Summary Molecular
Genetics
Genome replication
If DNA repair were perfect (no mutations ever), there would be no genetic variation, and this variation is
the raw material of evolution
Semi-conservative = The DNA strands would be separated and each act as a template for the synthesis of
a new DNA molecule
But there was an topological problem: how do you unwind the helix without knotting? →
topoisomerases: prevent the overwinding during the DNA replication, and prevents knotting
Types of topoisomerases:
- Type 1A: uses an ss-break as a gate for the uncut DNA strand
- Type 1B: relaxing the torsional stress by swirling the cut strand around the uncut one
- Type 2: ds-break created in the phosphate backbone
The linking number is the number of times either strand crosses the other completely in a circular
molecule
0
2 complications with DNA polymerase:
1) It only synthesizes in ‘5 → 3’ direction: Okazaki fragments
2) Needs a primer:
Prokaryotes: primase, DNA polymerase III
Eukaryotes: primase bound to polymerase alfa, primase synthesizes RNA primer and polymerase
alfa extends this (resulting RNA-DNA primer). Finished by DNA polymerase ε on the leading
strand, polymerase δ on the lagging strand
Fork progression in both bacteria and eukaryotes is done by helicase and topoisomerases, the
ssDNA in the replication fork is sticky, SSBs (RPAs) bind to this and form a channel: preventing ss-
DNA re-associating and degradation
,DNA synthesis and Okazaki fragments
Prokaryotes: two DNA polymerase III molecules are linked together (dimer), the lagging strand spools
through its DNA polymerase III (replicating both strands at the same time). The clamp loader opens the
clamp and attaches it to the DNA, the sliding clamp binds the DNA polymerase
When DNA polymerase III meets a RNA primer that needs to be removed it releases the lagging strand
and polymerase I will take over (exonuclease activity) and extends the 3’end, ligase finishes it
Eukaryotes: don’t have DNA polymerase with exonuclease activity, solution: flap endonuclease (FEN1)
DNA polymerase δ and helicase will push away the primer and synthesize a new part,
after this FEN1 will cut at branch point, ligase finishes it
Termination
Prokaryotes: terminated by termination utilization substance (Tus) on termination sequences, when Tus is
bound to a terminator, it allows a fork to pass from one direction, but the other fork from the other
direction cannot pass
Eukaryotes: less known, synthesize occurs in replication foci, these are immobile region with all the
necessary proteins, this model explains why daughter molecules don’t tangle as much
Other protein involved in the prevention of entanglement: Cohesions. Cohesions are multisubunit
proteins that form a ring structure that are attached to daughter molecules after the fork passed and stay
until anaphase. They keep the chromatids aligned and will be cut after by proteases
Telomeres
Consist of multiple copies of a short repeat motif (tandem repeats), shortening of telomers in two ways:
1) Extreme 3’end of lagging strand might not be copied because the priming site is beyond the
template
2) The primer is placed at the extreme 3’ end, and the RNA primer cannot be converted into DNA
because it requires extension of 3’end of Okazaki, which is not possible
Telomerase: contain RNA and protein, his 5’end is reverse complementary with the telomere repeat,
telomerase can extend telomeres by using its RNA as template: RNA dependent DNA polymerase.
Critical factor is not losing genes, but maintaining the protein cap to protect it from repair
enzymes
Telomerase will produce a G-rich strand that can invade the double helix and pair with the
complementary sequence on the C-rich strand (TRF2) → stabilization of the chromosome end (T-
loop). This structure is done by Shelterin complexes (telosome)
Shelterin complexes regulate telomerase activity, protect against degradation by nucleases and
DNA repair mechanisms
Telomere shortens → TRF1 proteins decrease → telomerase can attach and extend the telomeres
→ TRF 1 reattach → telomerase disappears (negative feedbackloop)
, Mutations
Causes of mutations:
1) Spontaneous mutations during replication,
evade the proofreading capacity by DNA polymerase
In the closed formation the nucleotide is placed onto the template, if it is incorrect, the
nucleotide is rejected before it is attached. If this goes wrong, this will result in an inheritable ds
version of a mutation
These error rates are decreased do to: nucleotide selection process of DNA polymerase,
proofreading activity, and the mismatch repair system
Two types:
- Base tautomerization: a tautomeric shift itself is not a mutation (it’s a chemical change), but a
transient change to an alternative from of the molecule will ultimately result in a mutation
A---C
T ---G
G---T
- Replication slippage: are seen by DNA molecules with short repeated
- sequences, this can result in insertions or deletions
2) Chemical and physical mutagens
are chemical or physical agents that cause mutations
Four types chemical agents:
- Base analogs: purine and pyrimidine bases are mistakenly incorporated (tautomeric shift)
e.g. 5-bromouracil’s enols form will pair with g → leading to mutations
- Deaminating agents: deamination happens spontaneously (losing an amino group), but the
rate is increased by chemicals like nitrous acid and sodium bisulfite
e.g. deamination of adenine: hypoxanthine pairs with C instead of T, this leads to point
mutations during replication
- Alkylating agents: an important alkylation is methylation, certain alkylating agents are
mutagenic, like alkyl halides (pesticides) → leads to point mutations and cross-linking of DNA
- Intercalating agents: flat molecules that can creep between basepairs, and slightly unwind
the double helix and causing insertion mutations
Three types of physical mutagens:
- Ionization radiation: various effects depending on the type and intensity
- UV radiation: results in dimerization of pyrimidines bases, and will usually lead to deletions
upon replication
- Heat: stimulates the cleavage of beta-N-glycosidic bond resulting in baseless
(AP=apurinic/apyrimidinic site)
Transversions usually have a more pronounced effect because transitions are more likely to encode for
the same amino acid (wobble) (degeneracy of the code)
Genetics
Genome replication
If DNA repair were perfect (no mutations ever), there would be no genetic variation, and this variation is
the raw material of evolution
Semi-conservative = The DNA strands would be separated and each act as a template for the synthesis of
a new DNA molecule
But there was an topological problem: how do you unwind the helix without knotting? →
topoisomerases: prevent the overwinding during the DNA replication, and prevents knotting
Types of topoisomerases:
- Type 1A: uses an ss-break as a gate for the uncut DNA strand
- Type 1B: relaxing the torsional stress by swirling the cut strand around the uncut one
- Type 2: ds-break created in the phosphate backbone
The linking number is the number of times either strand crosses the other completely in a circular
molecule
0
2 complications with DNA polymerase:
1) It only synthesizes in ‘5 → 3’ direction: Okazaki fragments
2) Needs a primer:
Prokaryotes: primase, DNA polymerase III
Eukaryotes: primase bound to polymerase alfa, primase synthesizes RNA primer and polymerase
alfa extends this (resulting RNA-DNA primer). Finished by DNA polymerase ε on the leading
strand, polymerase δ on the lagging strand
Fork progression in both bacteria and eukaryotes is done by helicase and topoisomerases, the
ssDNA in the replication fork is sticky, SSBs (RPAs) bind to this and form a channel: preventing ss-
DNA re-associating and degradation
,DNA synthesis and Okazaki fragments
Prokaryotes: two DNA polymerase III molecules are linked together (dimer), the lagging strand spools
through its DNA polymerase III (replicating both strands at the same time). The clamp loader opens the
clamp and attaches it to the DNA, the sliding clamp binds the DNA polymerase
When DNA polymerase III meets a RNA primer that needs to be removed it releases the lagging strand
and polymerase I will take over (exonuclease activity) and extends the 3’end, ligase finishes it
Eukaryotes: don’t have DNA polymerase with exonuclease activity, solution: flap endonuclease (FEN1)
DNA polymerase δ and helicase will push away the primer and synthesize a new part,
after this FEN1 will cut at branch point, ligase finishes it
Termination
Prokaryotes: terminated by termination utilization substance (Tus) on termination sequences, when Tus is
bound to a terminator, it allows a fork to pass from one direction, but the other fork from the other
direction cannot pass
Eukaryotes: less known, synthesize occurs in replication foci, these are immobile region with all the
necessary proteins, this model explains why daughter molecules don’t tangle as much
Other protein involved in the prevention of entanglement: Cohesions. Cohesions are multisubunit
proteins that form a ring structure that are attached to daughter molecules after the fork passed and stay
until anaphase. They keep the chromatids aligned and will be cut after by proteases
Telomeres
Consist of multiple copies of a short repeat motif (tandem repeats), shortening of telomers in two ways:
1) Extreme 3’end of lagging strand might not be copied because the priming site is beyond the
template
2) The primer is placed at the extreme 3’ end, and the RNA primer cannot be converted into DNA
because it requires extension of 3’end of Okazaki, which is not possible
Telomerase: contain RNA and protein, his 5’end is reverse complementary with the telomere repeat,
telomerase can extend telomeres by using its RNA as template: RNA dependent DNA polymerase.
Critical factor is not losing genes, but maintaining the protein cap to protect it from repair
enzymes
Telomerase will produce a G-rich strand that can invade the double helix and pair with the
complementary sequence on the C-rich strand (TRF2) → stabilization of the chromosome end (T-
loop). This structure is done by Shelterin complexes (telosome)
Shelterin complexes regulate telomerase activity, protect against degradation by nucleases and
DNA repair mechanisms
Telomere shortens → TRF1 proteins decrease → telomerase can attach and extend the telomeres
→ TRF 1 reattach → telomerase disappears (negative feedbackloop)
, Mutations
Causes of mutations:
1) Spontaneous mutations during replication,
evade the proofreading capacity by DNA polymerase
In the closed formation the nucleotide is placed onto the template, if it is incorrect, the
nucleotide is rejected before it is attached. If this goes wrong, this will result in an inheritable ds
version of a mutation
These error rates are decreased do to: nucleotide selection process of DNA polymerase,
proofreading activity, and the mismatch repair system
Two types:
- Base tautomerization: a tautomeric shift itself is not a mutation (it’s a chemical change), but a
transient change to an alternative from of the molecule will ultimately result in a mutation
A---C
T ---G
G---T
- Replication slippage: are seen by DNA molecules with short repeated
- sequences, this can result in insertions or deletions
2) Chemical and physical mutagens
are chemical or physical agents that cause mutations
Four types chemical agents:
- Base analogs: purine and pyrimidine bases are mistakenly incorporated (tautomeric shift)
e.g. 5-bromouracil’s enols form will pair with g → leading to mutations
- Deaminating agents: deamination happens spontaneously (losing an amino group), but the
rate is increased by chemicals like nitrous acid and sodium bisulfite
e.g. deamination of adenine: hypoxanthine pairs with C instead of T, this leads to point
mutations during replication
- Alkylating agents: an important alkylation is methylation, certain alkylating agents are
mutagenic, like alkyl halides (pesticides) → leads to point mutations and cross-linking of DNA
- Intercalating agents: flat molecules that can creep between basepairs, and slightly unwind
the double helix and causing insertion mutations
Three types of physical mutagens:
- Ionization radiation: various effects depending on the type and intensity
- UV radiation: results in dimerization of pyrimidines bases, and will usually lead to deletions
upon replication
- Heat: stimulates the cleavage of beta-N-glycosidic bond resulting in baseless
(AP=apurinic/apyrimidinic site)
Transversions usually have a more pronounced effect because transitions are more likely to encode for
the same amino acid (wobble) (degeneracy of the code)
