L10 - DNA replication 2 Flashcards
how many origins of replication does E Coli have and why
1
circular genome = 2 replication forks
1 clockwise + 1 anticlockwise
describe e.colis replisome - complex of proteins fro DNA synthesis
sliding clamp ascociated with 2x DNA polymerases held on template strands
= 1 works clockwise the other anticlockwise round circular genome
= 2x repliaction forks
helicase unwinds strand with help of DNA gyrase - supecoiling modifier
primase produces short RNA primers for lagging strand
single stranded binding proteins (SSBs) bind to open ssDNA to protect them
what is DNA polymerase 3 holoenzyme and the componenets
main enzyme complex for DNA replication in prokaryotes
- 2-3x core DNA polymerases
- sliding clamp (b subunit)
- clamp loader (y complex)
- flexible linkers (t proteins)
describe the componets of the holoenzyme in prokaryotes -polymerases,clamp,clamp loader and flexible linkers
DNA pol 3:
hand shaped enzyme with 3 subunits - alpha,epsilon and O
clamp loader - y:
uses ATP as energy to open beta clamp onto DNA at primer-template junction
sliding clamp - b:
donut shaped protein that cicles DNA tethering polymerase
flexible linkers - t:
link each core polymerase to clamp loader , very flexible
= allow binding of polymerases
describe DNA polymerase 3 core subunits
alpha:
catalytic subunit for polymerase activity
epsilon:
proofreading mechanisms with 3’-5’ exonuclease activity
O:
stimulates epsilon subunit
describe the sliding clamp
ring/donut shaped protein that surround dsDNA
pore in the middle with a layer of water mooecules inside
= easy slding of DNA
descibe mechansism of clamp loader to load clamp onto DNA
claw like pentamer protein
- ATP binds opening claw with high affinity to beta-clamp
- protein interactions with loader opens sliding clamp = No ATP required
- complex has high affinity for junction/meeting point of single + dsDNA = replication fork
- ATP hydrolysis on clampmloader releases slidng clamp + loader discociates
describe the trombone model of DNA replication in E.coli
- dsDNA is unwound by helicase
- leading strand has 3’ open ready to be added to
- lagging strand (bottom of trombone) is added to by RNA primers coming out of the helicase
- loading clamp loads sliding clamp + polymerase onto RNA primer (clamp binds with high affinity to ds-ss junctiuon)
- pol works BACKWARDS extending the ‘trombone’ with ocazaki fragments on RNA primer
= extending area of dsDNA being newly syntheised and the ssDNA just been unwound between pol 3 + helicase
- once fragment completed polymerase discociates from clamp
- new clamp at clamp-loader is loaded onto the next RNA primer = trombone retracts
what if DNA polymerase maes an undeited mistake
incorrect base pairng in one of the 2 dsDNA helices produced
2nd round of replication adds base pairng to the wrongly added base
= forms a natural base pair
= apart of the genome now and impossible to know what was a mutation and what wasn’t
name the complex in E.coli that recognises mismtached bases - mismatch repair
MutS
= homodimer with DNA binding domain at bottom
= scans for distortions
doesnt directly read bases detctes structural changes
mechanism of mismatch repair in E.coli - MutS
- MutS detcts mismcth by distortions in dsDNA
- binds via DNA binding domain
- recruits MutL + MutH
- nick created in newly synthesised daughter strand
- endonuclease cuts strand TOWARDS MutS + fragment removed
- 3’ is left to be acted on by polymerase + correct bases added
how does mismatch repair mechansism/proteins know whether to cut in the template strand or the newly synthesied strand that contains the mismatched base
methylation patterns
= all adenines in DNA are methyltated
newly synthesied stsrands have short period whete A is unemthylated
MutH recgonsies lack of methyl on A
= cuts nick in strand
