L9 - DNA replication 1 Flashcards
how many families of DNA polymerases are there and what is different about them
8
= core catalytic domains are different due to difference in folding of active site
what general structure do all DNA polymerases have
right hand shaped structure
= finger, thumb and palm domains
which polymerase is the main replicating enzyme
DNA polymerase 3
- 3 subunits/polypetides
= although 1 was dicovered first
which domain does the site of reaction take place in DNA polymerases
palm domain
= finger domains cause bendinga round them to direct DNA into palm/active site
= hydrogen bonds/non-specific bonding to minor grooves of DNA in palm
define proscessivity
ability of a protein to stay ascociated to somthing
what is the role of the sliding clamp in DNA replication
Sticks to DNA and acts as an assembly point for proteins
= if polymerase falls off while replicating
= due to being ascoiated with clamp it can reassemble and continue
how does the “hand” structure of DNA polymerases work when adding a nucleotide
- ‘hand’ openes allowing a single base of the template strand into active site
= in palm domain
- complimentary nucleotide comes in and hand closes causing catalysis . phosphodiester bond
- hand opens and pyrophosphate released
= only 1/3 of the triphosphate nucletide remains in the DNA stucture
name of the molecule released when ‘hand’ openes in DNA replication
pyrophosphate
= 3’ hydroxyl attacks 5’ phosphate
= bond formed between the 1st phoshate in triphosphate
= other 2 are released
you ca make the same number of hydrogen bonds between any of the base pairs - so why does a C bond to a G and T to a A
catalytic selectivity
= when fingers close to forming catyltic site
= same shape every time
complimnetary base pairs fit together perfectly and bind fastest
what happens when 2 non-complimnetary base pairs are added in the catalytic site of DNA polymerase
steric clashes
= bases bash inton active site
= active site is unstable and caanot cuase catalysis of bases
describe how catalytic slectivity prevents addition of rNTPs (Ribo-nucletides triphosphate - Uracil)
rNTPs have an additional hydroxyl - 2’-OH group on ribose sugar
discrimnator amino acids active sites streiccaly clash with the hydroxyl
= Uracil/rNTP does not fit into active site properly
= the 3’ primer -OH in strand cannot form phosphodiester bonbd with the nucleotide
name the 2 ways that errors are reduced in DNA replication
Catalytic selectivity - DNA polymerase
Enzymatic proofreading
mismatch repair
if DNA polymerase has catalytic selctivity why are there errors in replication
chemistry of bases
= tortemisation
what is the tautomer form of a nucletide and how does this cause errors in DNA replication
tortemisation
= nucleotides change around their hydorgens forming isomers = tautomers
cytosine - imino form - hydorgen falls from NH2 group and binds to another
changes which base pairngs fit togther in active site due to different side chains
how long do tautormers last/imino form of cytosine
very rare for hydorgen to fall off NH2
rapidly goes back to amino group
= changes which side chaiuns are hydrogen donors or acceptors
how do tautomers affect DNA replication
incorrect base added
tautomer changes back to stable form
= causes distortion in DNA
= dealth with by enzymatic proofreading
mechanism of enzymatic proofreading
distortion of DNA due to incoreect base recognised by hydorgen bonds with minor groove by ‘palm’ domain
= 3’ OH in incorrect position
DNA forced into exonuclease domain
unwinding of 3-4 base pairs and chews up in 3’-5’
= most recently added is chewed first
DNA forced back into palm to comtinue replication
what the roles of the palm,fingers and thumb domain in DNA polymerase
palm:
contains active site
- checks minor groove via -H bonding for incoorect structure
fingers:
positition the incoming nucletide into active site
- help form active site amd ensure only 1 added at a time by ‘opening’ + ‘closing’
thumb:
stability of structure - holds newly synthesised strand
enzymatic proofreading simple mechanism
- incorrect base added and detected by palm
- exonuclease chews back in 3’-5’ way
- DNA polymerase continues replication
which polymerases have no proofreadinga dn do not stick onto the DNA well/high affinity
polymerase 4 + 5
= Y-family transeslion polymerases
what are Y-family translesion polymerases
polymerase 3 cannot bypass bulky DNA damage
= beta clamp + pol dissociate
clamp rejoins with translesion enzyme
adds random bases ro strand
= discociates and pol 3 rejoins
= damage will be repaired later
what do transeslion polymerases have that allows them to add random bases
larger and more open catalytic site
= flexible on what bases can be added
what does the main copying error in DNA replication come from
base stuctural isomerisation
= tatutomers due to hydrogens switching about groups on base
