DNA replication Flashcards
How did the Meselson-Stahl experiment lead to semi-conservative replication over conservative and dispersive?
3 possible mechanisms of DNA replication:
- Semi Conservative - Parental dsDNA melted into 2 strands - 1 serves as template for new strand so both offspring cells inherit and old strand and a new strand
- Conservative - dsDNA of parent cell inherited by one of the offspring cells. Other offspring cell inherits brand new DNA
- Dispersive - dsDNA somehow chopped up into segments, each offspring gets 50:50 mixture of new and old sections of dsDNA
Meselson-Stahl Experiment - → demonstrated that DNA is replicated SEMI CONSERVATIVELY through DNA polymerase
- E.coli on 15N source integrates this nitrogen in DNA molecule
- Transfer E.Coli onto 14N source medium so new DNA formed of lighter isotope
- Take a sample every generation, extract DNA and use a density-gradient centrifugation to separate DNA into bands according to their density
SEMI CONSERVATIVE = heavy 15N dsDNA of the parent is melted into 2 ss and serves as template for new light strand - both generation 1 cells contain a half light and half heavy hybrid
CONSERVATIVE = heavy dsDNA of the parent is inherited wholesale by 1 of the generation of the offspring cells. - other generation 1 cell inherits bran new light 14N DNA
DISPERSIVE = ds DNA is chopped up into segments, each offspring in generation 1 gets a 50:50 mixture of heavy and light DNA - for every generation, the amount of 15N DNA is halved, so you have an asymptotic approach to fully 14N so only a single band going lighter down the generations
How does replication initiation occur in Prokaryotes?
- DNA replication is initiated by proteins melting AT rich origins of replication (2 H bonds → easier to melt) ONLY 1 origin (ori) on Prokaryotic Chromosome
- Origin regions recruit initiator proteins which are topology altering enzymes that allow DNA to melt below 90degrees celsius
- Helicase continue to melt DNA using ATP
- FORMS replication bubble with replication forks
ARCHAEA use enzymes that are more similar to eukaryotes - have circular genomes
PROKARYOTES have stepped replication - can start one replication bubble before another is finished
In prokaryotic cells, there is only one point of origin, replication occurs in two opposing directions at the same time, and takes place in the cell cytoplasm
- possess 2 types of DNAP (DNAP1 and 3)
How does replication initiation occur in Eukaryotes?
same as prokaryotes but have multiple origins of replication per chromosome so multiple replication bubbles are formed
- uni directional replication within nucleus of cell
- Contain 4 or more types of DNAP
Describe bacterial DNAP1 and 3
Bacterial DNAP3 - used in replication
- 2 active sites
- 5’ to 3’ polymerase
- proofreading 3’ to 5’ exonuclease (cuts end of sequence)
DNAP1 - used in repair
- 3rd active site - arm domain - 5’ to 3’ exonuclease activity (moves ahead of polymerase and destroys any damage DNA which polymerase cannot fix)
KLENOW FRAGMENT = first 2 active sites without the arm)
When can replication go wrong and how does proofreading lower error rates?
During DNA synthesis, mutations can be introduced by transient base tautomery
- if dsDNA, not as large effect as guanine can bind to get it back to normal form
- if ssDNA, can have large effect as can bind to adenine and can cause mutation which is introduced to next cell
THEREFORE needs proofreading and correcting
- All DNAPs synthesise new strand 5’ → 3’
- Binding of new nucleotides has error of c.10^-4 bp-1
- DNAP chews back mismatched pairs with 3’ → 5’ exonuclease site → decreases error rate to 10^-6 bp-1
- BUT mutation rate in inherited DNA is only 10^-9 bp-1 therefore…
→ if imino form binds to adenine, can tautomerise back to cytosine form but then A→C binding is wrong so cytosine pushes back and comes out of alignment → causing it to move to proofreading exonuclease site so it can be cleaved off
How does replication elongation occur in prokaryotes and eukaryotes?
Elongation - DNAP does NOT bind to ssDNA
- RNA primers are added on one leading strand and many on lagging strand to allow DNAP to bind
- therefore left with RNA sections in DNA which are removed by RNAse H in eukaryotes which degrades RNA primer so that DNAP can bind to form ds DNA - falls off when reaches other dsDNA - DNA ligase produces phosphodiester bonds to ligate fragments together using ATP
in PROKARYOTES: DNAP1 has primer removing nuclease activity part of it
Describe the replisome (single strand binding proteins, ssbp, topoisomerase, primosome, primase, helicase, sliding clamp, leading, lagging strand)
Single Stranded Binding Protein: stabilise ssDNA and will eventually be displaced when the lagging DNAP starts making next Okazaki fragment. Prevents random c.b.p within lagging strand - prevents tangle
Primosome: Primase + Helicase
Primase: synthesises RNA primer
Topoisomerase: supercoiling strain relief - enzymes that participate in the overwinding or underwinding of DNA.
What are telomeres in eukaryotes and how do telomerases maintain them? (Hayflick limit)
Telomeres = repeats at the end of DNA strand
DNAP cannot replicate the telomeres at very 3’ end of a eukaryotic linear chromosome
without Telomerase, there will always be a 3’ DNA overhang on the parental strand
Telomerase = a reverse transcriptase with an internal RNA template which extends telomeres in germ line and cancer cells
- adds a sequence to telomere which is complementary to internal RNA template
- DNA primase then synthesises an RNA primer at 3’ prime end
- and DNA polymerase fills in vacant region between primer and new generation strand
→ Therefore RNAse H degrades it and it is lost in next generation
→ Means that we dont need to have shortening of DNA
Hayflick limit is the number of times a normal human cell population will divide before cell division stops
