L8 - DNA replication Flashcards
describe process of DNA replication
- DNA helicases catalyse breaking of H bonds between bases - unwinding the DNA strand and creating forks (using ATP)
- DNA pol 3 catalyses addition of new complimentary bases
how are new nucleotides added?
- DNA pol 3 will bind the phosphate group of deoxy nucleoside triphosphate to the 3’ carbon of the growing DNA strand
- DNA pol 3 cleaves off PPi, because only 1 is needed
what happens to the PPi (2xphos) that is cleaved off?
hydrolysed to 2x Pi to provide some energy for the nucleotide addition
describe DNA pol 3
holoenzyme - multiple subunits with different functions
dimer - one molecule for each DNA strand
contains a ‘core’ polymerase
function of primase
enzyme that creates a short RNA primer to provide free 3’OH (on sugar backbone) for DNA pol to bind to
why is primase needed
DNA pol 3 cant start to elongate the backbone without it
describe the polarity problem
due to the polarity (5’ to 3’) of the strands and polymerase action, we have a leading and lagging strand
how is lagging strand synthesised
in fragments
- the lagging strand is looped back on itself
- DNA pol 3 can synthesise in fragments (as the looped region is now 5’ to 3’) (okazaki fragments)
- DNA pol 1 removes the RNA primers put in to aid DNA pol 3
- ligase joins up okazaki fragments
describe the supercoiling problem
unwinding of the DNA causes supercoiling in the strand before the fork
function of ligase in DNA replication
joins up okazaki fragments of lagging strand after primers have been removed
function of DNA pol 1 in DNA replication
removes RNA primers and replaces with DNA
what fixes the supercoiling problem
Topoisomerases 1 & 2 relieve supercoiling
topoisomerase 1 cuts one strand
topoisomerase 2 cuts both strands
why is proofreading important?
- incorrect bases can lead to mutations
function of single stranded binding protein
prevents the strands from reannealing before replication is complete
function of the sliding clamp
Keeps DNA pol 3 firmly attached to DNA
what removes incorrectly paired bases? and in what direction
exonucleases
3’ to 5’
how are incorrect bases able to be removed?
incorrect bases wont bond as closely/correctly and exonuclease can detect this
what is repair machinery
processes that remove incorrect bases that aren’t corrected by exonucleases
what is repair machinery called in E.coli
mismatch repair
explain mismatch repair mechanism
a group of proteins:
- identify incorrect pairing due to bonding distortion
- remove DNA from the new (incorrect) strand)
- synthesise replacement DNA
how does mismatch machinery in E.coli identify the new strand from the original?
the original is methylated, the new strand isn’t yet
differences between eukaryotic and prokaryotic DNA replication
- in E, replication is part of cell cycle
- more enzymes in E (pol a, B etc)
- E have independently replicated mtDNA
- E has to remove and re add histones
- P only need 1 replicon, E have many
