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Flashcards in DNA Replication Deck (44):
1

How is replicating DNA conserved

Semi-conserved.
One parent (original) strand, one daughter (new) strand.

2

What is different about the origin of DNA replication in Prokaryotes and Eukaryotes

Prokaryotes - Only one origin
Eukaryotes - Multiple origins

3

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

4

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.

5

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.

6

What is the main structural difference between prokaryote and eukaryote DNA

Prokaryote - naked DNA
Eukaryote - as chromatin, wrapped around a histone.

7

What proteins are involved in the initial stages of DNA replication

Initiator proteins (P) / ORC complex (E).
Helicase

8

How does a helicase function

Unwinds DNA by breaking the hydrogen bonds that hold the two strands together (hydrolysis)

9

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.

10

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.

11

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)

12

What is the structure of a eukaryotic primase enzyme

4 subunits.
1 alpha primase subunit which has DNA polymerase activity

13

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.

14

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)

15

How accurate is DNA replication

Very. Replicases have proofreading activity. Mutations occur in ~ 1 in 10^-8/10.

16

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

17

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.

18

What is the structure of DNA pol III

An asymmetric dimer.

19

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

20

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

21

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.

22

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.

23

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

24

What is the structure of the beta subunit of DNA pol III in e.coli

Ring shaped dimer

25

What is the eukaryotic homologue of the beta subunit clamp in e.coli

PCNA

26

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' )

27

What is the purpose of telomeres

They fill the 'primer gap' at the end of a lagging strand.
To attempt to maintain DNA length

28

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.

29

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)

30

What are the main stages of the eukaryotic cell cycle

Mitosis (Pro/Meta/Ana/Telo) <- DNA replication

31

At what point in the cell cycle are the origins (of replication) active

S-phase (once)

32

What factor regulates origin (of replication) activation, and how

Licensing factor.
Present at origins that haven't fired; absent from origins that have.

33

When in the cell cycle is DNA replication initiated

As soon as a G1 nucleus enters S-phase

34

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

35

How does a licencing factor become attached to DNA

Its a component of the pre-replicative complex that assembles at origins (ORC)

36

What does ORC stand for

Origin Recognition Complex

37

Name proteins involved in formation of the pre-initiation complex

Cdc 6/18 , Cdt1 (which recruit:)
MCM2-7
ORC

38

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.

39

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.

40

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.

41

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.

42

At which point in the cell cycle do CDK and DDK both act as a checkpoint

Initiation of DNA replication - beginning of S-phase.

43

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.

44

What role does GINS have in DNA replication

Required for formation of active helicase CMG
Stabilises the MCM complex