DNA replication Flashcards
what are the Three possible models for DNA replication
-conservative
-semi conservative
-dispersive- double strand chunks of original and new DNA next to another one (go, new, go kind of appearance)
what did Matthew Meselson and Franklin Stahl do
-Meselson and Stahl used differences between isotopes to do their experiment.
-In this case, Nitrogen: DNA contains lots of Nitrogen atoms.
-relies on isotopes of nitrogen
what are the different isotopes of nitrogen
-14N = 7 protons + 7 neutrons, atomic weight= 14
-15N = 7 protons + 8 neutrons, atomic weight= 15
-The extra neutron means that 15N is heavier than 14N.
DNA molecules containing the different isotopes can therefore be separated by their weight.
-not radioactive- NB
what did Matthew Meselson and Franklin Stahl do to answer their question of is the mechanism of DNA replication conservative, semi-conservative, or dispersive?
1)Grow bacteria in media containing 15N: make “heavy” DNA
2)Transfer them to media containing 14N: new DNA will be “light”
3)Separate heavy and light molecules by ultracentrifugation
Caesium chloride gradients: centrifugation
-sample of CaCl and EtBr centrifuged
-low density at top
-high density at bottom
-lighter= supernatent
-heavier= pellet (at bottom)
what does 2 bands in the generations show us
-its semi-conservative model
what are the origins of replication
-A single origin of replication in the E. coli genome, vs
tens of thousands of origins of replication in the human genome
-Bidirectional replication forks
-replication proceeding in both directions
-replication bubble
what are the Enzymatic activities for DNA replication
-primase
-DNA polymerase
-DNA ligase
-single-stranded binding protein
-helicase
-topoisomerase (unwinds DNA in orderly fashion)
what’s the mechanisms for DNA polymerase
-Add nucleotides one at a time, in a 5’-3’ direction-nucleotides per second
always added onto the 3’ carbon
-Using template strand to form H-bonds: tells which base to add next
-Tens or hundreds of nucleotides per second
what does primase do in replication
-generates the primer (RNA), ligase then joins the stretches of the new DNA together
what does ligase do in replication
-joins the loose ends together into single strand of DNA
-Base-pairing between nucleobases but gaps in the sugar-phosphate backbone need to be joined up
why is topoisomerase important in DNA replication
-Topoisomerase relieves pressure from overwinding around the replication bubble by making and resealing breaks in the DNA
why is helicase important for DNA replication
-Helicase breaks hydrogen bonds between the two DNA strands, separating them
why is single-strand binding protein important in DNA replication
-SSB binds to the separated strands, preventing them from reannealing
what’s Replication on leading and lagging strands like
-Leading strand: 5’-3’ DNA synthesis points towards the replication fork and can proceed continuously
-Lagging strand: 5’-3’ DNA synthesis points away from the replication fork and must therefore be discontinuous (primed numerous times)
-Okazaki fragments = the pieces of DNA that are stuck together to make up the lagging strand of replication
whats Semidiscontinuous DNA replication
-Continuous replication of the leading strand
-Discontinuous replication of the lagging strand
-lagging strand is the discontinuous strand because it has its of different pieces
-look at slide32
how does Erosion of genetic material at ends of linear chromosomes occur
-Primer removal at the end of the chromosome leaves a gap that can’t be filled in (there is no DNA polymerase coming along to fill in that piece).
-So on every round of replication, a little piece is lost from the end of the chromosome.
-This is a problem for the lagging strand at each end of the linear DNA.
what solves the problem of erosion of genetic material at ends of linear chromosomes
-telomeres
-Telomeres = short DNA sequences that are repeated over and over at the ends of the chromosomes.
-Short stretches are lost from telomeres at each round of replication.
-But that’s alright because there is an enzyme called TELOMERASE that can replenish the telomeres from an RNA template in some cell types
-The repetitive nature of telomeres also allows them to bind the shelterin complex of proteins, protecting the vulnerable ends of the chromosome and differentiating them from DNA breaks.
