Lecture 3 - Genetic Code and DNA Replication

Question 1

What are the 3 types of mutations

Answer 1

Substitution
Deletion
Addition

Question 2

What are the 3 outcomes of mutations

Answer 2

No change in AA sequence - Neutral
* Change in AA sequence that doesn’t affect its function
* Change in AA sequence that affects its function

Question 3

What is a nucleoside

Answer 3

Base and sugarW

Question 4

What is a nucleotide

Answer 4

Base and sugar and Pi

Question 5

What is the proof for semi-conservative replication

Answer 5

• Meselson-Stahl experiment (1958)
• Radiolabeled N
• Measured ratio of heavy/light N in successive generations of bacteria

Question 6

What are the stages for DNA Replication

Answer 6

Initiation
* Proteins bind to DNA and open the double helix
* Prepare DNA for complementary base pairing
* RNA primers bind first to start the process

Elongation
* Proteins join incoming nucleotides together into continuous new strands
* Always polymerise 5’ to 3’ so one strand is discontinuous
* Proof-reading takes place to ensure fidelity of sequence
* RNA primer replaced with DNA

Termination
* Proteins release the replication complex

Question 7

What are the enzymes involved in DNA Rep

Answer 7

• DNA helicase breaks H-bonds between base pairs at origins of replication
• DNA primase assembles and catalyses synthesis of short RNA primers on DNA template
• DNA polymerase adds nucleotides to the primer to form a new strand, joining sugar backbones of newly added bases
• DNA ligase joins discontinuous strands
• Topoisomerase relieve physical tension that could cause DNA damage

Question 8

What is the difference between leading strand and lagging strand

Answer 8

• Leading strand – synthesised CONTINUOUSLY from replication fork, starts with an RNA primer
• Lagging strand – synthesized DISCONTINUOUSLY in Okazaki fragments – 100-200 base pairs long, and each fragment starts with a primer

Question 9

How does synthesis of bases occur in DNA rep

Answer 9

• Incoming dNTP has PPPi on the 5’ carbon atom in the ribose group
• Phosphodiester bond forms with 3’ OH group on the end of the DNA chain
• Catalytic energy comes from hydrolysis of PPi

Question 10

How does the Single strand binding and sliding clamp work

Answer 10

• Single Strand Binding (SSB) proteins prevent ssDNA from base pairing with other template strand
• Sliding clamp keeps DNA polymerase from falling off the strand, assembled at replication fork by clamp-loader complex to ensure efficient replication

Question 11

How are mismatches sorted

Answer 11

Mis-matched bases impair polymerisation reaction
Mis-matches are removed by exonuclease domain