Molecular
genetics
Introduction:
Im going out on a limb here and say that
you have a pretty good understanding of
the prior knowledge necessary for this
course. But, here are some things to refresh your mind:
The structure of DNA and RNA;
As shown on the picture on the right.
Synthesis of RNA or DNA is always in the 5’ to 3’ direction. And one of the
differences between DNA and RNA is that RNA ribose has an extra -OH groups
on the 2’ carbon.
DNA polymerase always needs primers to start synthesis, and cannot start of a
naked strand of DNA. While RNA polymerase doesn’t need primers.
PCR is the multiplication of DNA, and is in the order of; denature, annealing,
synthesis. Annealing is the binding of the primers to the DNA.
Palindromic sequences are a sequences in DNA that reads the same for both
the 5’ to 3’
direction and
the 3’ to 5’
direction.
Like;
GGATCC.
Restriction
enzymes can cut in this
to make a sticky overhang.
This can be a 5’ overhang or a 3’ overhang;
A cell of which a gene is taken out, is called a knock out.
Transcription makes RNA from DNA, it uses RNA polymerase on a DNA
template.
reverse transcription makes DNA from RNA, it uses DNA polymerase on a RNA
template.
1
,Translation makes protein from RNA,
it uses ribosomes on a RNA template.
Genome replication:
In 1953, Watson and Crick theorized that DNA replication is semi-
conservative. This means that after the first replication cycle, both DNA
strands have halve of the original DNA strand.
Opposed to the conservative replication, in which after one replication cycle,
one would be the exact replica of the original while the other would consist of
newly synthesized DNA.
The semi-conservative replication was put into question, because people didn’t
understand yet that DNA could unwind. The topological problem states that it is
impossible to unwind DNA, and thus DNA replication would not be possible in
the semi-conservative way.
To proof that DNA replication is semi-conservative, they did the Meselson-Stahl
experiment in 1958. In the experiment, they made an E.coli culture with the
isotope N15, then they transferred this into an N14 medium. And observed the
density of the first generation and the second generation. They found that this
matched exactly to the semi-conservative replication.
This still did not explain how the DNA solves the topological problem. Topology
of DNA is defined by how the two DNA strands are intertwined. DNA can be
overwound (<10.5 bp per turn), or be underbound (>10.5 bp per turn).
DNA can also be in a positive or a negative supercoil.
If nothing would happen when DNA replicated in a circular (or very very long
linear DNA), an overwound area would be created.
But don’t worry, this is solved using topoisomerase. These are enzymes that
catalyze changes in the topological state of DNA by cutting into the strands.
They relax positive and negative supercoils.
Topoisomerase I; single strand cuts
Topoisomerase II; double strand cuts
There is a special kind of topoisomerase II, which is gyrase. Gyrase deserves
respect because she introduces negative supercoils. This relaxes and prevents
overwinding during DNA replication. She does need ATP for this, but it is
definitely worth it.
2
,Initiation of DNA replication:
Replication starts at the origin of replication (ori), this is where two DNA Strats
are separated generating a replication bubble that contains two replication
forks, where replication occurs.
A replicon is a unit of the genome that is replicated by a single ori.
Bacterial circular chromosomes contain a single ori, and thus only have a single
replicon
Eukaryotic linear chromosomes have multiple ori, and thus have multiple
replicons.
There is a specific ori in E.coli that has been researched for its regulation; oriC.
The oriC contains 11 copies of the palindromic sequence; GATCCTAG. The
enzyme dam methylase methylates the adenines of the sequence. Only when
both strand of the DNA is methylated, does the replication start.
So, the replication is regulated by methylation.
Hemimethylated origins (only one strand of the DNA is methylated) are
inhibited from replicated. This is to protect the DNA, because shortly after
replication, you don’t want another replication to start.
When the oriC is fully methylated, 6 proteins are involved in the formation of
the replication fork;
- dnaA, is an ATP-binding protein and it is only activated when it is bound to
ATP. dnaA-ATP binds to the fully methylated oriC, on the high affinity sites,
and later on the low affinity sites* when the DNA is wrapped around.
- HU, this helps the DNA twist around itself.
- dnaB, is an ATP hydrolysis-dependent 5’-3’ helicase, that unwinds the DNA
by breaking the hydrogen bonds.
- dnaC, is a chaperone. They form the two replication forks.
- Gyrase, as stated before, she relaxes the DNA supercoils.
- SSB, single strand binding protein, stabilizes the DNA, keeps the replication
fork open and degradation of ssDNA from ss-specific-nucleases.
* low affinity means the DNA has high AT content.
3
, To put this all together is a summery;
DNA polymerases:
In both eukaryotes and prokaryotes, are DNA polymerases used in all sorts of
processes, like; DNA repair and replication. But they all have common features;
1. DNA synthesis from 5’ to 3’
2. DNA polymerase always needs primers to start synthesis, and cannot start
of a naked strand of DNA.
3. Proofreading and error control is done by exonuclease activity (3’ to 5’).
There are different types of DNA polymerase, and they are differ between
prokaryotes and eukaryotes.
Prokaryotes:
DNA poly- Function:
merase:
I* Synthesis of DNA, DNA repair and removal of RNA primers and replacing
them by DNA
II DNA repair
III** Main polymerizing enzyme
IV DNA repair
V DNA repair and SOS response
* has both 5’-3’ and 3’-5’ exonuclease activity.
** has only 3’-5’ exonuclease activity.
Eukaryotes:
DNA poly- Function:
merase:
𝛼 Primase and elongation of RNA primers
𝛽 DNA repair
𝛾 Mitochondrial DNA synthesis
4
genetics
Introduction:
Im going out on a limb here and say that
you have a pretty good understanding of
the prior knowledge necessary for this
course. But, here are some things to refresh your mind:
The structure of DNA and RNA;
As shown on the picture on the right.
Synthesis of RNA or DNA is always in the 5’ to 3’ direction. And one of the
differences between DNA and RNA is that RNA ribose has an extra -OH groups
on the 2’ carbon.
DNA polymerase always needs primers to start synthesis, and cannot start of a
naked strand of DNA. While RNA polymerase doesn’t need primers.
PCR is the multiplication of DNA, and is in the order of; denature, annealing,
synthesis. Annealing is the binding of the primers to the DNA.
Palindromic sequences are a sequences in DNA that reads the same for both
the 5’ to 3’
direction and
the 3’ to 5’
direction.
Like;
GGATCC.
Restriction
enzymes can cut in this
to make a sticky overhang.
This can be a 5’ overhang or a 3’ overhang;
A cell of which a gene is taken out, is called a knock out.
Transcription makes RNA from DNA, it uses RNA polymerase on a DNA
template.
reverse transcription makes DNA from RNA, it uses DNA polymerase on a RNA
template.
1
,Translation makes protein from RNA,
it uses ribosomes on a RNA template.
Genome replication:
In 1953, Watson and Crick theorized that DNA replication is semi-
conservative. This means that after the first replication cycle, both DNA
strands have halve of the original DNA strand.
Opposed to the conservative replication, in which after one replication cycle,
one would be the exact replica of the original while the other would consist of
newly synthesized DNA.
The semi-conservative replication was put into question, because people didn’t
understand yet that DNA could unwind. The topological problem states that it is
impossible to unwind DNA, and thus DNA replication would not be possible in
the semi-conservative way.
To proof that DNA replication is semi-conservative, they did the Meselson-Stahl
experiment in 1958. In the experiment, they made an E.coli culture with the
isotope N15, then they transferred this into an N14 medium. And observed the
density of the first generation and the second generation. They found that this
matched exactly to the semi-conservative replication.
This still did not explain how the DNA solves the topological problem. Topology
of DNA is defined by how the two DNA strands are intertwined. DNA can be
overwound (<10.5 bp per turn), or be underbound (>10.5 bp per turn).
DNA can also be in a positive or a negative supercoil.
If nothing would happen when DNA replicated in a circular (or very very long
linear DNA), an overwound area would be created.
But don’t worry, this is solved using topoisomerase. These are enzymes that
catalyze changes in the topological state of DNA by cutting into the strands.
They relax positive and negative supercoils.
Topoisomerase I; single strand cuts
Topoisomerase II; double strand cuts
There is a special kind of topoisomerase II, which is gyrase. Gyrase deserves
respect because she introduces negative supercoils. This relaxes and prevents
overwinding during DNA replication. She does need ATP for this, but it is
definitely worth it.
2
,Initiation of DNA replication:
Replication starts at the origin of replication (ori), this is where two DNA Strats
are separated generating a replication bubble that contains two replication
forks, where replication occurs.
A replicon is a unit of the genome that is replicated by a single ori.
Bacterial circular chromosomes contain a single ori, and thus only have a single
replicon
Eukaryotic linear chromosomes have multiple ori, and thus have multiple
replicons.
There is a specific ori in E.coli that has been researched for its regulation; oriC.
The oriC contains 11 copies of the palindromic sequence; GATCCTAG. The
enzyme dam methylase methylates the adenines of the sequence. Only when
both strand of the DNA is methylated, does the replication start.
So, the replication is regulated by methylation.
Hemimethylated origins (only one strand of the DNA is methylated) are
inhibited from replicated. This is to protect the DNA, because shortly after
replication, you don’t want another replication to start.
When the oriC is fully methylated, 6 proteins are involved in the formation of
the replication fork;
- dnaA, is an ATP-binding protein and it is only activated when it is bound to
ATP. dnaA-ATP binds to the fully methylated oriC, on the high affinity sites,
and later on the low affinity sites* when the DNA is wrapped around.
- HU, this helps the DNA twist around itself.
- dnaB, is an ATP hydrolysis-dependent 5’-3’ helicase, that unwinds the DNA
by breaking the hydrogen bonds.
- dnaC, is a chaperone. They form the two replication forks.
- Gyrase, as stated before, she relaxes the DNA supercoils.
- SSB, single strand binding protein, stabilizes the DNA, keeps the replication
fork open and degradation of ssDNA from ss-specific-nucleases.
* low affinity means the DNA has high AT content.
3
, To put this all together is a summery;
DNA polymerases:
In both eukaryotes and prokaryotes, are DNA polymerases used in all sorts of
processes, like; DNA repair and replication. But they all have common features;
1. DNA synthesis from 5’ to 3’
2. DNA polymerase always needs primers to start synthesis, and cannot start
of a naked strand of DNA.
3. Proofreading and error control is done by exonuclease activity (3’ to 5’).
There are different types of DNA polymerase, and they are differ between
prokaryotes and eukaryotes.
Prokaryotes:
DNA poly- Function:
merase:
I* Synthesis of DNA, DNA repair and removal of RNA primers and replacing
them by DNA
II DNA repair
III** Main polymerizing enzyme
IV DNA repair
V DNA repair and SOS response
* has both 5’-3’ and 3’-5’ exonuclease activity.
** has only 3’-5’ exonuclease activity.
Eukaryotes:
DNA poly- Function:
merase:
𝛼 Primase and elongation of RNA primers
𝛽 DNA repair
𝛾 Mitochondrial DNA synthesis
4
