiparallel?

Answer 12

• The two strands of DNA that make up the double helix are parallel but are oriented in opposite directions

Question 13

What does it mean to call the DNA strands complementary?

Answer 13

• The two strands that create the double helix are related by the rules of base pairing, pairs are opposites

Question 14

Replication takes place where in a Eukaryotic cell? Prokaryotic cell?

Answer 14

• The nucleus in eukaryotic
• Cytoplasm in prokaryotic

Question 15

Why is DNA replication called semi-conservative?

Answer 15

• Half of the strand used when attaching to the new base pairs

Question 16

What is a replication bubble?

Answer 16

• Where DNA starts to replicate along the strand
• There are many of these along a piece of DNA

Question 17

Origin of replication?

Answer 17

• Where replication bubbles start

Question 18

Replication fork

Answer 18

• Where new DNA is polymerized by DNA Polymerase

Question 19

What are Okazaki fragments? Why are these created?

Answer 19

• DNA polymerase can only add on to the 3 prime end
• So when synthesizing the lagging strand it has to read from inside as the strand opens up (copies backwards to the direction it’s opening), which means that it has to copy in fragments as the lagging strand opens up bit by bit
• And since it can only add onto 3’ without ligase, it doesn’t connect making fragments

Question 20

What are RNA primers? What enzyme creates these?

Answer 20

• RNA primers are short strands of RNA or DNA that serve as starting points for DNA synthesis on the lagging strand
• Primase makes these

Question 21

Helicase

Answer 21

• Unwinds the helix during DNA replication

Question 22

Primase

Answer 22

• Sets down RNA Primers for DNA Polymerase

Question 23

DNA polymerase

Answer 23

• Adds new DNA nucleotides to the parent strand

Question 24

Ligase

Answer 24

• Covalently bonds Okazaki fragments

Question 25

How does replication proceed from a replication fork?

Answer 25

• Proceeds until it reaches another replication bubble, DNA replication is complete

Question 26

Differences between leading and lagging strands

Answer 26

• The leading strand gets polymerized continuously 5—>3, while the lagging strand gets polymerized 3–>5 overall in the 5–>3 direction in Okazaki fragments