Lahiri Flashcards
how much heat energy does the double helix formation release
250 kJ/mol
what is the overall reaction of adding nucleotide
(DNA)n +dNTP <–> (DNA)n+1 + PPi
what is template directed DNA synthesis
adding nucleotides to a strand done by DNA polymerases
what coordinates in the incoming nucleotide in DNA polymerase
two Asps and two Mg2+s
how does the double helix form despite the decrease in entropy
there is a -ve dG due to the enthalpy of H bond formation between complimentary bases
name of PPi
pyrophosphate
what happens to pyrophosphate when it is released
it is unstable so hydrolysed to form 2 Pi
what coordinates incoming dNTP
two Ds (aspartic acid) two Mg2+
what domain is the DNA polymerase active site in
palm domain, made of beta sheets
DNA polymerase mechanism
a transient water molecule acts as a base, abstracts a hydrogen from the last NT of the primer strand
pushing e- to the alpha-P of the incoming nucleotide
this then pushed the e- to break the bond between the O and the beta, gamma-phosphate
what is the role of the metal ions in the 2 metal ion DNA polymerase mechanism
Mg2+A stabilised the deprotonation and the electron transfer
Mg2+B stabilised the -ve charge on the beta, gamma-P
what is the role of the third metal ion in the 3
metal ion DNA polymerase mechanism
Mg2+ acts as an acid, accepts the e- from the O-beta, gamma-P bond breakage
how does DNA polymerase prevent non Watson and crick base pairing
fingers do not fully close when their is non watson and crick base pairing
Mg2+ will not be stabilised, inefficient catalysis
how does DNA polymerase discriminate between dNTP and NTP
phenylalanine residue from the fingers stacks against the sugar of the incoming dNTP, if there was a 2’ -OH there would be a steric clash - steric gate residue
what happens if an error occurs in DNA polymerase
it is sent from the polymerase to the exonuclease site
mistake sensing in DNA polymerase
done by different AAs for different mistakes
what happens in the exonuclease of DNA polymerase, what is its effect on the error rate of DNA polymerase
the P=3’O bond needs to be broken and the mistake NT removed
Tyr acts as a base not water this time
error rate decreased from 1 in 10^6 to 1 in 10^9
what is done to continue polymerisation what DNA has been damaged
use lesion bypass polymerase which has a less tight active site (?) to allow polymerisation to continue then other DNA repair systems can step in
why are there many different polymerases
they have to do different jobs, 1 polymerase couldn’t do them all accurately
what is different about DNA polymerase compared to other enzymes
it must hold on to one of its products (the new primer NT)
translocation machinery moves the strand along
