(10-11) The functions and mechaisms of DNA polymerase

What 3 things is a nucleic acid made of?

• Phospahte group

• Purine or Pyridime Base

• Pentose sugar

How many rings are in a purine?

2

How many rings are in a pyrimidine?

1

What are the Purine bases?

• Adenine

• Guanine

What are the Pyrimidine bases?

• Uracil

• Thymine

• Cytosine

What are the nucleotides bound to each other by?

Phosphodiester linkage

How is the DNA helix formation spotaneous?

• The formation of the helix releases ~250KJ/mol

• This means that despite the reduction in entropy delta-G (free enery, is still negative)

What does it mean that DNA is semi-conservative?

• Parent strand acts as a template for the daughter strands

• Each new strand i half made up of the old strand (semi-conservative)

Why is accurate replication essential?

• low to mid level stress = genomic instability → tumerogenesis

• High level stress = Mitotic catastrophe → cell death

What are some causes of Replication stress?

• ROS

• Insufficient dNTP

• Oncogene activation

• TSG (tumor suppresor gene) inactivation

What is the overall reaction that DNA pol catylieses, and what class is it?

(DNA)n + dNTP → (DNA)n+1 + PPi

• SN2

What is the structural analogy of a DNA polymerase?

Right hand

What are the 3 parts of the right hand analogy and what does each bit do?

• Palm - Catalyitic site (Mg2+, Asp residues)

• Fingers - Binds incoming dNTP and positions it

• Thumb - Holds the DNA in place and maintains processivity

What happens conformationaly when DNA binds the Polymerase?

The protein conformation changes to facilitate substrate binding, ‘fingers’ curl in, Tip of thumb contacts the DNA duplex. Essential for efficient catalysis

How are different DNA polymerases convergently evolved?

• Very different overal structures and evolutionary origins,

• Active sites are very similar, with similar catlyitic mechanism

• Catalytic geometry is mechanisticly optimal for DNA synthesis

What do the 2 Metal ions do in DNA polymerase?

• Metal A: Activates the 3’ OH of primer strand, lowering pKa. Transient water acts as a base.

• Metal B: Stabilises the negative charge on the triphophate group of the incoming dNTP and the leaving PPi group

What dos the 3rd metal do in DNA polymerases with 3 metal ions?

• Metal C: Stabilises the leaving PPi further, facilitates product release, prevents reverse reaction. Acts as an acid

What is the error rate of DNA polymerase?

Average error rate of 1 in every 1,000 to 100,000 nucleotides added

How is dNTP selected for over NTP?

Searic gate residue discriminates between dNTP and NTP,

What makes DNA polymerase so accureate?

• The fingers closing align the 3’ OH and incoming dNTP, active site is restricted only allowing WC pairing

• WC pairing forms stable H-bonds with active site amino acids

• Alignments of the reactants facilitate metal a binding, facilitating catalysis

Waht happens when a rare mistake happens in DNA pol?

• The DNA primer strand moves from Pol to Exo site through conformational changes

How does a polymerase know that a misincorporation has happened?

• The thumb region tracks the minor groove

• Interacting with the sugar phosphate backbone and minor groove of the DNA

• Tracks the periodic interactions

How does Polymerase switch to Exo domain on recognition of a misincorporation?

• The mismached base slows down the polymerase as DNA geometry is distorted

• The 3’ OH is mis aligned, causing melting/fraying exposing the 3’ end

• Conformational shift or DNA translocation occurs, passing the frayed 3’ end to the Exo domain

• Incorect base removed

What is pressent at the Exonuclease site that is also present at the Pol site?

2 metal ions, working in revese to remove incorrect Nucleotide

What is the problem with DNA polymerases being high fidelity?

They can only accomodate WC base pairs, meaning if damadged bases occur then dosen’t work.

• DNA undergoes damadge on a daily basis

Which type of Polymerase can accomodate damadged bases?

Translesion synthesis polymerases, accomodate lesions

What does DNA pol I do in Prokaryotes?

Removes primer and fills gaps on lagging strand

What does DNA pol II do in Prokaryotes?

DNA repair (error prone polymerase)

What does DNA pol III do in Prokaryotes?

Primary enzyme of DNA synthesis

What do DNA pol IV and V do in Prokaryotes?

Translesion synthesis polymerases

Explain the ratchet like mechanism in Pol II

The DNA stars bound to the Pol:

• Nucleotide added

• confomational reset

• Ratchet forwards

Dosent ever move back (like a ratchet)