DNAP

Question 1

How is DNA different to RNA?

Answer 1

2’ C on sugar ring has an OH attached in RNA, but just a H attached in DNA

Question 2

How are 2 nucleotides connected?

Answer 2

By a phosphodiester bond
- 5’ phosphate bound to 3’ OH

Question 3

What is meant by semi-conservative DNA duplication?

Answer 3

Parent strands act as template strands for new (daughter) strands

Question 4

What complex facilitates replication?

Answer 4

Replisome which consists of multiple proteins/enzymes

Question 5

What is added to the template strand to initiate synthesis?
What is added to this?

Answer 5

Primer base
Each base is then added to the primer strand, complementary to that of the template strand

Question 6

What happens when a dNTP is reacted with template strand?
- Products?

Answer 6

(DNA)n + dNTP <–> (DNA)n+1 + PPi (inorganic pyrophosphate

Pyrophosphate is quickly hydrolysed to 2 inorganic phosphates

Question 7

What is DNAP shaped like?
What is the common structure of DNAP?
- How many domains etc?

Answer 7

Shaped like a hand

2 catalytic regions
- Top half (3 domains) - DNA synthesis
- Bottom half (1 domain) - Exonuclease activity

Question 8

How does the conformation of DNAP change when substrate binds? (hint - talk in relation to ‘hand)

Answer 8

‘Fingers’ and ‘thumb’ of ‘hand’ curl in to wrap around substrate for effective binding
‘Palm’ contains the active site residues
- Consists of β-sheet made from anti-parallel β-stands

Question 9

What are the key aspects of the DNAP active site and their functions?
- 2 residues
- Metal ions

Answer 9

2 catalytic Aspartates
- Also stabilise Mg2+
2 Mg2+ ions
- Positions the triphosphate of dNTP

Question 10

What’s the difference between the 2 major groups of polymerase active sites?
- Classical
- β-Nucleotidyl Transferase (βNT)

What must be consistent?

Answer 10

Classical - 2 aspartates on 2 different β-strands
βNT - 2 aspartates on the same β-strand

3D positioning of 2 aspartates in active site must be the same
- These enzymes have different conformations to ensure this

Question 11

2 metal ion catalytic mechanism (look at); Describe in words too
- Name function of all the

Answer 11

Transient water acts as base and attacks 3’ OH

Bond between 1st and 2nd phosphate in dNTP is cleaved
- Leaves pyrophosphate; -ve

2 Mg2+ stabilise the -ve pyrophosphate
- Aspartate stabilises 2 Mg2+

Question 12

What was their discovered to be another one of involved in some enzymes DNA synthesis mechanism?
What would it do?

Answer 12

Another divalent metal ion; Most likely Mg2+
- Acts as an acid; Accepts electrons

Question 13

How does 3rd metal ion act as an acid in some polymerases?

Answer 13

Electrons from bridging oxygen (between 1st and 2nd phosphate) attack metal
- Dissipates -ve charge and stabilises pyrophosphate

Question 14

Why does DNAP need to be so accurate?
How is it? (hint - think about H-bonds and the ‘hand’)

Answer 14

To minimise chance of mutations in the daughter strand

It is accurate due to its active site
- H-bonds are formed when W-C base pairing occurs
- Causes ‘fingers’ to close correctly, stabilising metal ion and facilitating catalysis

Question 15

How does DNAP ensure integration of dNTP and not NTP?

Answer 15

Steric gate which consists of Phenylalanine
- Clashes with sugar if it contains 2’ OH; Prevents RNA nucleotide stabilisation and it is rejected

Question 16

What happens when an incorrect base is integrated into the daughter strand? (3 main steps)
- What happens to base in duplex region of primer strand?

Answer 16

Daughter and template strand are separated at incorrect base

Daughter strand is then moved from polymerase domain to exonuclease domain
- Bases in the duplex region of the primer will also separate; Polymerase then moves in reverse direction

Domain senses misincorporated base and removes it

Question 17

Look at exonuclease domain mechanism; Describe in words too

Answer 17

2 metal ion mechanism in reverse:

Tyrosine acts as base instead of water
3 Aspartates (instead of 2) stabilise the 2 Mg2+
Leucine and Phenylalanine optimally position primer strand substrate

Question 18

Why does high accuracy come at a cost? (hint - damage)

Answer 18

Polymerase cannot recognise damaged bases
If a damaged base enters active site, it becomes stuck due to constriction
Leads to disassembly of replication machinery and apoptosis

Question 19

How does a cell overcome the drawbacks of highly accurate DNAP?
Characteristics of this method?
- What do they ensure?

Answer 19

Utilises specialised DNAP called Translesion Synthesis Polymerases

These have a wider active site so can easily accommodate a damaged nucleotide
- Ensure DNA replication doesn’t stop and continues

Question 20

What is the role of DNAP I->V

Answer 20

I - Removes primer and fills gaps in lagging strand
II - DNA repair
III - Primary enzyme of DNA synthesis
IV and V - Translesion synthesis polymerases

Question 21

How is DNAP different to the standard enzyme in how it treats its substrate/product?

Answer 21

DNAP doesn’t let go of the substrate as its bound so tightly
It moves along the DNA to catalyse the next dNTP addition