Lecture 22

Question 1

What are the 3 hypothesized mechanisms for DNA replication

Answer 1

• conservative replication
• dispersive replication
• semiconservative replication

Question 2

What is conservative replication

Answer 2

• one daughter DNA molecule is all new and the other is all old DNA

Question 3

What is dispersive replication

Answer 3

• each daughter DNA molecule contains a mixture of old and new DNA

Question 4

What is semiconservative replication

Answer 4

• each daughter DNA molecule contains one new strand and one old strand
• all living organisms

Question 5

The meselson-stahl experiment provided biochemical evidence that DNA replication is

Answer 5

• semiconservative

Question 6

How was the Meselson-Stahl experiment done

Answer 6

• grow bacteria in medium with 15N isotope (heavy)
• separate DNA by density centrifugation
• switch bacteria to medium with 14N isotope (light)
• separate DNA by density centrifugation

Question 7

What results would you expect for conservative replication of the Meselson-Stahl experiment

Answer 7

• original DNA would still be there and new would also all be there, would not see hybrid/mixture of both

Question 8

What are DNA building blocks

Answer 8

• nucleotides (chemistry must be same to create new DNA)

Question 9

How many phosphates do dNTPs have

Answer 9

3
- alpha, beta, gamma

Question 10

What kinds of bonds are formed in polymerizing DNA? Between which atoms?

Answer 10

• phosphodiester bond
• always place dNTPs on 3’ end
• 3’ OH and phosphate

Question 11

Are there any required cofactors for DNA synthesis

Answer 11

• Mg2+
• help to position the incoming nucleotide for nucleophilic attack by the 3’-OH

Question 12

The raw materials for DNA synthesis/replication are

Answer 12

1. a DNA template
2. dNTPs
3. a DNA or RNA ‘primer’ to provide the first 3’-OH
4. Mg2+ ions (cofactor for the polymerase)
5. DNA polymerase - a large protein complex that includes the enzyme that catalyzes the addition of dNTPs on the 3’ OH end of the primer

Question 13

How do the materials work together

Answer 13

• The DNA polymerase reaction adds a deoxynucleotide to the 3’ end of the
  growing DNA chain.
• The Mg2+ ions stabilize the negative charges on the deoxynucleotide and assist in deprotonation of the 3’-OH by a base
  (shown as a water molecule).
• The 3’-oxygen of the growing DNA strand serves as the nucleophile in this reaction, displacing the pyrophosphate from the
  deoxynucleoside triphosphate (dNTP) in the active site.
• The product is a DNA strand that has been extended by one nucleotide in the 3’ direction.

Question 14

How does the shape of DNA polymerase affect base pairs

Answer 14

• the shape of the DNA polymerase active site facilitates formation of the correct watson-crick base pairs
• correct base pairs fit well into the DNA polymerase active sire, mismatches do not
• high fidelity, base selection, steric clashes

Question 15

What are the major E. coli DNA polymerases

Answer 15

• Pol 1 (proofreading, repair, primer removal)
• 5’—>3’ exonuclease and 3’ —>5’ exonuclease
• Pol 3 (main polymerizing enzyme)
• no 5’ —> 3’ exonuclease but does have 3’ ->5’ exonuclease

Question 16

What is endonuclease

Answer 16

• within
• cuts from within (inside)
• phosphodiester bond gets hydrolyzed

Question 17

What is an exonuclease

Answer 17

• outside
• cuts outside
• one at a time from edge from either side

Question 18

What is a 5’ —> 3’ exonuclease

Answer 18

• cuts/hydrolyzes phosphodiester link from outside from 5’ to 3’ direction

Question 19

What is a 3’ —> 5’ exonuclease

Answer 19

• cuts/hydrolyzes phosphodiester link from outside from 3’ to 5’ direction

Question 20

How does DNA synthesis occur at the replication fork

Answer 20

• occurs simultaneously for both parent strands (always in the 5’ —> 3’ direction)
• DNA is partially unwound at “replication forks.” Both strands are used as templates for the synthesis of new strands in a 5’-> 3’ direction.
• The leading strand is synthesized continuously, whereas the lagging strand is, by necessity, is synthesized in short pieces termed Okazaki fragments (100s to 1000s of nucleotides).
• Both new strands are synthesized in a
  coordinated fashion by a single multimeric DNA polymerase III complex (DNA pol III).
• The template for the lagging strand loops so that both template strands are oriented in the same direction, and both daughter strands, the leading and lagging strands, are synthesized in the same direction by the DNA polymerase III complex.
• The requirement for looping means that
  lagging strand synthesis cannot occur continuously.

Question 21

Explain the process of DNA replication

Answer 21

• In all cells (eukaryotic, prokaryotic) with circular or linear DNA molecules, DNA
  replication begins at defined sequences termed “origins of replication” (ori).
• Replication is initiated by proteins that recognize and bind to the DNA sequence of the origin of replication (ori):
• often there are multiple origins
• prokaryotes, like E. coli, have
  circular chromosomes with a
  single origin of replication -
  “oriC”
• the ori opens up (DNA strands
  are separated by a helicase) to
  form 2 replication forks
• In E. coli, replication is
  bidirectional, with a DNA
  polymerase III complex
  operating at each of the 2
  replication forks