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3382 > DNA Replication > Flashcards

DNA Replication Flashcards

1
Q

the — is clean in prokaryotes and not in eukaryotes

A

origin of replication

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2
Q

RNA primers have

A

less fidelity for DNA than DNA polymerase

primers added to 5’ end

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3
Q

what purpose of sliding clamp?

A

what all other replication proteins attach to

PCNA

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4
Q

DNA polymerases start at

A

the end of the primer

5’ to 3’

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5
Q

DNA polymerase in E.coli vs eukaryotes?

A

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

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6
Q

Type – is favoured in eukaryotes and prokaryotes for topoisomerase

A

II

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7
Q

purpose of SSB?

A

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

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8
Q

how many DNA Pols are active in replication?

A

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

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9
Q

how does E.coli deal with okazaki fragment maturation?

A
  • Pol III disociates when runs into next primer
  • Pol I digests primer while synthesizing (then ligase)
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10
Q

how do eukaryotes deal with okazaki fragment maturation?

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

Fen1 recognizes 3-way junction of DNA and RNA

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11
Q

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

A

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

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12
Q

how is unwinding favoured in vivo?

A

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

favours replication and requires less type II topoisomerases + ATP

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13
Q

what does prokaryotic ori look like?

A

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

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14
Q

what are the important components of prokaryotic ori?

A
  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

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15
Q

What are the steps of E.coli replication initiation?

A
  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
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16
Q

why must the cell control replication initiation?

A

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

17
Q

how is E.coli replication regulated?

A
  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

18
Q

What does eukaryotic replication involve?

A
  • controlled by CDKs that require cyclins for activation
  • cyclin only expressed during given phases
  • stalled replication forms trigger DNA damage or apoptosis signal
19
Q

what are eukaryotic oris like?

A

many
3-300 kbp apart
no strict consensus sequence

20
Q

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

A

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

21
Q

what are the 2 stages of eukaryotic replication initiation?

A
  1. licensing: recruit helicases (G1)
  2. activation of helicases (S)
22
Q

What are the steps of licensing?

A
  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
23
Q

How is licensing controlled in S Phase?

A
  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

24
Q

why is none of the licensing regulations not on helicase?

A

has many other purposes

25
Q

How is activation of eukaryotic replisome work?

A
  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!

3382 (20 decks)

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