DNA Replication

• What did Levene (1909) determine correctly about DNA

  • DNA is made of of chains of nucleotides
  • DNA is made up of a phosphate, Sugar and a nitrogen base
  • Nucleotide backbone = Phosphate + Sugar
• What did Levene (1909) determine Incorrectly about DNA

  • Tetranucleotide Hypothesis
  • All nitrogen bases are in equal ratios
• What did Chargaff (1947) determine about DNA?

  • The Base Pairs
  • A = T, G=C
• What did Franklin & Wilkins (1952) determine about DNA?

  • X-Ray crystalline DNA fibers
  • X-Ray crystallography
• What is X-Ray crystallography?

  • Physics approach in examining a biological molecule
  • Taking a pure crystallized sample of DNA
  • X-Rays bombard the sample and different patterns are examined
• What is a way to remember X-ray Crystallography?

  • Throwing a tennis ball covered in paint at an invisible object and examining the parts that are covered in paint
• What can you see with the X-ray?

  • Double Helix Structure
  • Phosphate + Sugar backbone
  • Nitrogen bases oriented in the middle of the molecule and at a right angle
• What did Pauling (1953) determine correctly?

  • The alpha Helix in the secondary structure of a protein
• What did Watson & Crick (1953) determine about DNA?

  • Determined the rules for base pairing
  • H-Bonds between the nitrogen bases
  • Width of purine + pyrimidine and why they need to be paired like that and not any other way
• Why do nitrogen bases need to be paired Purine + Pyrimidine?

  • Purine - Purine Pair = Not Enough Space 
  • Pyrimidine - Pyrimidine = Too Much SPace 
  • Purine - Pyrimidine = Just Right
• What is the DNA Helix Structure?

  • B-DNA is right handed 
  • Bases are stacked on top of each other in a parallel direction
  • The spaces between the DNA strands have 2 grooves with different widths 
  • Major 
  • Minor
  • 1 full helical turn 360 degrees
• What is the correct DNA Replication model?

  Semi - Conservative
• In which phase of the DNA cycle does DNA replication happen in?

  S Phase
• What did Meselson and Stahl’s Experiment determine?

  • The correct DNA Replication model
• What is the first step of Meselson and Stahl’s Experiment?

  • Grew E.coli in 15Nitrogen  
• What did they choose to grow E.coli in 15N instead of the normal 14N?

  B/C 15N is heavier that 14N

  • When they put the DNA thru centrifugation… the DNA will sink to the bottom on the density gradient 
• What is the second step of Meselson and Stahl’s Experiment?

  •  transferred the E.coli into another tube with 14Nitrogen
  • Allowed it to undergo 1 replication
  • It was then put back into centrifugation
• How many bands did they see?

  • Saw 1 band which eliminates the possibility of the DNA model being conservative B/C that one has 2 bands 
• What is the third step of the E.coli experiment?

  • Cells underwent another replication cycle in the 14Nitrogen
  • Went back into centrifugation for a final time
• What did they see after the final centrifugation?

  2 bands which confirms the correct model is semi - conservative
• What is the first step of DNA Replication?

  Initiation
• Where does initiation begin?

  At the Origin of Replication (ORI)
• Where is the ORI located?


  In the DNA strands
• Is the ORI A&T or G&C Rich?

  A&T
• How many ORIs are there in prokaryotes?

  1
• Which direction does replication go in the ORI in prokaryotes?

  Bidirectionally
• How many ORIs are there in Eukaryotes?

  There is always more than 1
• What does the ORI have in eukaryotes that prokaryotes don't have?

  Replication bubbles and replication forks
• How do replication bubbles form?

  DNA strands separate which makes a bubble
• When initiation is finished, where does the replication bubble go?

  It elongates and fuses
• What are the 3 proteins involved in replication initiation?

  • Helicase
  • Single Stranded Binding Proteins (SSBP)
  • Topoisomerase
• What does Helicase do?


  Unzips DNA strands by disrupting the H-Bonds

  This allows them to access the template at the replication fork
• What does SSBP do?

  Binds to the single stranded part of the DNA and keeps it from closing during replication
• What does topoisomerase do?

  It breaks bonds and reforms new bonds at a different location

  This is crucial b/c it releases the tension from the double stranded part of the DNA
• Why is priming DNA important?

  B/C RNAP has different abilities than DNAP
• What is a primer?


  A primer is a short segment of RNA that initiates DNA replication
• What is Primase?


  Primase is an enzyme that synthesizes RNA primers during DNA replication.
• How does Primase synthesize RNA primers?


  By adding ribonucleotides which are complementary in the DNA template
• What can RNAP do that DNAP cannot?

  • Start a new chain
• Why can't DNAP start a new chain?

  B/C it doesn't have the OH on 3' so it can only add nucleotides to an existing chain