MB - DNA Replication errors I

Question 1

What are 3 types of mutations or errors in DNA synthesis?

Answer 1

• Incorrect nucleotide addition
• RNA nucleotide incorporation instead of DNA
• Nicks in the DNA backbone

Question 2

How does DNA polymerase ensure the correct nucleotide is added during DNA synthesis?

Answer 2

Through induced fit and steric exclusion

• Involve the proper base pairing and exclusion of incorrect nucleotides

Question 3

How does proofreading activity of Pol III reduce error rates?

Answer 3

Allows it to remove incorrect nucleotides that were added

Question 4

What happens when a mismatched nucleotide is present at the 3’ end of a growing DNA strand?

Answer 4

DNA polymerase stalls

• The exonuclease activity can remove the incorrect nucleotide

Question 5

Why can’t rNTPs (ribonucleotides) be directly incorporated into growing DNA strands?

Answer 5

The extra hydroxyl (OH) group in ribose causes a steric clash

Question 6

Does Pol III bind to nicks?

Answer 6

No, only Pol I does

Question 7

Why do Okazaki fragments have RNA at their 5’ end and a nick at their 3’ end?

Answer 7

• Okazaki fragments have RNA at the 5’ end because DNA polymerase cannot add rNTPs directly to DNA
• The nick at the 3’ end allows DNA polymerase to remove and replace the ribonucleotides with deoxyribonucleotides

Question 8

What is the problem with having uracil (U) in DNA?

Answer 8

Suggests damage and can lead to mutations

Question 9

Why is U a problem?

Answer 9

U accidentally added opposite A –> Not a problem

U formed by deamination of C –> Mutation

Question 10

What seals nicks in the DNA?

Answer 10

DNA Ligase

Question 11

How is U removed from DNA? (3)

Answer 11

1. Removed by Uracil-N-glycosylase
2. Baseless nt recognised and phosphodiester backbone cleaved by AP endonuclease
3. Forms Nicked DNA

Question 12

What are the first 5 steps in Initiation?

Answer 12

1) Multiple DnaA products bind to 9bp repeats

2) Use ATP to separate(melt) the duplex at the 13bp repeats

3) A DnaC protein binds to ssDNA and loads a DnaB helicase onto one strand, facing →3’. DnaC detaches and helicase moves to fork.

4) After ~65 nt have been unwound by the helicases, DnaG primase enzymes bind to them, forming a primosome

5) Primase synthesizes a ~10nt RNA primer and detaches from helicase

Question 13

What are the next 5 steps on initiation?

Answer 13

6) Single stranded binding protein (SSB) binds to exposed ssDNA preventing re-annealing

7) First primer and SSB trigger arrival of the Pol III holoenzyme at the 3’ end of the primer

8) The clamp loader loads a β clamp onto the DNA. Pol III core binds to the β clamp

9) Pol III travels to replication fork, synthesizing the leading strand as it goes and displacing SSB

10) As it catches the helicase, a replisome forms