Lecture 2- Eukaryotic DNA replication

Question 1

what is the eukaryotic replication phase

Answer 1

S phase

Question 2

how many replication forks are there in bacterial DNA replication?

Answer 2

2

Question 3

how many replication forks are there in eukaryotic DNA replication?

Answer 3

can be many- form ‘replication bubbles’

Question 4

first protein complex which binds to DNA to initiate replication

Answer 4

ORC- origin recognition complex

Question 5

2nd proteins to bind to the DNA

Answer 5

DNA helicase Mcm2-7

Question 6

what proteins facilitate helicase loading?

Answer 6

Cdc6, Cdt1

Question 7

what activates DNA helicase?

Answer 7

CDK and DDK- these lead to the generation of ssDNA which can be replicated by pols

Question 8

when do polymerases load to DNA?

Answer 8

late G1 phase into S

Question 9

features of pol alpha

Answer 9

acts as a primase (produces primers), doesn’t do a lot of replicating

Question 10

pol epsilon and delta- features compared to alpha

Answer 10

better error rates and proofreading activity than pol alpha

Question 11

which polymerase stays on the strand longest?

Answer 11

epsilon- keeps replicating until it hits another fork as it takes the leading strand

Question 12

pol delta activity

Answer 12

takes on the lagging strand, so stops and starts more

Question 13

which proteins join Okazaki fragments?

Answer 13

Fel1 and DNA ligase I

Question 14

what happens once the helicases reach another primer?

Answer 14

helicase switches to encompass both strands

Question 15

what triggers the removal of the replication complex from the DNA?

Answer 15

ubiquitilation of Mcm7 (helicase)- but is not broken down- complex removed from chromatin by p97 ATPase

Question 16

example of how replication origins can be specified

Answer 16

AT rich region in S pombe, epigenetic marks and G4 structures in mammals

Question 17

why might it be advantageous to lack specific replication sequences?

Answer 17

allows change throughout the lifetime, e.g. during development vs adulthood- may be useful to have more origin points when there is a lot of cell division and specialisation

Question 18

how does a cell ensure there is no replication in G0

Answer 18

not synthesising the necessary proteins, not activating CDK etc, degrading and inactivating proteins during S phase

Question 19

example of protein deactivation during S as regulation

Answer 19

geminin deactivates DNA replication factor CDT1, which acts as a ‘licencing factor’ in the ORC. also ubiquitinated and broken down following formation of a replication fork

Question 20

what is a ‘temporal program’?

Answer 20

different clusters of replication happening at different times throughout S phase

Question 21

how can we identify temporal programs?

Answer 21

denaturing and staining at a specific point, or looking at shifts from euchromatin to heterochromatin

Question 22

why might temporal programs exist?

Answer 22

to reduce the energy/dNTP/etc burden on cells, or to alter copy number- more early genes should be replicated than late