8 - DNA Replication

Question 1

How is bacterial DNA compacted

Answer 1

Looping and supercoiling

Question 2

How is eukaryotic DNA organised

Answer 2

Tightly coiled and packed together

Question 3

Error rate of DNA replication in eukaryotic cells

Answer 3

1 in 10^9 chance

Question 4

Two prediction in Watson-Crick DNA model

Answer 4

1. DNA strands are held together by hydrogen bonds between complementary base pairs
   A-T - 2 H bonds
   C-G - 3 H bonds
   - to make this work, the strands must be anti-parallel
2. Each strands is therefore complementary to the other
   - so each DNA strand can act as a template for DNA replication
   - so DNA replication should be semi-conservative

Question 5

3 models for testing DNA replication

Answer 5

1. Semi-conservative - one parental and one new strand in DNA molecule
2. Conservative - parent helix conserved, daughter helix completely new
3. Dispersive - parent helix broken into fragments, dispersed, copies and then assembled into two new helices
   - new and old DNA completely dispersed

Question 6

Results of Meselson and Stahl experiment in 1958

Answer 6

• used CsCl equilibrium density gradient centrifugation to separate molecules on basis of their densities
• found 3 different forms of DNA strands after multiple replications and generations:
• 14N/14N light DNA
• 14N/15N hybrid DNA
• 15N/15N heavy DNA
• showed that DNA replication is semi-conservative

Question 7

Arthur Kornberg DNA polymerase experiment 1957 - info

Answer 7

• he wanted to work out procedure and conditions required for DNA replication
  Procedure:
• added multiple chemicals to E. Coli protein extracts
• template DNA required to provide something to copy
• deoxynucleotide triphosphtaes (dNTPs, dATP, etc.) added
• co factor Mg2+ added
• ATP added as energy source
• Primer added - small piece of DNA with free 3 OH group needed

Question 8

Where does energy for DNA replication come from
- what does this produce

Answer 8

2 phosphate groups removed from phosphate backbone of DNA to release chemical energy from bond
- produces pyrophosphate
- free nucleotide is joined to new DNA strand

Question 9

DNA replication mechanism

Question 10

DNA polymerase editing in DNA replication info

Answer 10

• wrong nucleotide can be incorporated at a low frequency
• this leaves an unpaired 3’ OH- end that blocks further elongation by DNA polymerase
• DNA polymerase 3’ —> 5’ exonuclease activity removes the mismatch to leave a base paired 3’ OH end
• DNA replication can now resume

Question 11

Conclusion of DNA replication by 1960

Answer 11

• DNA strands are anti-parallel
• 5-3 and 3-5
• Watson-Crick complementary base pairing is correct
• DNA replication is semi-conservative
• new DNA made in the 5-3 direction and is mediated by polymerase enzymes that can edit error
• polymerases however, require priming
• DNA polymerase has a proof-reading 3’ to 5’ exonuclease activity
• DNA synthesis is semi-continuous, leading and lagging strand

Question 12

what are primers and functions

Answer 12

short, single-stranded DNA sequences that are used as a starting point for DNA synthesis in PCR and DNA replication

Question 13

why are primers needed for DNA replication and polymerase

Answer 13

DNA polymerase can only build nucleotides onto an existing DNA strand