17751008
Description
Flashcards by Sophia Puliasis, updated more than 1 year ago
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Created by Sophia Puliasis
about 6 years ago
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| Question | Answer |
| How was it determined and that DNA replication is conservative? |
E. coli cells. Grown and transferred.Analysed by density centrifugation
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| How many hydrogen bonds between G&C, and A&T? | Three and two, respectively |
| Principles of DNA synthesis | - Primer must expose 3' hydroxyl - DNA polymerase is synthesised in the 5' to 3' direction (leading Strand same direction as fork, lagging strand opposite direction – Okazaki fragments |
| How long are Okazaki fragments? | Eukaryotes: 160 – 200 nucleotides bacteria: 1000 – 2000 nucleotides |
| Which RNA polymerase are used by bacteria and eukaryotes respectively? | Bacteria: DNA polymerase three eukaryotes: DNA polymerases alpha, delta, Epsilon |
| What do the three copies of DNA polymerase three do in bacteria during replication? | One is associated with the leading strand while the other two may take it in turns to replicate the Okazaki fragments |
| Name and give the functions of the DNA polymerases involved in eukaryotic replication | Alpha: primates subunit – primes Okazaki fragments and leading strand during initiation, extends RNA primer with a few DNA bases Delta: binds PCNA and finishes synthesising the Okazaki fragment Epsilon: binds PCNA and synthesises the leading strand |
| How to DNA polymerase as remain associated with replication forks? | By binding to processivity factors (processivity factors stay associated with DNA by clamping around it) |
| What is PCNA? | A processivity factor |
| As DNA is unwound, the adjacent parts experience torsional stress/super coiling. How was this relieved? | By topoisomerases |
| What is the function of single-stranded DNA binding proteins (SSBs)? | Stabilise unwound conformation, protect ssDNA from hydrolysis and formation of hairpin helices |
| What are DNA primases? | RNA polymerases that make new RNA strands for elongation by DNA polymerases |
| What's in the CMG complex ? (A.k.a. helicase in eukaryotes) | CDC 45, MCM 27, GINS |
| Describe what is going on at the eukaryotic replication fork |
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| Which two processing factors that every polymerase need? What are their functions? | PCNA: sliding clamp (encircles double-stranded DNA and tethers polymerase to DNA) RFC: loader (installs PCNA onto DNA/ATP -dependent, opens up PCNA, ATP hydrolysis sparks its dissociation) |
| During what Stage is the replication origins fire at in eukaryotes? | S phase |
| How did blow and Lasky, (1988) discover the licensing factor? |
Isolated nuclei from frog eggs at different stages of the cell cycle, transfer them to fresh extract.
Result: interphase nuclei did not re-replicate. Entry into mitosis or artificial nuclear envelope permeabilitsation allowed for replication
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| Describe the licensing factor model |
– Licensing factor binds stably to replication origins
– LC can't access DNA once nuclear assembly has occurred
– LC required for replication initiation
– LC inactivated as it drives replication
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| Why does licensing not occur in S and G2 phase in metazoans? | Low Cdt1 activity |
| What is Geminin? | A metazoan licensing inhibitor. (Active in nuclei in late G1, S, G2 – destroyed during late mitosis, binds and inhibits Cdt1) |
| Describe the organisation of the replication origin in prokaryotes | Single origin of replication per circular chromosome, sequence specific (DnaA = replication initiator protein) |
| How is the replication of large eukaryotic genes facilitated? | Large number of replication origins that may fire at different times |
| What was the basis of the initial licensing factor model? | the essential replication factors are unable to penetrate the nucleus unless the nuclear membrane is broken (only at M phase), and hence the re-replication is prevented, due to absence of replicating factors throughout interphase |
| Describe the licensing cycle | Late mitosis and G1: replication origins licensed by loading Mcm2-7 complexes. Other stages: licensing inhibited |
| What happens during the timing decision point? | Different chromosomal regions become programed to replicate at different stages of S-phase |
| What is the pre-replication complex composed of? | -ORC (origin replication complex – already bound to origin and doesn't leave throughout cell cycle) - Cdc6 - Cdt1 - these then load Mcm2-7 |
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