Module 2 - DNA replication Flashcards
why is DNA replication important
DNA needs to double to pass on equal amount of DNA
essential for reproduction and continued life
defects in DNA rep can cause disease (e.g. cancer)
general idea of DNA replication
- DNA strands seperate
- DNA copied using parent strand as template
- comp base pairing
- semi cons replication
= forms 2 identical strands same as parent DNA
each daughter strand has half a strand from parent
other than semi conservative, what other ways could DNA possibly replicate
Conservative - one fully original, one fully new
Dispersive - both strands formed are a combo of old and new
Conservative replication
In theory:
- semi cons rep occurs
- 2 strands that are half old half new are made as per usual
- but then some kind of detachment and reassociation occurs where:
old strands combine w/ each other
new strands combine w/ each other
Dispersive replication
in theory:
two strands seperate
as the new strand moves along the template strand, the template switches repeatedly
- creates daughter molecules that are a combo of old and new
in 1950s, this was what most accepted
why did scientists think semi cons was impossible?
- DNA is plectonemic = strands cannot seperate without unwinding
- and cuz many DNA are circular, so cannot theoretically unwind
e.g. bacteria DNA, mitochondria DNA, plasmids etc.
unless you cut it
BUT
- bacteria chromosome is ~4mill bp
- if this was converted to linear wouldnt work theoretically cuz
- bac divide every 20 mins
- so must replicate 200,000 bp per min (10bp per turn of helix)
- so 20,000 revs per min
so impossible, will pretty much explode
Explain how Meselson - Stahl experiement proves semi cons
Cultured E.coli bacteria in medium with HEAVY isotope of NITROGEN (15^N) using heavy NH4CL (ammonium chloride just in case u forgot basic chem)
N is component of DNA bases
so gets used by bacteria in their DNA
so to distingush between DNA w/ heavy or lght nitrogen,
= density gradient centrifugation
50,000x for 48hrs
- low dens at top
- hevy dens at bottom
due to BUOYANT DENSITY
explain method of mehselon-stahl’s experiement
E.coli cultured in heavy NH4Cl
transferred to light NH4Cl culture
grow for diff amounts of time:
- after 20mins, isolate dna (=one cell division
- do dens gradient centrifugation
= ended up with intermediate band (so half and half) - after 40mins, isolate dna (=two cell divisions)
- do dens grad centrifugation
= ended up with one intermediate (same as prev)
and one light (two strands of dna both with light N cuz thats what it was cultured in)
what are the predicted bands for all types of replication
Conservative =
after 1 rep
1 molecule with all heavy
1 molecule with all light
Dispersive =
after 1 rep
exact same as semi cons (cuz has an intermediate strand)
BUT
after 2 rep
stays intermediate (so stays as one intermediate band)
so how tf does circular DNA go through semi cons replication?
TOPOISOMERASES hehheh
Role of topoisomerases
prevents supercoils during replication
supercoils - get super knotted and replication cant go on with them
Types of DNA topoisomerase: TYPE 1
TYPE 1 DNA topoisomerase:
- ‘nick’ a strand, moves the other strand through the nick, gets rid of knot by ‘removing a helix’
- opens up structure
look at diagram if ur lost idk how else to explain it
Types of DNA topoisomerase: TYPE 2
TYPE 2 DNA topoisomerase:
major enzyme
- so there’s two segments of DNA supercoiled
- cuts BOTH strands (so like cuts through whole thing)
- G segment = DNA strand that gets cut so other strand can pass through
- T segment = DNA strand that gets passed through
see diagram in onenote its clearer
Why are DNA polymerases important
- cellular enzymes that make DNA strands from nucleotides
- mutations in these can cause colorectal cancer
what do DNA helicases do
break aprt the base pairs by hydrolysing the H bonds
how is DNA synthesised at the replication fork and what is the process called?
Template-dependant DNA synthesis
what direction does DNA synthesis go in and what is required to initiate synthesis?
5’ to 3’
a primer is required
What is exonuclease activity
DNA polymerases degrading DNA
2 types of exo nuclease activity:
3’->5’
basically means if theres a mistake in the nucleotide, it can reverse itself and go back to correct it
this is called PROOFREADING
5’ to 3’
can displace DNA that’s ahead of it
all DNA polymerase types involved in replication
see table in onenote
how are the seperated single strands protected
Single-strand binding proteins (SSBs)
- prevents them reattching to each other
- prevents them being attacked by nucleases
e.g. in eukryotes = replication protein A
if one strand is the template strand, what’s the other one called?
leading strand - has continuous synthesis
what is the primer made out of
made of RNA by the primase enzyme
it’s 4-15 nulceotides in length
once primer comes in, DNA pol III makes the new strand
process in eukaryotes
sry for the poo questions idk how to word it
- RNA primer extended by DNA pol ALPHA
- alpha adds about 20 nucleotides
- then DELTA makes the rest of the strand
BECAUSE
alpha doesn’t have the 3’->5’ exonuclease mechanism that is required for proof reading
so it switches to delta
DNA synthesis at site
beta and gamma phosphates are removed from nucleotide
on one end alpha phos binds to the sugar on the previous nucleotide AT 3’ OH terminus
bases join together on the other end
see diagram
lagging strand and Okazaki fragments
because DNA synth always has to be 5’ -> 3’
lagging strand is on the other side
and because synthesis always starts with a primer
it has to be made in sections
sections = Okazaki fragments
these must be joined together and RNA primers must be removed
2 ways to remove RNA primers, in bacteria and in wukaryotes
in lagging strand in bacteria, how are the Okazaki fragments combined?
when the DNA pol III reaches the primer, it stops and switches to DNA pol I, then continues to synthesise
cuz
DNA pol III has no 5’->3’ exonuclease activity
and DNA pol I does
then, DNA ligase ‘glues’ the two ends of DNA fragments together
in lagging strand in eukaryotes, how are the Okazaki fragments combined?
DNA pol delta + helicase push the primer aside
FEN1 (an ENDONUCLEASE) cuts at the branch point
DNA ligase links the fragments
cant just get rid of primer CUZ
non of the eukaryotic polymerases have 5’-3’ exonuclease
why in a linear molecule, would the dna molecules get shorter and shorter gradually
the final okazaki fragment can’t be made
priming site would be after the end of the parents molecule
so it would just cut short
hence why we need telomerase
what does telomerase do
extends the parent DNA by adding TTAGGG several times so the final okazaki fragment can be primed
almost like a buffer zone
what is telomerase made of
is an RNA/protein complex
few thousand telomere repeats at the end of human chromosoms
what is the name and properties of the organism that has the telomere sequence of TTGGGG
Tetrahymena thermophilia
single cell ciliated organism
has 40,000 chromosomes
obvs needs lots of telomerase
used in discovery of telomerase cuz theres so much
which cells have telomerase
only stem cells
or some cancer cells - which is why they divide continuously
what is senescence
cells die
can divide ~50x
becuase normal cells dont have telomerase, the ends of chromosomes shorten
so they die rip
what phase does dna replication occur
S phase
how do we syncrhonise cells during experimentation
use chemicals to bring them all to beginning of S phase
how do we visiualsie and detect DNA replication sites
modify thymidine nucleotides with Br
then can detect using antibodies that are fluorecently labelled
and antibodies attach to the Br nucleotides
how many patterns of DNA replication can be observed?
5 patterns
they relate to the types of DNA being replicated:
-Euchromatin first
-Heterochromatin next
can be observed during S phase - see onenote for diagram
