DNA Replication Flashcards
What are the 4 stages of the eukaryotic cell cycle?
- G1
- S
- G2
- M
What happens at the G1 stage?
Cell with chromosome in the nucleus
What happens at the S stage?
DNA replication = chromosome duplication occurs
What happens at the G2 stage?
Cell with duplicated chromosomes in nucleus
What happens at the M stage?
Mitosis = chromosome seperation
1 cell becomes 2
What does the chromosome state reflect?
The need to replicate and partition the genetic material prior to cell division
What are three important areas of a chromosome?
- Telomeres
- Centromeres
- Origin of replication
Where do you find the telomeres on the chromosome?
At the ends of the chromosome
What is the telomeres?
areas of highly repetitive DNA that protect chromosome ends from DNA degradation, recommendation and end fusion with other chromosomes
What is the centromere on a chromosome?
Repetitive DNA which forms the spindle attachment sites in mitosis
What is the origin of replication?
Special sequence where duplication of the DNA begins; each chromosome will have many origins
At which stage is the DNA long and not condensed?
Interphase
At which stage is the chromosome condense and visible underneath a microscope?
Mitosis
Who discovered semiconservative replication?
Meselson and Stahl 1958
What is semiconservative replication?
Two stands of parental double helix unwind (by DNA helicase) and each specifies a new daughter strand by base-pairing rules
Describe Meselson and Stahl 1958 experiment:
- Density ultra centrification
- molecules of different densities with sediment at different levels in the column
- Grew bacteria in medium N-15
- Extracted the DNA from N-15
- N-15 centrificated - sediment was lower than N-14
- Shared N-15 into N-14 medium and found as replication went on DNA became more and more like N-14
Which is heavier N-15 or N-14?
N-15
What can we concluded from Meselson and Stahl 1958 experiment?
N-14 is integrated in DNA
What is required for DNA synthesis?
-Enzyme (DNA polymerase) + Mg2+ dNTPs (dATP, dCTP, dGTP, dTTP) -Single stranded template DNA -Primer 3’-OH -DNA synthesis 5’ to 3’ direction
What is the problem in DNA replication?
That there is not enough time to replicate from start to finish
What solves the problem of not having enough time to replicate from start to finish?
The origins of replication break up the time
What does the involvement of the origin of replication make DNA replication?
bidirectional
What does bidirectional DNA replication form?
Replication bubbles
Which is bigger eukaryotic or bacterial genome?
eukaryotic
What is the shape of the eukaryotic genome?
Linear
What is the shape of the bacterial genome?
Circular
Is the bacterial genome compact?
Yes
What is the amount of base pairs in the human genome?
3000 million bp
What is the amount of base pairs in the e.coli genome?
5 million bp
Describe the replication of bacterial DNA:
- Starts at the only origin of replication
- forms a theta structure
- 2 circular daughter DNA molecules are produced
Describe the replication of DNA in a eukaryotic cell?
- Both strands are copied at a replication fork
- Synthesis in a 5’ to 3’ direction
- leading strand: replicates easily as it is going in the 3’ to 5’
- lagging strand: hard to replicate as strand is going in 5’ to 3’ so synthesis is discontinuously so gaps left
What is caused in the DNA replication fork of the lagging strand?
Okazaki fragments
Describe the lagging strand synthesis:
- Primase synthesis short RNA oligonucleotides (primer) copied from DNA
- DNA polymerases elongate RNA primers with new DNA
- Nucleases remove RNA at 5’ end of neighbouring fragment and DNA polymerase fills the gap
- DNA ligase connects adjacent Okazaki fragments
What was the enzyme at the replication fork required to unwind the DNA?
DNA helicase
What was the enzyme at the replication fork required to stabilise ssDNA?
ss-binding protein
What was the enzyme at the replication fork required to synthesis RNA primer?
primase (RNA polymerase)
What was the enzyme at the replication fork required to synthesise new DNA strand?
DNA polymerase
What was the enzyme at the replication fork required to proof-read?
DNA polymerase
What was the enzyme at the replication fork required to keep DNA polymerase on the DNA?
Sliding clamp
What was the enzyme at the replication fork required to trim the Okazaki fragements??
Nuclease
What was the enzyme at the replication fork required to join the Okazaki fragements?
DNA ligase
What was the enzyme at the replication fork required for the other factors?
Replisome
What does DNA helicase do?
Unwinds double-stranded DNA
What does ssDNA do?
Prevents base-pairing until DNA polymerase arrives
What is primase?
Specialised RNA polymerase
What does sliding clamp do?
Attached to DNA polymerase which encircles double stranded DNA
What does DNA ligase do?
joins Okazaki fragments
What does nuclease do?
trims Okazaki fragments
How is the sliding clamp loaded around the DNA?
By a clamp loader
How are histones loaded onto newly synthesis DNA?
By histones chaperones
What is the problem with DNA helicase unwinding DNA?
Causes supercoilling
What enzyme unwinds the supercoiling?
Topoisomerase
What is the total error possible in DNA replication?
1 in 10^10
What is the error per nucleotides added during 5’ to 3’ polymerisation?
1 in 10^5
What is the error of not being corrected in 3’ to 5’ exonucleolytic proofreading?
1 in 10^2
What is the error of not being corrected in strand-directed mismatch repair?
1 in 10^3
What is more accurate DNA polymerase or RNA polymerase?
DNA polymerase
Why is DNA polymerase more accurate than RNA polymerase?
-Proof-read (incorrect nucleotides removed by 3’ to 5’ exonuclease)
What detects incorrect base-paring in newly-synthesised DNA?
Mismatch repair protein MutS
What are mutations in human Mismatch Repair genes associate with?
Predisposition to some cancers
