Lecture 10b Flashcards
What are the three initial models of DNA replication?
- Semi-conservative: the double helix of each daughter
DNA molecule contains one strand from the original DNA molecule and one newly synthesized strand - Conservative: the parent DNA molecule is conserved, and a single daughter double helix is produced consisting of two newly synthesized strands
- Dispersive: daughter molecules consist of strands each
containing segments of both parental DNA and newly synthesized DNA
Describe the experiment that demonstrated DNA replication is semi-conservative.
Allow parental DNA molecules containing nucleotides of one density to replicate in medium containing nucleotides of different density.
Experiment: Grow E coli in medium containing heavy 15N isotope so that it is incorporated in nitrogenous bases –> Cells moved into medium with light 14N –> cell division –> DNA isolated and separated based on density
How was DNA separated by density?
Cesium chloride gradient centrifugation: CsCl is spun in centrifuge at high speed for hours which creates gradient of density in the tube. Larger density at the bottom. DNA is centrifuged with CsCl and forms band at position identical with it’s density.
What are the band position predictions for each model?
SC: 1st gen - middle, 2nd gen - middle and top
C: 1st gen - bottom and top, 2nd gen - bottom and top
D: 1st gen - middle, 2nd gen - between middle and top
What are the 3 activities of DNA pol I?
- Polymerase activity in 5’ to 3’ direction
- 3’ to 5’ exonuclease activity to remove mismatched bases
- 5’ to 3’ exonuclease activity to degrade single strands of DNA or RNA (replaces RNA primer)
DNA pol I is much slower than pol III
Describe DNA replication in prokaryotes.
- One ori per chromosome
- Occurs in cytoplasm
- DnaA is a protein that recognizes the oriC and pulls apart strands (opens helix)
- Helicase then unzips the rest of the DNA. Helicase forms complex with replisome
- Two replication forks
- Bidirectional
- Forms theta structure
What is a replisome?
“Molecular machine” that carries out DNA replication. It is composed of DNA pol I, DNA pol III, helicase, gyrase, ligase, SSB, and other proteins.
What are helicases?
Enzymes that disrupt the hydrogen bonds that hold the two strands of the double helix together. It unzips the DNA helix ahead of DNA synthesis.
What are single-strand binding proteins (SSB)?
Stabilize unwound DNA by binding to single-stranded DNA and preventing the duplex from re-forming.
What are topoisomerases?
Relax supercoiled DNA by breaking either a single DNA strand or both strands, which allows DNA to rotate into a relaxed molecule. Topoisomerases finish by rejoining the strands of the now relaxed DNA molecule.
AKA DNA gyrase
Describe DNA replication in eukaryotes.
- Multiple ori per chromosome
- Multiple replication bubbles (thus multiple replication forks)
- YEAST: Presence of origin recognition complex (ORC) recruits Cdc6 and Cdc1 proteins. All together, they recruit helicase and replisome assembles. DNA replication is restricted to the S phase in cell cycle because G1 is the only time Cdc6 and Cdc1 are produced. They are destroyed after synthesis.
What what direction is DNA made?
5’ to 3’
What end are nucleotides added to?
3’ OH
What is the difference between the leading and lagging strand?
Leading: made continuously, 5’ to 3’ synthesis occurs in same direction as fork migration
Lagging: made discontinuously, 5’ to 3’ synthesis occurs in opposite direction as fork migration, creates series of small okazaki fragments (fragments are longer in prok than euk)
What are the 6 most important enzymes of DNA replication?
Helicase, SSB, primase, DNA pol III, DNA pol I, DNA ligase