L9 - DNA replication 1

Question 1

how many families of DNA polymerases are there and what is different about them

Answer 1

8

= core catalytic domains are different due to difference in folding of active site

Question 2

what general structure do all DNA polymerases have

Answer 2

right hand shaped structure

= finger, thumb and palm domains

Question 3

which polymerase is the main replicating enzyme

Answer 3

DNA polymerase 3
- 3 subunits/polypetides

= although 1 was dicovered first

Question 4

which domain does the site of reaction take place in DNA polymerases

Answer 4

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

Question 5

define proscessivity

Answer 5

ability of a protein to stay ascociated to somthing

Question 6

what is the role of the sliding clamp in DNA replication

Answer 6

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

Question 7

how does the “hand” structure of DNA polymerases work when adding a nucleotide

Answer 7

1. ‘hand’ openes allowing a single base of the template strand into active site

= in palm domain

2. complimentary nucleotide comes in and hand closes causing catalysis . phosphodiester bond
3. hand opens and pyrophosphate released

= only 1/3 of the triphosphate nucletide remains in the DNA stucture

Question 8

name of the molecule released when ‘hand’ openes in DNA replication

Answer 8

pyrophosphate

= 3’ hydroxyl attacks 5’ phosphate
= bond formed between the 1st phoshate in triphosphate
= other 2 are released

Question 9

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

Answer 9

catalytic selectivity

= when fingers close to forming catyltic site
= same shape every time

complimnetary base pairs fit together perfectly and bind fastest

Question 10

what happens when 2 non-complimnetary base pairs are added in the catalytic site of DNA polymerase

Answer 10

steric clashes

= bases bash inton active site
= active site is unstable and caanot cuase catalysis of bases

Question 11

describe how catalytic slectivity prevents addition of rNTPs (Ribo-nucletides triphosphate - Uracil)

Answer 11

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

Question 12

name the 2 ways that errors are reduced in DNA replication

Answer 12

Catalytic selectivity - DNA polymerase

Enzymatic proofreading

mismatch repair

Question 13

if DNA polymerase has catalytic selctivity why are there errors in replication

Answer 13

chemistry of bases

= tortemisation

Question 14

what is the tautomer form of a nucletide and how does this cause errors in DNA replication

Answer 14

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

Question 15

how long do tautormers last/imino form of cytosine

Answer 15

very rare for hydorgen to fall off NH2

rapidly goes back to amino group

= changes which side chaiuns are hydrogen donors or acceptors

Question 16

how do tautomers affect DNA replication

Answer 16

incorrect base added

tautomer changes back to stable form

= causes distortion in DNA
= dealth with by enzymatic proofreading

Question 17

mechanism of enzymatic proofreading

Answer 17

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

Question 18

what the roles of the palm,fingers and thumb domain in DNA polymerase

Answer 18

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

Question 19

enzymatic proofreading simple mechanism

Answer 19

1. incorrect base added and detected by palm
2. exonuclease chews back in 3’-5’ way
3. DNA polymerase continues replication

Question 20

which polymerases have no proofreadinga dn do not stick onto the DNA well/high affinity

Answer 20

polymerase 4 + 5

= Y-family transeslion polymerases

Question 21

what are Y-family translesion polymerases

Answer 21

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

Question 22

what do transeslion polymerases have that allows them to add random bases

Answer 22

larger and more open catalytic site

= flexible on what bases can be added

Question 23

what does the main copying error in DNA replication come from

Answer 23

base stuctural isomerisation

= tatutomers due to hydrogens switching about groups on base