Test 1: Nucleic acid enzymes

Question 1

All polymerases synthesize in what direction?

Template is read by them in what direction?

Answer 1

5’ - 3’ (keep adding to free 3’ OH)

3’ - 5’ (as strands are antiparallel)

Question 2

DNAse 1:

- Is what?

Answer 2

Endonuclease which creates a nick between 3’ OH and the 5’ phosphate.
It is NOT site specific, and creates nicks at low concentrations.

Question 3

Ligase requires what to work?

Answer 3

5’ phosphate and 3’ OH

Question 4

How does ligase work?

Answer 4

It binds with ATP, losing two phosphates. -> binds phosphate of AMP to the phosphate from the nucleotide which needs to be attached to 3’ OH -> unbinds AMP and enzyme, attaching the nucleotides

Question 5

Phosphatase works how?

Implications?

Answer 5

Cuts 5’ and 3’ connection to phosphate, the phosphate is completely removed.
Ligase can’t fix this.

Question 6

T4 kinase:

Answer 6

Phosphorates 5’ OH using ATP

Question 7

RNAse A:

Answer 7

Endonuclease which cuts the 5’ OH from the phosphate, leaving the phosphate on the 3’OH

Question 8

RNAse T1:

Answer 8

Endonuclease which cuts after the guanine 3’ phosphate, leaving phosphate attached to guanosine’s 3’ OH

Question 9

pX vs Xp?

Answer 9

pX means the phosphate is on the 5’ side, Xp means phosphate is on the 3’ side.

Question 10

Snake venom phosphodiesterase:

Answer 10

Exonuclease, requires 3’ OH and produces pX

Question 11

Spleen phosphodisterase

Answer 11

Exonuclease, requires 5’ OH and produces Xp

Question 12

RNase is stable after boiling and cooling. What is the significance of this?

Answer 12

Can destabilize other enzymes, making the activity of RNAse pure.

Question 13

May not be completed:

Answer 13

See table of page 18 (23) of NucleicAcids2018, ask if you need to know it.

Question 14

Restriction enzymes:

Answer 14

Cut at specific sites of DNA
Leave 5’ phosphates
Some will not cut methylated sites

Question 15

4 types of restriction enzymes:
I:
II:
III:
IV:

Answer 15

I: Cut away from restriction site
II: Cut at or in restriction site
III: Exist in complex with methylase
IV: Target modified DNA

Question 16

Draw a blunt cut, 5’ overhang cut, and a 3’ overhang cut from an enzyme.

Answer 16

Check online

Question 17

An enzyme with an:
5’ overhang:
blunt:
3’ overhang:

Answer 17

5' overhang:
EcoR1
blunt:
EcoR5
3' overhang:
Pst1