This summary includes all the information that you need for the course Molecular Genetics. The summary is 129 pages long, because it contains a lot of information, but also a lot of describing pictures which means that you can easily go through it. By reading and understanding this summary you don'...
➢ How genomes replicate and evolve
Primary function of genome is to specify biochemical signature of cell in which it resides
→ The metabolome = all the metabolites that are present in the cell at a given moment
→ It seems essential that copying of the genome is flawless → Important for the survival of
an individual that should be genetically stable
→ And indeed: DNA sequences are replicated almost perfectly → What this lecture goes
about
→ Survival of a species, in the long run, benefits from incidental genetic changes
(mutations)
→ Eg, in E. coli: an average gene (1000 bp) obtains a mutation ~1 x per million divisions
➢ Not all mutations are bad
Many mutations can cause cancer
→ DNA repair is crucial for eukaryotic (human) cells
→ However, if DNA repair were perfect (no mutations ever)
→ No genetic variation - while this variation is the raw material for evolution
Successful organisms have evolved means to repair DNA efficiently (but not too efficiently,
with just enough genetic variability for evolution to continue)
➢ DNA replication, as predicted by Watson and Crick
→ You have to pull the 2 strands apart and then you have the substrate to obtain a new
strand
→ Each strand produces 1 new strand = semi-conservative replication
→ How does one prove that?
→ And, there seems to be a topological problem:
- How to pull the two polynucleotides of the parent helix apart
- How to unwind the helix (without pulling it into a knot)
➢ The Meselson-Stahl experiment, 1958
Replication is semi-conservative:
,→ They grew them in a medium of 15Nh4Cl → Then they transferred a part of the culture to
a fresh medium with a normal 14NH4Cl and then they started growing these cells for 20 or
40 minutes
→ E.coli divides once every 20 minutes → So after 1 cell division they extracted the DNA and
did a density gradient centrifugation and obtained one DNA band → SO this DNA contains
an equally amount normally synthesized normally N14 DNA and parental DNA which is
labelled with N15
→ When they grew 20 minutes more, so after 40 minutes and did the same thing they
obtained 2 bands in the gradient → The same band with N14/N15 band and a new band
with N14/N14 DNA
→ There are 3 possible schemes for replication:
1. Conservative replication → You have the parent DNA that leads to new DNA and the
parents strands stick together
2. Dispersive replication → The old and the new DNA is dispersed over the 2 new
daughter molecules
3. Semiconservative replication → One of the DNA strands contains old DNA and the
other strand contains new DNA
→ Expected bands for conservative replication would be 2 while the obtained bands will be
1 → In other words it can’t be conservative replication
→ Then if you look at what you expect with dispersive and semiconservative replication you
can’t make the distinction between the 2 after 1 generation, but you can after the second
generation → By dispersive replication you would still have 1 band of mixed N15/N14 DNA,
but what you in fact get is the 2 bands as predicted
→ So this means that dispersive replication is also not the scheme
→ This is how they proved that in fact DNA is replicated semiconservative
➢ The topological problem of DNA replication
→ If it’s semiconservative how do you prevent the DNA from becoming one big knot if you
start pulling the strands apart?
→ That problem was solved with the discovery of DNA topoisomerases which prevent over
winding during DNA replication
→ They are in fact DNA breakage and reunion enzymes
→ Type I enzymes that make a single-strand cut in the phosphate backbone
→ Type II makes double-strand cut in phosphate backbone
→ Linking number = number of times one strand crosses the other in a circular molecule →
So when the green strand winds around the grey one → So the upper left DNA strand has a
linking number of 2
,→ The topoisomerase type IA makes a Nick and ultimately you end up with the upper right
molecule and you can see that you go from a linking number of 2 to a linking number of 1
→ The topoisomerase type IB makes a single strand and then in this case it’s swirled around
the other uncut strand and that also leads to a reduction of the linking number to 1
→ So these enzymes take care that while the DNA is being replicated and while there is
being a pull on the 2 strands, that the whole thing doesn’t become one big knot by taking
out the knots or lowering the linking number
➢ Initiation of bidirectional genome replication
→ To start replication the double helix needs to be opened
→ And this is the case for a circular bacterial chromosome and the replication of that → The
DNA helix is open and in bacterial chromosomes you have 1 origin of replication (ori) and
then you have a replication fork → So you open it up and then you can start replication in
the left and right direction
→ The replication fork is the place where the replication machine is assembled
→ The start of replication is not just random, but it starts at an origin of replication
→ In circular bacterial chromosomes the ori is 1 so you have 2 replication forks, but the
replication of linear eukaryotic chromosomes has multiple origins
➢ Escherichia coli origin of replication, oriC
→ oriC = the origin of E.coli
→ You see it’s a structure of about 245 base pairs and it has a number of specific regions
→ It has a grey area called the DNA unwinding element → Then it has a few boxes in red
and blue that are binding sides for the protein DnaA
→ The DNA has a number of high affinity binding sides indicated in red → Those sides are
always occupied by dnaA
→ The other, the blue ones are low affinity DNA sides and they are filled just prior to
replication
→ These elements are important for the melting of the helix and the formation of the
replication forks
→ So DNA binding to its binding sides makes it that DnaA proteins form a barrel that
together with the help of HU proteins DUE (DNA Unwinding Elements) is melting
,→ This allows the attachment of the prepriming complex which formed by the proteins
DnaB and DnaC
→ DnaC is a helper protein and helps DnaB attach to the side over here and DnaB is a
helicase which is an enzyme that breaks base pairs and so together B on both sides extend
the melted region and this then allows elongation enzymes to bind and for the replication
forks to progress and that’s the start of replication
➢ Understanding replication = understanding DNA polymerases
DNA polymerases are the central players in genome replication
→ Complication with DNA replication is that DNA polymerases:
o Only synthesize in 5ʹ → 3ʹ direction
o Need a primer to initiate synthesis of a new polynucleotide
➢ Priming is necessary for DNA replication → RNA polymerase (Primase)
→ Primase makes an RNA primer of ~ 10 nt
NB: It is NOT involved in transcription…!
→ You have a DNA template that needs to be copied → First a primer is made on this DNA
template and this primer is a RNA primer made by primase and the primase makes a RNA
primer of about 10 nucleotides
→ You have to realize that primase makes RNA and is not involved in transcription, but
only in making the RNA primer needed for replication
→ LEFT: Situation in bacteria where a primase just made a small primer and then that end
over here in the RNA structure can be used in a polymerase couple nucleotides to it and
make new DNA in the 5’ to 3’ direction
→ RIGHT: This situation is a little bit different, because the primase is tightly bound to the
DNA polymerase and together they cooperate to make a RNA primer first of 7-12
nucleotides and then the primase hands over the DNA polymerase alpha which extends the
primer with a little bit of new DNA and then after completion of this extended primer then
DNA polymerase that actually does the replication takes over and start synthesizing the
whole stretch of DNA in the 5’ to 3’ direction
➢ DNA synthesis only proceeds in 5’ to 3’ direction
→ On the left strand you can see that you make a primer and then you synthesizes the DNA
polymerase and start synthesizing while the DNA strand is being opened you can synthesis
in one genuine flow
→ If you look at the right strand it’s a problem because it’s the other way around → So
synthesis can only take place in the direction to left up or right bottom
,→ It can only start when the DNA opens up → So you get an opening of the helix and a little
bit of the synthesis on the leading (left) strand and then the helix opens up further allowing
of a primer to be made here and now synthesis can also take place on the lagging (right)
strand → In this way you have discontinuous copying because the strand first needs to be
opened up
→ Okazaki fragments are the result of discontinues copying
→ Leading strand = strand that can be copied continuously
→ Lagging strand = strand that can only be copied discontinuously by Okazaki fragments
→ Priming: only once on the leading strand, (very) often on lagging strand
➢ Progress at the replication fork
→ In both bacteria and eukaryotes, fork progression is maintained by:
- Helicase, melting out the base pairing
- Topoisomerases, relieving torsional stress from unwinding of ds-helix ahead
→ The helicase pulls the strands apart which actually takes away these H-bonds
→ Topoisomerase takes care that you don’t get a knot over here → So it takes care of the
linking numbers while the process is going on
→ The primase at this position makes a new RNA primer for a next polymerase for
synthesizing this little bit over here
→ This is how the progression of the fork is maintained by enzymes in bacteria and
eukaryotes
➢ Single-strand binding proteins (SSBs)
→ Single-stranded DNA in replication fork (obtained by helicase) is “sticky”
→ SSBs (RPAs) bind to phosphate backbone forming a channel to:
o prevent ss-DNA from re-associating
o protect against degradation by e.g. single-strand-specific nucleases
→ You can also realize that while the DNA strand is being opened up there is a little bit of
single stranded DNA before it’s made double stranded again by the polymerase
→ First of all the DNA is single stranded so it could stick on itself
→ It’s also quite sensitive to degradation
→ So that’s why single strand binding proteins are very important because they protect
the DNA from reassociation or degradation
→ RPA’s = eukaryotic SSBs
, ➢ Parallel synthesis of DNA leading- and lagging-strands
→ The E. coli model:
- DNA synthesis done by two DNA polymerase III molecules linked together (dimer)
→ One for the leading strand and one for the lagging strand
- One Pol-III copies leading strand, the other copies lagging strand
- Lagging strand spools through its DNA Pol-III monomer
→ So 1 dimer can replicate both strands at the same time
→ A few proteins are involved in attaching and detaching of DNA polymerase from the
template:
- Primarily during lagging strands replication
- “Clamp loader” protein
- “Sliding clamp” protein → Binds the DNA polymerase
➢ 1st-year BCMB stuff – Molecular Biology of the Cell
→ Here you see the sliding clamp (circular molecule) that can be opened up by the clamp
loader and held of ATP → So ATP bound to the clamp loader opens the sliding clamp which
holds the DNA to go in to the donut → Then upon ATP hydrolysis the sliding clamp is closed
again so it’s now a full closed donut containing the DNA and the clamp loader is released
and goes back and is recycled for the next round
→ The sliding clamp is binding DNA polymerase so that it can start replication
→ So in the DNA replication fork of a prokaryotic cell → We have the double strand DNA
helix and we see it’s opened and ready for replication → We see a helicase breaking the H-
bonds and that single stranded DNA is protected by Single-stranded binding proteins (SSBs)
→ Then the sliding clamp to which the polymerase bind and you see that the synthesis on
the leading strand goes on continuously and on the lagging strand the primase that is
making a small RNA primer so that again sliding clamp and the DNA polymerase can start
from this RNA primer to synthesize small Okazaki fragments on the lagging strand
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