DNAP Flashcards
How is DNA different to RNA?
2’ C on sugar ring has an OH attached in RNA, but just a H attached in DNA
How are 2 nucleotides connected?
By a phosphodiester bond
- 5’ phosphate bound to 3’ OH
What is meant by semi-conservative DNA duplication?
Parent strands act as template strands for new (daughter) strands
What complex facilitates replication?
Replisome which consists of multiple proteins/enzymes
What is added to the template strand to initiate synthesis?
What is added to this?
Primer base
Each base is then added to the primer strand, complementary to that of the template strand
What happens when a dNTP is reacted with template strand?
- Products?
(DNA)n + dNTP <–> (DNA)n+1 + PPi (inorganic pyrophosphate
Pyrophosphate is quickly hydrolysed to 2 inorganic phosphates
What is DNAP shaped like?
What is the common structure of DNAP?
- How many domains etc?
Shaped like a hand
2 catalytic regions
- Top half (3 domains) - DNA synthesis
- Bottom half (1 domain) - Exonuclease activity
How does the conformation of DNAP change when substrate binds? (hint - talk in relation to ‘hand)
‘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
What are the key aspects of the DNAP active site and their functions?
- 2 residues
- Metal ions
2 catalytic Aspartates
- Also stabilise Mg2+
2 Mg2+ ions
- Positions the triphosphate of dNTP
What’s the difference between the 2 major groups of polymerase active sites?
- Classical
- β-Nucleotidyl Transferase (βNT)
What must be consistent?
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
2 metal ion catalytic mechanism (look at); Describe in words too
- Name function of all the
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+
What was their discovered to be another one of involved in some enzymes DNA synthesis mechanism?
What would it do?
Another divalent metal ion; Most likely Mg2+
- Acts as an acid; Accepts electrons
How does 3rd metal ion act as an acid in some polymerases?
Electrons from bridging oxygen (between 1st and 2nd phosphate) attack metal
- Dissipates -ve charge and stabilises pyrophosphate
Why does DNAP need to be so accurate?
How is it? (hint - think about H-bonds and the ‘hand’)
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
How does DNAP ensure integration of dNTP and not NTP?
Steric gate which consists of Phenylalanine
- Clashes with sugar if it contains 2’ OH; Prevents RNA nucleotide stabilisation and it is rejected
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?
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
Look at exonuclease domain mechanism; Describe in words too
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
Why does high accuracy come at a cost? (hint - damage)
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
How does a cell overcome the drawbacks of highly accurate DNAP?
Characteristics of this method?
- What do they ensure?
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
What is the role of DNAP I->V
I - Removes primer and fills gaps in lagging strand
II - DNA repair
III - Primary enzyme of DNA synthesis
IV and V - Translesion synthesis polymerases
How is DNAP different to the standard enzyme in how it treats its substrate/product?
DNAP doesn’t let go of the substrate as its bound so tightly
It moves along the DNA to catalyse the next dNTP addition
