L8 - DNA replication

Question 1

describe process of DNA replication

Answer 1

1. DNA helicases catalyse breaking of H bonds between bases - unwinding the DNA strand and creating forks (using ATP)
2. DNA pol 3 catalyses addition of new complimentary bases

Question 2

how are new nucleotides added?

Answer 2

1. DNA pol 3 will bind the phosphate group of deoxy nucleoside triphosphate to the 3’ carbon of the growing DNA strand
2. DNA pol 3 cleaves off PPi, because only 1 is needed

Question 3

what happens to the PPi (2xphos) that is cleaved off?

Answer 3

hydrolysed to 2x Pi to provide some energy for the nucleotide addition

Question 4

describe DNA pol 3

Answer 4

holoenzyme - multiple subunits with different functions
dimer - one molecule for each DNA strand
contains a ‘core’ polymerase

Question 5

function of primase

Answer 5

enzyme that creates a short RNA primer to provide free 3’OH (on sugar backbone) for DNA pol to bind to

Question 6

why is primase needed

Answer 6

DNA pol 3 cant start to elongate the backbone without it

Question 7

describe the polarity problem

Answer 7

due to the polarity (5’ to 3’) of the strands and polymerase action, we have a leading and lagging strand

Question 8

how is lagging strand synthesised

Answer 8

in fragments

1. the lagging strand is looped back on itself
2. DNA pol 3 can synthesise in fragments (as the looped region is now 5’ to 3’) (okazaki fragments)
3. DNA pol 1 removes the RNA primers put in to aid DNA pol 3
4. ligase joins up okazaki fragments

Question 9

describe the supercoiling problem

Answer 9

unwinding of the DNA causes supercoiling in the strand before the fork

Question 10

function of ligase in DNA replication

Answer 10

joins up okazaki fragments of lagging strand after primers have been removed

Question 11

function of DNA pol 1 in DNA replication

Answer 11

removes RNA primers and replaces with DNA

Question 12

what fixes the supercoiling problem

Answer 12

Topoisomerases 1 & 2 relieve supercoiling

topoisomerase 1 cuts one strand
topoisomerase 2 cuts both strands

Question 13

why is proofreading important?

Answer 13

1. incorrect bases can lead to mutations

Question 14

function of single stranded binding protein

Answer 14

prevents the strands from reannealing before replication is complete

Question 15

function of the sliding clamp

Answer 15

Keeps DNA pol 3 firmly attached to DNA

Question 16

what removes incorrectly paired bases? and in what direction

Answer 16

exonucleases

3’ to 5’

Question 17

how are incorrect bases able to be removed?

Answer 17

incorrect bases wont bond as closely/correctly and exonuclease can detect this

Question 18

what is repair machinery

Answer 18

processes that remove incorrect bases that aren’t corrected by exonucleases

Question 19

what is repair machinery called in E.coli

Answer 19

mismatch repair

Question 20

explain mismatch repair mechanism

Answer 20

a group of proteins:

1. identify incorrect pairing due to bonding distortion
2. remove DNA from the new (incorrect) strand)
3. synthesise replacement DNA

Question 21

how does mismatch machinery in E.coli identify the new strand from the original?

Answer 21

the original is methylated, the new strand isn’t yet

Question 22

differences between eukaryotic and prokaryotic DNA replication

Answer 22

1. in E, replication is part of cell cycle
2. more enzymes in E (pol a, B etc)
3. E have independently replicated mtDNA
4. E has to remove and re add histones
5. P only need 1 replicon, E have many