DNA replication

Question 1

Semi-conservative replication of DNA
Matthew Meselson and Franklin Stahl 1958

Answer 1

Want to know which mechanism used for DNA replication
Used differences in nitrogen isotopes to do experiment
DNA contains lots of nitrogen
^14N = 7 protein, 7 neutron, atomic weight 14
^15N = 7 proteins, 8 neutrons, atomic weight = 15
15N heavier, DNA containing different isotopes separated by weight
Possible models DNA replication:
- Conservation - 1 completely new and one original molecule
- Semi-conservative - 1 strand old with 1 strand new for both molecules
- Dispersive - Chunks of old and new together throughout both molecules

Question 2

Method

Answer 2

1. Grow E coli in media 15N
2. Transfer to media 14N - new DNA will be light
3. Separate heavy and light molecules by ultrcentrifuge

Question 3

Caesium chloride gradient

Answer 3

Need to do after looking back at lecture

Question 4

Look at DNA using UV light

Answer 4

After one generation - Can only see one band, so eliminates conservative replication
After two generations - Can see two bands, so matches semi-conservative model
After four generations - Can see two bands, very strong 14N band and weak mixed band, supports semi-conservative model

Question 5

Process of replication - Semi-conservative replication
Enzymes involved in replication

Answer 5

DNA polymerase
Primase
DNA ligase (Okazaki fragments)
Topoisomerase
Helicase
Single-strand binding protein

Question 6

Mechanism DNA polymerase

Answer 6

Add nucleotides one at a time 5’ to 3’ direction
Use template strand (original strand), form H-bonds, tells which base add next
Tens or hundreds of nucleotides added per second

Question 7

Primase

Answer 7

Generates the primer (made of RNA) - short stretch DNA allows replication to occur on lagging strand
Can act several times along chromosome
Primers easily recognised and removed by enzymes, gap left over easily filled in nucleotides and ligase joins stretches of new DNA together

Question 8

Ligase

Answer 8

Joins loose ends in backbone together into single strand DNA
Ligating together - technical term for them joining together

Question 9

Helicase

Answer 9

Breaks H-bonds between DNA strands to separate them

Question 10

Topoisomerase

Answer 10

Relieves pressure from over winding around replication bubble
Makes little breaks, relieves pressure, reseals break in DNA

Question 11

Single-strand binding protein

Answer 11

Bind to separated strands
Prevents them reanealing

Question 12

Okazaki fragments

Answer 12

Pieces of DNA that get stuck together to make lagging strand

Question 13

Leading and lagging strands of DNA replication
Replication of leading and lagging at replication fork

Answer 13

Leading strand - 5’ to 3’ synthesis points towards replication fork, proceed continuously
Lagging strand - 5’ to 3’ synthesis points away from replication fork, discontinuous, has to be primed multiple times
DNA replication is semi-discontinuous
There is bidirectional replication

Question 14

Erosion of genetic material at end of linear chromosomes

Answer 14

Primer removal at end of chromosome leaves gap that can’t be filled in
On every round replication, little pieces lost from end of chromosome
This is problem for lagging strand at each end of linear DNA
Can lead to that bit of DNA being lost or degraded

Question 15

Telomeres

Answer 15

Solve erosion problem
Are short DNA sequences that repeat over and over at end of chromosome
Short stretch lost from telomeres at each round replication
Ok because enzyme telomerase can replenish telomeres from DNA template
Repetitive nature of telomers allows binding to specific proteins, protect vulnerable end chromosomes

Question 16

Shelterin complex

Answer 16

Group specialised proteins bind telomere DNA and each other
For a cap on telomer to:
- Differentiate from DNA breaks
- Recruit telomerase - the enzyme that adds new telomere sequence
- Protect from nucleases - enzyme degrading DNA