DNA Replication Flashcards
the — is clean in prokaryotes and not in eukaryotes
origin of replication
RNA primers have
less fidelity for DNA than DNA polymerase
primers added to 5’ end
what purpose of sliding clamp?
what all other replication proteins attach to
PCNA
DNA polymerases start at
the end of the primer
5’ to 3’
DNA polymerase in E.coli vs eukaryotes?
E.coli = DNA Pol III is replicative
Eukaryotes = δ DNA Pol
Type – is favoured in eukaryotes and prokaryotes for topoisomerase
II
purpose of SSB?
stops unwound DNA from acting like RNA and rebase pairing or forming 2º structures
how many DNA Pols are active in replication?
NOT SURE
- 2 DNA pols; one for each strand
- may have a 3rd for okazaki fragments
how does E.coli deal with okazaki fragment maturation?
- Pol III disociates when runs into next primer
- Pol I digests primer while synthesizing (then ligase)
how do eukaryotes deal with okazaki fragment maturation?
- pol δ displaces primer during synthesis
- primer removed by flap endonuclease (Fen 1)
Fen1 recognizes 3-way junction of DNA and RNA
what are the local differences of supercoiling in a replication fork?
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
how is unwinding favoured in vivo?
bacteria: DNA gyrase intros ive supercoiling
euk: DNA/histone makes underwound state
favours replication and requires less type II topoisomerases + ATP
what does prokaryotic ori look like?
in circular genome, only 1, has 245 bp specific sequence in ori
what are the important components of prokaryotic ori?
- DNA unwinding DNA element: 13 A-T rich repeats
- 9 mer-sites recognized and binds DnaA
- Dam sites: GATC
Dam site should be every 256, but is more in ori -> not coincidence
What are the steps of E.coli replication initiation?
- DnaA-ATP and HU protein binds to mer-site in ORI
- DNA is in coiled form
- DNA stressed –> unwinds A-T rich region to makes some ss
- recruit DnaC (loading protein for DnaB)
- DnaB = helicase –> expand replicaion fork
- DnaA triggered to inactivate by hydrolyzing to DnaA-ADP
why must the cell control replication initiation?
don’t want to start replication when replication is already happening or over replicate
how is E.coli replication regulated?
- Regulatory Inactivation of DnaA (RIDA) hydrolyzes DnaA-ATP to ADP
- IHF protein guides free DnaA to bind to datA locus and encourages hydrolysis to ADP
- DARS sites: in genome makes DnaA release ADP to make DnaA (nucleotide exchange)
- Dna added to ATP to reinitiate
- hemimethylated Dam sites blocked by SeqA so DnaA can’t bind
full methylation in 10 mins
What does eukaryotic replication involve?
- controlled by CDKs that require cyclins for activation
- cyclin only expressed during given phases
- stalled replication forms trigger DNA damage or apoptosis signal
what are eukaryotic oris like?
many
3-300 kbp apart
no strict consensus sequence
why is there no strict consensus seq for euk. oris?
recognize DNA in chromatin form where some oris might be covered by histone – need to access nucleosome free regions
what are the 2 stages of eukaryotic replication initiation?
- licensing: recruit helicases (G1)
- activation of helicases (S)
What are the steps of licensing?
- origin of replication complex (hexamer) (ORC) binds DNA
- Cdc6 binds ORC
- MCM2-7 binds Cdt1
- Cgt1 binds Cdc6
- MCM2-7 loaded to ds DNA
- MCM207 hydrolyzes ATP so Cdc6 & Cdt1 released
- repeat of same of 2nd ORC to have 2 MCM2-7 complexes
How is licensing controlled in S Phase?
- Cdc6 phosphorylated and exported to cytosol
- Cdt1 phosphorylated and degraded
- ORC2 phosphorylated -> lower affinity to chromatin and falls off DNA
- ORC 1 phosphorylated and degraded
- Cdt1 bound by geminin to stop Cdc1 interaction with MCM
geminin degraded in G1
why is none of the licensing regulations not on helicase?
has many other purposes
