DNA Replication Flashcards
How is replicating DNA conserved
Semi-conserved. One parent (original) strand, one daughter (new) strand.
What is different about the origin of DNA replication in Prokaryotes and Eukaryotes
Prokaryotes - Only one origin
Eukaryotes - Multiple origins
Why is necessary for a DNA replication origin to occur
Binding sites for initiator proteins/origin recognition proteins
Something to unwind/distort the DNA
Sometimes need binding sites for transcription activation proteins.
Overall sequence is AT rich
What is the mechanism by which DNA polymerase makes a polymer
DNA strand separation exposes the hydrogen bond donor/acceptor groups on each base.
DNA pol uses this to add free nucleotides to the 3’ OH end of the chain.
The new nucleotide loses 2 phosphate groups in this.
What is semi-discontinuous DNA replication and why does it occur
Antiparallel strands of DNA.
DNA pol can only code in the 3’ direction.
The 5’-3’ (leading) strand can be synthesised straight away.
The 3’-5’ (lagging) strand is semi-discontinuous. Requires specialist bits to recruit DNApol to site further ahead, and it codes backwards. Creates Okazaki fragments which are stuck together by DNA ligase.
What is the main structural difference between prokaryote and eukaryote DNA
Prokaryote - naked DNA
Eukaryote - as chromatin, wrapped around a histone.
What proteins are involved in the initial stages of DNA replication
Initiator proteins (P) / ORC complex (E). Helicase
How does a helicase function
Unwinds DNA by breaking the hydrogen bonds that hold the two strands together (hydrolysis)
How can a helicase be identified in vitro
Radioactively label DNA (if helicase present DNA will unwind)(observed in gel electrophoresis)
Fragment displacement assay. radioactively labelled DNA is hybridised to ss circular DNA (complementary). If helicase present DNA unwinds and fragment dissociates from the circular DNA.
What is the importance of single strand (ss) DNA binding proteins in DNA replication
They assist in helix-opining processes, then stabilise the unwound DNA.
They bind to DNA without shielding bases.
What is the point of a priming event
To provide a 3’ OH group on the template DNA strand. Allows DNA pol to work.
Single primer for leading strand; multiple needed on the lagging strand.
Carried out by a primase enzyme (DNAG in prokaryotes)
What is the structure of a eukaryotic primase enzyme
4 subunits.
1 alpha primase subunit which has DNA polymerase activity
What is required to catalyse primase activity
A template to bind to Nucleotide binding Synthesis initiation Extension to a functional primer Transfer to DNA pol.
What is the process of DNA replication (in prokaryotes/eukaryotes)
ABF1 binds to origin (E)
Initiator complex binds (DNA(A/C)/ ORC)
Helicase binds (DNAB/ MCMs)
Replication initiated (SSB, Gyrase/ R(P/F)A, Topo 2)
Replication elongation (DNAG, RNA primer / DNA pol 1, RNA primer, Initiator DNA)
Elongation stage 2 (DNA pol 3/DNA pol 2)
How accurate is DNA replication
Very. Replicases have proofreading activity. Mutations occur in ~ 1 in 10^-8/10.
How does DNA polymerase act as a proof-reader
Nucleotide enters vacant site of DNA pol.
DNA pol advances 1 nucleotide.
If nucleotide is not correct, polymerase activity is inactivated; DNA pol goes back. Exonuclease activity activated. Removes nucleotide.
DNA pol tries again
Describe the three types of DNA polymerase present in e.coli
DNA pol I (most common);
Monomer, polymerase, exonuclease (both direction). Involved in DNA replication and repair.
DNA pol II;
Monomer, polymerase, 3’-5’ exonuclease activity. Involved in DNA repair
DNA pol III;
Multimer, polymerase, 3’-5’ exonuclease. Involved in DNA replication.
What is the structure of DNA pol III
An asymmetric dimer.
What roles do specific DNA pol III subunits have
Alpha - polymerase (DNA synthesis)
Beta - DNA Clamp
Epsilon - 3’-5’ exonuclease (proofreading)
Gamma - displaces primase from the RNA primer
What roles do specific higher eukaryotic DNA polymerase subunits have
Alpha - DNA replication Beta - DNA repair Delta - DNA replication Gamma - mitochondrial DNA replication Epsilon - DNA replication and repair
How are the alpha, delta and gamma subunits of eukaryotic DNA pol involved in DNA replication
Alpha (complex with primase; initiator), Delta (links lagging strand primer region), Epsilon (leading strand) synthesise DNA at the replication fork.
What is the mechanism by which DNA ligase joins okazaki fragments
Catalyses the formation of a phosphodiester bond between 3’ OH and 5’ P on adjacent nucleotides.
Why is a clamp (accessory protein) required for DNA replication
It keeps the polymerase on the DNA when it is synthesising, then facilitates polymerase release when replication is finished
What is the structure of the beta subunit of DNA pol III in e.coli
Ring shaped dimer
What is the eukaryotic homologue of the beta subunit clamp in e.coli
PCNA
Suggest a way that the leading and lagging strand can be coordinated as one action
Lagging strand template may be looped. Would enable polymerase at replication fork to synthesise both strands. (lagging strand would be in reverse 5’-3’ )
What is the purpose of telomeres
They fill the ‘primer gap’ at the end of a lagging strand.
To attempt to maintain DNA length
What is the function of telomerase
To resynthesise telomeric sequences - extend the 3’ end of a DNA strand.
Acts as a reverse transcriptase (makes DNA from RNA)
Less telomerase leads to telomere shortening.
How is DNA replication and the cell cycle linked
Replication MUST only occur once per cell cycle.
Replication initiation commits the cell to division (which occurs after replication is complete)
What are the main stages of the eukaryotic cell cycle
Mitosis (Pro/Meta/Ana/Telo) <- DNA replication
At what point in the cell cycle are the origins (of replication) active
S-phase (once)
What factor regulates origin (of replication) activation, and how
Licensing factor.
Present at origins that haven’t fired; absent from origins that have.
When in the cell cycle is DNA replication initiated
As soon as a G1 nucleus enters S-phase
How is DNA re-replication blocked
Either; An inhibitor binds to the DNA after replication
Or; A licence factor required for DNA replication is removed when replication is complete
How does a licencing factor become attached to DNA
Its a component of the pre-replicative complex that assembles at origins (ORC)
What does ORC stand for
Origin Recognition Complex
Name proteins involved in formation of the pre-initiation complex
Cdc 6/18 , Cdt1 (which recruit:)
MCM2-7
ORC
How do CDC6/18 and CDT1 act as licencing factors
CDC6 is phosphorylated/degraded after it has recruited MCM2-7/ORC to the origin.
CDT1 undergoes ubiquitin-mediated degradation. Activity is inhibited by geminin.
How can the MCM complex act as a licencing factor
Required for DNA replication.
Can be phosphorylated and dissociate from chromatin. This correlates with the removal of the licence for chromosomes to replicate.
How can the MCM complex act as a replicative helicase
Possess DNA-dependent ATPase motifs.
Similar structure to other known helicases
Shown to leave origins when ssDNA binding protein does. Appears to move with the replication fork.
How are CDK and DDK involved in replicative helicase activation
CDK required for formation of pre-loading complex and its association with pre-replicative complex at the origin.
DDK phosphorylates MCM complex. Results in conformational change. Helps convert inactive MCM complex into an active helicase.
At which point in the cell cycle do CDK and DDK both act as a checkpoint
Initiation of DNA replication - beginning of S-phase.
What role does cdc45 have in DNA replication
Binds to the pre-replicative complex.
Stimulates helicase activity of the MCM complex.
Interacts with DNA pol delta/epsilon and bridges with helicase; important in elongation.
What role does GINS have in DNA replication
Required for formation of active helicase CMG
Stabilises the MCM complex
