Molecular genetics Summary
Lecture 1 Genome replica on
Ques ons
DNA lacks an oxygen at the 2’ posi on of the sugar.
Hydrogen bonds are present between two DNA strands or when RNA is folding in on itself. The
linkage of nucleo des is between the 3’ of the first nucleo de and the 5’ of the second nucleo de.
This has nothing to do with the direc on of the polymerase chain. The direc on of DNA synthesis is 5’
to 3’.
The only thing going in the 3’ to 5’ direc on are exonuclease enzymes, which are degrading enzymes.
Primary: the joining of different amino acids into a polypep de chain. Secondary: the conforma on
taken by the polypep de chain (α-helix or β-sheet).
Degeneracy of the gene c code means that the same amino acid can be encoded by mul ple triplets
(codons). The role of degeneracy is to be a buffer for muta ons. When there is a muta on that
causes a change in the codon this might be detrimental. Degeneracy causes flexibility and therefore
some protec on.
A PCR reac on needs the following components: template DNA, DNA polymerase, dNTPs, and
primers. DNA polymerase needs a free 3’ OH, so therefore primers are needed. RNA polymerase is
capable of synthesis from scratch, so it doesn’t need a primer. This is a key difference between DNA
polymerase and RNA polymerase.
The order of the stages of a PCR reac on is: denatura on, annealing, synthesis. Annealing is when
the primers will base pair to the template. Every enzyme has an op mal temperature to work at, but
it will s ll work at a subop mal temperature. Then it is just a bit slower, but it is s ll ac ve.
A restric on endonuclease leaving a s cky 5’ overhang creates DNA molecules with single-stranded
nucleo des at their 5’ end. Endonuclease cuts the DNA in the middle and not at the ends. Restric on
enzymes are enzymes that recognize polydromic sequences. A polydromic sequence is a sequence of
which the read in the 5’ to 3’ direc on on either strand is the same. It is called a s cky end, because it
will s ck together with another piece of DNA cut by the same enzyme.
A clone is a group of “gene cally iden cal” cells. The sheep Dolly was a clone of another sheep,
because it was gene cally iden cal.
A reverse transcriptase is an RNA-dependent DNA polymerase. Reverse transcriptase is an enzyme
that reads RNA and synthesizes DNA, so it needs an RNA template. Reverse transcriptase can be
found in viruses, e.g. HIV.
What happens during splicing? Splicing occurs on RNA not on DNA. The introns are the non-protein
coding part, so they need to be removed. The introns are removed from the pre-mRNA and the exons
are rejoined.
Lecture
All the organisms have their gene c informa on stored in DNA sequences. The genome entails all
gene c informa on of an organism. Part of the genome will be transcribed into RNA by a process
called transcrip on. Not all of the DNA sequences are transcribed to RNA. The RNA transcripts
,cons tute the transcriptome of the organism. The
transcriptome is a set of all RNA transcripts from a
genome. Some of the RNA transcripts will be translated
into amino acids in a process called transla on. Animo
acids will form polypep des, which will form proteins.
The proteome is a set of all proteins expressed by an
organism. The metabolome is a set of all metabolites in
an organism. This represents all these processes. These
processes are connected to the environment. To be
able to con nue the gene c flow of DNA over mul ple
genera ons, DNA replica on is needed. DNA replica on is the process of making copies of the
genome to transfer to daughter cells.
Key concepts of genome replica on:
DNA replica on is semi-conserva ve.
The topological problem.
Ini a on of DNA replica on.
Elonga on of DNA replica on.
Termina on of DNA replica on.
The end problem.
The fidelity problem.
The phases of DNA replica on are ini a on, elonga on, and termina on.
DNA replica on is semiconserva ve. In 1953, Watson and Crick proposed a method for DNA
replica on. They proposed that DNA replicates in a semiconserva ve way. Semiconserva ve means
that each parental DNA strand serves as a template to create a new daughter DNA strand. However,
at this me scien sts had a hard me believing this theory as the two DNA strands of the genome
had to be separated completely. This seemed impossible as it results in posi ve supercoils, which
creates tangles in the DNA. The topological problem arose from this, which entails how to unwind the
DNA. Other replica on mechanisms were described: conserva ve and dispersive replica on
mechanisms. The conserva ve mechanism is when the whole DNA molecule serves as a template to
generate an en rely new DNA molecule. So the original DNA strand is undisturbed. This is repeated in
the second replica on cycle. The dispersive mechanisms is when DNA is randomly distributed of the
old and new DNA strands.
,In 1958, Ma hew Meselson and Franklin Stahl proved experimentally that DNA replica on is
semiconserva ve in the Meselson-Stahl experiment. In this experiment, E. coli bacteria are used. A
culture of E. coli is grown in a medium containing a heavy isotope of nitrogen, N15. The hypothesis
was that E. coli will incorporate the heavy isotope of nitrogen into the nitrogen bases in its DNA. This
was checked by taking a sample of the culture and extrac ng DNA. A er this a density gradient
centrifuga on was performed. In the end, the DNA was separated into different density gradients.
This was used to visualize if the DNA contains a heavy, normal or light density. The density depends
on whether the heavy isotope of nitrogen is incorporated or not. All of the DNA in the sample
contained a heavy nitrogen isotope, so both DNA strands contained a heavy isotope.
A er this, the E. coli culture was passed into a new medium containing a normal nitrogen isotope,
N14, which has a lighter density. The experiment was repeated with a normal nitrogen molecule in
two genera ons. A er one genera on
all the DNA molecules contained an
intermediate nitrogen density, so half
of the DNA was light and half of the
DNA was heavy. A er two rounds of
replica on, half of the DNA molecules
had only a light density and the other
half had an intermediate density. This
proves the proposed DNA replica on
method by Watson and Crick. With
only two cycles of replica on it was
proven that DNA replica on is
semiconserva ve.
Watson and Crick were able to prove DNA replica on is semiconserva ve as the expected results of
all three possibili es were known before doing the experiment. If it was conserva ve, the density
gradient expected in the fist genera on is half light and half heavy. So with the first genera on it was
already known that DNA replica on is not conserva ve. It could s ll be dispersive a er the first
genera on, because intermediate density was expected. This is because the DNA would be randomly
distributed between the two strands. For dispersive in the second genera on was intermediate
density expected as well, but this was not the case. Therefore, DNA replica on is semiconserva ve
and this could be proven with only two genera ons.
The topology of DNA is defined by how the two DNA strands are intertwined. It plays an important
role processes such as replica on, recombina on, transcrip on, etc. The topological problem arises
as the DNA strands need to be separated, but an overwound region is
created, called a supercoil. The chromosomes are fixed at the end, so they
cannot rotate freely when entangled. In a relaxed state one whole turn of
the DNA strand has a distance of more or less 10 base pairs. When the
DNA is twisted one whole turn of the DNA strand is more than 10 base
pairs, this is overwound DNA. The DNA turns in on itself because of the
tension in the DNA. This is a posi ve supercoil. When the DNA is
underwound one whole turn of the DNA strand is less than 10 base pairs.
The lack of tension needs to be compensated, which creates supercoils in
the opposite direc on. The topology of the DNA is constantly changing.
Chromosomes contain both nega ve and posi ve supercoils in different
regions.
, Topoisomerases are enzymes that catalyse changes in the topological state of DNA by cu ng DNA
strands. They relax posi ve and nega ve supercoils. Type I
topoisomerase performs single-strand breaks and type II
topoisomerase performs double-strand breaks. Type I topoisomerase
binds to the DNA and performs a single-strand break. Then the other
strand is passed over the nick a er which the DNA is ligated back
again. This causes the DNA strand to relax. Gyrase is a type II
topoisomerase and is crucial for DNA replica on. Gyrase is an enzyme that introduces nega ve
supercoils and it relaxes and prevents overwinding during DNA replica on. An
overwound region is created, so a er a while it is not possible to separate the
DNA anymore. Gyrase relaxes posi ve supercoils. It binds to the DNA at the
supercoils and performs a double strand break. Type II topoisomerase has the
same principle as type I topoisomerase, only with a double-strand break.
Posi ve supercoils are like a string that is twisted and twisted and then starts
turning in on itself.
Ini a on of DNA replica on
Genome replica on is bidirec onal. Replica on starts at very specific regions of the
genome, called origin of replica on (ori). Here the DNA strands are separated
genera ng a replica on bubble that contains two replica on forks, one to the right
and one to the le . At the replica on forks DNA replica on actually occurs. The
replica on forks move in opposite direc ons, so are bidirec onal. Bacterial circular
chromosomes contain a single ori of DNA replica on, which replicates the whole
chromosome. Eukaryo c linear chromosomes are bigger and more complex, so have
mul ple ori to cover the whole genome. A human chromosome, for example, has
between 30.000 and 50.000 ori. A replicon is a unit of the genome that is replicated
by a single origin of replica on, these can differ in sizes. E.g. one replicon in bacteria,
mul ple in eukaryotes. The origin of replica on is the sequence where DNA
replica on starts and a replicon is a unit of genome that is replicated by a single ori.
In the image, the second ori has created a smaller part of the DNA compared to the
first.
OriC of E. coli: methyla on regulates ini a on. The ori of E. coli is called OriC. OriC is 245 base pairs
long and contains mul ple recogni on sites for DNA binding proteins and enzymes. This cell has to
know that it has to start DNA replica on. Methyla on of the OriC regulates ini a on of replica on.
OriC contains 11 copies of a palindromic sequence (GATCCTAG), which are the black circles in the
image. Dam methylase enzyme methylates the adenines of the sequence. It adds a methyl group to
the adenines in both DNA strands of the sequence. When both DNA strands are methylated, oriC is
ac ve and replica on starts. A er replica on the daughter strands are not methylated yet, only the
parental strand is methylated. Hemimethylated origins (one strand methylated and other not) inhibit
ini a on of replica on, only fully methylated origins start DNA replica on
(regula on system). In this hemimethylated state the oriC is inac ve. A er some
me the daughter strands are methylated as well and then the OriC will be
ac ve again.
OriC of E. coli: ini a on of replica on. OriC contains regions for DNA binding proteins. When OriC is
fully methylated, 6 proteins are involved in the forma on of the replica on forks and the ini a on of
replica on: DnaA, DnaB, DnaC, HU, Gyrase, and SSB. The most important of these proteins is DnaA.
