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23 Cards in this Set

  • Front
  • Back

What are the 4 fundamental features of DNA replication in all organisms?

- It is semiconservative

- bidirectional (two replications folks per origin)

- semi-discontinuous (lagging strand - Okazaki)

- dependent on RNA primers

What does DNA polymerase need in order to synthesise DNA?

- All four deoxynucleoside triphosphates

- A template

- A primer

What is a primer?

- a short piece of nucleic acid base-paired to template

- acts as a start point

- must have 3'-OH group attached to a nucleotide base-paired to the template

In what direction is DNA synthesised?

- 5' to 3'

What is the role of Mg2+ ions in DNA replication?

- they are found at the active side of DNA polymerase

- one Mg2+ dissociation of the H from 3-OH to generate a nucleophilic O-

- the other neutralises the charge on the incoming triphosphatase

What does DNA polymerase resemble?

- a hand

What allows DNA polymerase to proofread?

- Most of them have an inbuilt 3'->5' exonuclease

- some cant - e.g. Taq DNA polymerase

How often does DNA polymerase incorporate an incorrect nucleotide?

- approximately once per 10^5 nucleotides

- proofreading removes 99% of those

- after replication mismatch repair removes 99.9% of the remaining ones

Why are there no DNA polymerases that synthesise from 3' to 5'?

- Once the mis-matched nucleotide is removed there is no triphosphate to form the next phosphodiester bond

What mechanism does RNA polymerase use?

- uses same Mg2+ dependent mechanism, but are structurally dissimilar and appear to have evolved separately

What is a steric gate?

- Amino acid that prevents bulkier rNTPs from entering so that the enzyme cannot synthesise RNA;

- found at the active site of DNA polymerase

What happens if you change the staric gate AA for a smaller one - tyr to gly?

- together with one other AA change alteration in "thumb" region allows DNA polymerases to synthesise RNA

The main DNA polymerases in both euk and prok utilise a sliding clamp:

- able to repeatedly add nucleotides to a DNA molecule without dissociating from template

- mediated by enzymes being attached to a sliding clamp

- β-subunites of DNA polymerase III in E. coli and proliferating cell antigen (PCNA) in eukaryotes

What are the primers synthesised by?

- In E. coli - primase

- in Eukaryoes - primase and DNA polymerase

What is leading strand synthesised by>

- In E. coli - DNA polymerase III

- In eukaryotes - DNA polymerase ε

What is the lagging strand synthesised by?

- In E. coli - DNA polymerase III

- in Eukaryotes - DNA polymerase δ

What removes the RNA primers and replaces them with DNA?

- In E. coli - DNA polymerase I

- in Eukaryotes - RNAse and DNA polymerase δ

What is the difference in speed of synthesis between eukaryotes and prokaryotes?

- prokaryotes are 10-20 times faster

What is the difference in nuber of Ori between prokaryotes and eukaryotes?

- In E. coli - 1

- in eukaryotes - 10 000 - 100 000

How many times is replication initiated per cell division in prok and euk?

- 1 in both (although in prok could be more than once)

What are the characteristics of the Ori sequence?

- In E. coli - AT rich

- in eukaryotes - no obvious consensus in mammals

When are different DNA segment of eukaryotic DNA replicated?

= they are not all activated simultaneously

- housekeeping genes are first

- heterochromatin is late

- tissue specific genes may replicate early in tissues where they are active and late where they are repressed

What is the "Window of opportunity" model?

- different proteins are present in the nucleus early and late during S-phase of the cell cycle

- e.g. acetylated histones - early

- deacetylated histones - late