DNA Replication

Question 1

the — is clean in prokaryotes and not in eukaryotes

Answer 1

origin of replication

Question 2

RNA primers have

Answer 2

less fidelity for DNA than DNA polymerase

primers added to 5’ end

Question 3

what purpose of sliding clamp?

Answer 3

what all other replication proteins attach to

PCNA

Question 4

DNA polymerases start at

Answer 4

the end of the primer

5’ to 3’

Question 5

DNA polymerase in E.coli vs eukaryotes?

Answer 5

E.coli = DNA Pol III is replicative
Eukaryotes = δ DNA Pol

Question 6

Type – is favoured in eukaryotes and prokaryotes for topoisomerase

Answer 6

II

Question 7

purpose of SSB?

Answer 7

stops unwound DNA from acting like RNA and rebase pairing or forming 2º structures

Question 8

how many DNA Pols are active in replication?

Answer 8

NOT SURE
- 2 DNA pols; one for each strand
- may have a 3rd for okazaki fragments

Question 9

how does E.coli deal with okazaki fragment maturation?

Answer 9

• Pol III disociates when runs into next primer
• Pol I digests primer while synthesizing (then ligase)

Question 10

how do eukaryotes deal with okazaki fragment maturation?

Answer 10

• pol δ displaces primer during synthesis
• primer removed by flap endonuclease (Fen 1)

Fen1 recognizes 3-way junction of DNA and RNA

Question 11

what are the local differences of supercoiling in a replication fork?

Answer 11

in ss region, its underwound (requires +ve supercoiling)
in ds region, its overwound (requires -ve supercoiling)

average overall twist is the same (ex. 40 bp/turn)

if less bp per turn, over wound

Question 12

how is unwinding favoured in vivo?

Answer 12

bacteria: DNA gyrase intros ive supercoiling
euk: DNA/histone makes underwound state

favours replication and requires less type II topoisomerases + ATP

Question 13

what does prokaryotic ori look like?

Answer 13

in circular genome, only 1, has 245 bp specific sequence in ori

Question 14

what are the important components of prokaryotic ori?

Answer 14

1. DNA unwinding DNA element: 13 A-T rich repeats
2. 9 mer-sites recognized and binds DnaA
3. Dam sites: GATC

Dam site should be every 256, but is more in ori -> not coincidence

Question 15

What are the steps of E.coli replication initiation?

Answer 15

1. DnaA-ATP and HU protein binds to mer-site in ORI
2. DNA is in coiled form
3. DNA stressed –> unwinds A-T rich region to makes some ss
4. recruit DnaC (loading protein for DnaB)
5. DnaB = helicase –> expand replicaion fork
6. DnaA triggered to inactivate by hydrolyzing to DnaA-ADP

Question 16

why must the cell control replication initiation?

Answer 16

don’t want to start replication when replication is already happening or over replicate

Question 17

how is E.coli replication regulated?

Answer 17

1. Regulatory Inactivation of DnaA (RIDA) hydrolyzes DnaA-ATP to ADP
2. IHF protein guides free DnaA to bind to datA locus and encourages hydrolysis to ADP
3. DARS sites: in genome makes DnaA release ADP to make DnaA (nucleotide exchange)
4. Dna added to ATP to reinitiate
5. hemimethylated Dam sites blocked by SeqA so DnaA can’t bind

full methylation in 10 mins

Question 18

What does eukaryotic replication involve?

Answer 18

• controlled by CDKs that require cyclins for activation
• cyclin only expressed during given phases
• stalled replication forms trigger DNA damage or apoptosis signal

Question 19

what are eukaryotic oris like?

Answer 19

many
3-300 kbp apart
no strict consensus sequence

Question 20

why is there no strict consensus seq for euk. oris?

Answer 20

recognize DNA in chromatin form where some oris might be covered by histone – need to access nucleosome free regions

Question 21

what are the 2 stages of eukaryotic replication initiation?

Answer 21

1. licensing: recruit helicases (G1)
2. activation of helicases (S)

Question 22

What are the steps of licensing?

Answer 22

1. origin of replication complex (hexamer) (ORC) binds DNA
2. Cdc6 binds ORC
3. MCM2-7 binds Cdt1
4. Cgt1 binds Cdc6
5. MCM2-7 loaded to ds DNA
6. MCM207 hydrolyzes ATP so Cdc6 & Cdt1 released
7. repeat of same of 2nd ORC to have 2 MCM2-7 complexes

Question 23

How is licensing controlled in S Phase?

Answer 23

1. Cdc6 phosphorylated and exported to cytosol
2. Cdt1 phosphorylated and degraded
3. ORC2 phosphorylated -> lower affinity to chromatin and falls off DNA
4. ORC 1 phosphorylated and degraded
5. Cdt1 bound by geminin to stop Cdc1 interaction with MCM

geminin degraded in G1

Question 24

why is none of the licensing regulations not on helicase?

Answer 24

has many other purposes

Question 25

How is activation of eukaryotic replisome work?

Answer 25

1. CDK2 and DDk (kinases) become active in S phase 2. MCM2-7 and other scaffold proteins are phosphorylated -> active to load Cdc45 and GINS 3. polyerase, PCNA, RFC, RPA recruited | most licensed origins do not fire!