Lecture 19: Enzymology of DNA Synthesis

Question 1

What are the two strands of a replication fork?

Answer 1

1. Leading

2. Lagging

Question 2

What are “Okazaki fragments” and where are they located?

Answer 2

Location: Lagging strand

Short sections of DNA on the lagging strand that get stitched together

Question 3

How are okazaki fragments formed?

Answer 3

The binding of primase to the lagging strand to add a RNA primer, this falls off and repeats

Question 4

What does processivity mean?

Answer 4

1. After one nucleotide is added to growing
   DNA, another is added or dissociates
2. Dissociation and association of
   polymerase can limit polymerisation rate
3. Avrg number of nucleotides added
   before polymerase dissociation defines
   “processivity”
4. Polymerases vary in their processivity

relevant when your talking about a whole genome

Question 5

What is DNA polymerase 1?

Answer 5

1. not the main enzyme for DNA synthesis
2. Performs several “clean-up” functions
3. Functions aided by its 5’-3’ exonuclease
   activity (distinct from 3’-5’ proof-reading
   activity)

Question 6

What is meant by error-prone DNA polymerases?

Answer 6

1. Damaged DNA causes Polymerase 3 to 
   stall during replication
2. DNA pol 4 or pol 5 can synthesis a 
    complement to damaged strands 
3. New DNA synthesised by these enzymes 
    often has errors

Question 7

How is fidelity improved during DNA synthesis?

Answer 7

1. Replication is highly accurate (1 mistake 
   every 10^10 nucleotides in E. coli)
2. Largely because active sites of DNA 
    polymerases prefer Watson-crick      
    base pairs
3. BUT; in vitro measurement show 
    enzymes make 1 mistake every ~10^4 
    nucleotides
4. So other processes improve accuracy:
   a. DNA repair strategies
   b. Proof-reading

Question 8

What is the role of Polymerase 1’s 5’-3’ exonuclease activity?

Answer 8

1. Can remove small sections of DNA or
   RNA
2. Removes RNA primers in lagging
   strand and replaces them with DNA

Question 9

How does the proof-reading property of Polymerase 1 work?

Answer 9

1. Polymerisation and proof-reading
   properties of Pol1 are separate
2. 3’-5’ exonuclease activity double-checks
   nucleotides after addition and removes
   mismatches
3. 3’-5’ exonuclease activity acts ahead
   of polymerase activity
4. mismatched BP impedes translocation
   of polym. to next site, so enzyme slides
   back and corrects before resuming