Molecular Genetics
LECTURE 1 GENOME REPLICATION ~ JAN KOK
How genomes replicate and evolve
Primary function of genome is to specify biochemical signature of cell in which it resides:
Het genoom van een organisme is het geheel van erfelijke informatie in een cel.
A gene undergoes transcription to a protein and if you look to a
genome it undergoes transcriptome and becomes a proteome. This all
leads to 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 (Survival of
an individual requires genetic stability).
Not all mutations are bad
Survival of a species, in the long run, benefits from incidental genetic changes (mutations).
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
Each strand of DNA produces 1 new strand (semi-conservative replication).
The Meselson–Stahl experiment, 1958: Replication is semi-conservative: The experiment done
by Meselson and Stahl shows that DNA replicated semi-conservatively, meaning that each strand in a
DNA molecule serves as a template for synthesis of a new, complementary strand, slide 7 and 8.
The topological problem of DNA replication
DNA topoisomerases prevent overwinding during DNA replication; DNA “breakage-and-reunion”
enzymes.
▪ Type I makes single-strand cut in
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 green is on the grey it is
one linking number. On the figure they
have a linking number of two.
Initiation of bidirectional genome replication
To start replication, double helix is opened, replication “machine” is assembled at replication forks.
Starting position is NOT random but at origin (ori) of replication.
1
, On imagine A you see one origen of replication and then to the left one
replication fork and to the right one replication fork, so you have a total of
2 replication forks.
On imagine B you see multiple origens of replication (30,000-50,000 in humans).
On the imagine below an origin of E coli. that consist of ~ 245 base pairs.
▪ The grey area is called the DNA winding element.
▪ The red are high affinity biding sites for DnaA, always occupied.
▪ The blue are low affinity binding sites filled just prior to replication.
DnaA binding + HU proteins ultimately cause melting
(unwinding) of DUE. Attachment of prepriming
complex (DnaB + DnaC) at each DUE end.
C “helps” B to attach; B is helicase (breaks base pairs),
extends melted region → Binding of elongation
enzymes, replication forks progress → DNA replication
Understanding replication = understanding DNA polymerases
DNA polymerases are the central players in genome replication, complication with DNA replication is
that DNA polymerases:
▪ Only synthesize in 5ʹ → 3ʹ direction
▪ 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!
▪ In eukaryotes the priming of DNA synthesis is a
little bit more complex, because the primase is in
fact tightly bind to the DNA polymerase. And
together they cooperate to make a DNA primer.
DNA synthesis only proceeds in 5ʹ → 3ʹ direction
▪ Leading strand starts with 3’ , continuous copying.
▪ Lagging strand starts with 5’ , discontinuous copying.
▪ Priming: only once on the leading strand, (very) often on
lagging strand.
▪ Okazaki fragments: are short sequences of DNA nucleotides
which are synthesized discontinuously and later linked together
by the enzyme DNA ligase to create the lagging strand during
DNA replication.
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.
2
,Single-strand binding proteins (SSBs)
Single-stranded DNA in replication fork (obtained by helicase) is “sticky” E
SSBs (RPAs) bind to phosphate backbone forming a channel to:
▪ prevent ss-DNA from re-associating s
▪ protect against degradation by e.g. single-strand-specific nucleases
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 Pol-III copies leading strand, the other copies lagging strand, lagging strand spools through its
DNA Pol-III → 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”
- “Sliding clamp” (binds the DNA polymerase)
Joining of adjacent Okazaki fragments
Initial copy of lagging strand = a series of Okazaki fragments:
-1- they contain RNA that needs to be removed
-2- they need to be coupled
Termination of genome replication
E. coli genome replication:
- Bidirectional
- One start point (ori)
- Replication faster than
transcription
- Replication fork pauses
behind RNA-Pol
- until trancript is completed
Replication terminator sequences:
- Prevent fork “overshoot”
- Bound by Tus
Tus (terminator utilization substance):
- Sequence-specific DNA binding protein
- Allows passage of replication fork only in 1 direction
Termination of replication in eukaryotes
▪ Replication in higher eukaryotes takes place in “replication foci”
▪ Discrete immobile regions with all necessary proteins
▪ ~ 10 replication origins in 1 focus; Termination is coordinated in
single focus
▪ Replicated DNA is looping out; Prevents entanglement of replicating chromosomes
Cohesin: Multi-subunit proteins forming ring structure
▪ Attach to DNA after replication fork has passed
▪ Keep sister chromatids aligned until anaphase
▪ Are then cut by proteases so daughter chromosomes can be separated
3
, Two reasons why linear DNA could become shorter after replication…
Primer removal:
requires extension of
3’-end of
adjacent Okazaki
fragment
Telomeres; Table 15.3
▪ Consist of multiple copies of a short repeat motif
▪ Several 100 copies in tandem repeats (total length: 2-15
kilobases in humans)
▪ A telomere is a region of repetitive nucleotide sequences at each end of a
chromosome, which protects the end of the chromosome from deterioration
or from fusion with neighboring chromosomes.
▪ Telomeres are extended by Telomerase
T-loop formation in mammalian cells
▪ Through invasion of free G-rich 3ʹ-end in double helix
▪ Basepairs with complementary sequence on C-rich strand
▪ Might provide additional stabilization of chromosome end
▪ At the very distal end of the telomere is a 300 base pair single-stranded portion, which forms the T-loop. This loop is
analogous to a knot, which stabilizes the telomere, preventing the telomere ends from being recognized as break
points by the DNA repair machinery.
Shelterin complexes are essential for formation of T-loop
Protein complex of telomere-binding proteins that:
▪ Binds to telomere repeat sequence
▪ Regulates telomerase activity
▪ Protects against degradation by nucleases
▪ Protects telomeres in many eukaryotes from DNA repair mechanisms
Absence of shelterin (telosome):
• Causes telomere uncapping
• Activates damage-signaling pathways
• May lead to non-homologous end joining (NHEJ; prevented by t-loop)
• Homology directed repair (HDR)
• End-to-end fusions
• Genomic instability
• Senescence, or apoptosis
Telomere length is implicated in cell senescence and cancer
Inhibition of telomerase as an anticancer treatment
Antibody against protein part of telomerase
Oligonucleotide complementary to RNA of telomerase
When telomeres are short, cells stop dividing and
undergo a growth arrest (called replicative
senescence).
4
LECTURE 1 GENOME REPLICATION ~ JAN KOK
How genomes replicate and evolve
Primary function of genome is to specify biochemical signature of cell in which it resides:
Het genoom van een organisme is het geheel van erfelijke informatie in een cel.
A gene undergoes transcription to a protein and if you look to a
genome it undergoes transcriptome and becomes a proteome. This all
leads to 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 (Survival of
an individual requires genetic stability).
Not all mutations are bad
Survival of a species, in the long run, benefits from incidental genetic changes (mutations).
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
Each strand of DNA produces 1 new strand (semi-conservative replication).
The Meselson–Stahl experiment, 1958: Replication is semi-conservative: The experiment done
by Meselson and Stahl shows that DNA replicated semi-conservatively, meaning that each strand in a
DNA molecule serves as a template for synthesis of a new, complementary strand, slide 7 and 8.
The topological problem of DNA replication
DNA topoisomerases prevent overwinding during DNA replication; DNA “breakage-and-reunion”
enzymes.
▪ Type I makes single-strand cut in
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 green is on the grey it is
one linking number. On the figure they
have a linking number of two.
Initiation of bidirectional genome replication
To start replication, double helix is opened, replication “machine” is assembled at replication forks.
Starting position is NOT random but at origin (ori) of replication.
1
, On imagine A you see one origen of replication and then to the left one
replication fork and to the right one replication fork, so you have a total of
2 replication forks.
On imagine B you see multiple origens of replication (30,000-50,000 in humans).
On the imagine below an origin of E coli. that consist of ~ 245 base pairs.
▪ The grey area is called the DNA winding element.
▪ The red are high affinity biding sites for DnaA, always occupied.
▪ The blue are low affinity binding sites filled just prior to replication.
DnaA binding + HU proteins ultimately cause melting
(unwinding) of DUE. Attachment of prepriming
complex (DnaB + DnaC) at each DUE end.
C “helps” B to attach; B is helicase (breaks base pairs),
extends melted region → Binding of elongation
enzymes, replication forks progress → DNA replication
Understanding replication = understanding DNA polymerases
DNA polymerases are the central players in genome replication, complication with DNA replication is
that DNA polymerases:
▪ Only synthesize in 5ʹ → 3ʹ direction
▪ 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!
▪ In eukaryotes the priming of DNA synthesis is a
little bit more complex, because the primase is in
fact tightly bind to the DNA polymerase. And
together they cooperate to make a DNA primer.
DNA synthesis only proceeds in 5ʹ → 3ʹ direction
▪ Leading strand starts with 3’ , continuous copying.
▪ Lagging strand starts with 5’ , discontinuous copying.
▪ Priming: only once on the leading strand, (very) often on
lagging strand.
▪ Okazaki fragments: are short sequences of DNA nucleotides
which are synthesized discontinuously and later linked together
by the enzyme DNA ligase to create the lagging strand during
DNA replication.
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.
2
,Single-strand binding proteins (SSBs)
Single-stranded DNA in replication fork (obtained by helicase) is “sticky” E
SSBs (RPAs) bind to phosphate backbone forming a channel to:
▪ prevent ss-DNA from re-associating s
▪ protect against degradation by e.g. single-strand-specific nucleases
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 Pol-III copies leading strand, the other copies lagging strand, lagging strand spools through its
DNA Pol-III → 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”
- “Sliding clamp” (binds the DNA polymerase)
Joining of adjacent Okazaki fragments
Initial copy of lagging strand = a series of Okazaki fragments:
-1- they contain RNA that needs to be removed
-2- they need to be coupled
Termination of genome replication
E. coli genome replication:
- Bidirectional
- One start point (ori)
- Replication faster than
transcription
- Replication fork pauses
behind RNA-Pol
- until trancript is completed
Replication terminator sequences:
- Prevent fork “overshoot”
- Bound by Tus
Tus (terminator utilization substance):
- Sequence-specific DNA binding protein
- Allows passage of replication fork only in 1 direction
Termination of replication in eukaryotes
▪ Replication in higher eukaryotes takes place in “replication foci”
▪ Discrete immobile regions with all necessary proteins
▪ ~ 10 replication origins in 1 focus; Termination is coordinated in
single focus
▪ Replicated DNA is looping out; Prevents entanglement of replicating chromosomes
Cohesin: Multi-subunit proteins forming ring structure
▪ Attach to DNA after replication fork has passed
▪ Keep sister chromatids aligned until anaphase
▪ Are then cut by proteases so daughter chromosomes can be separated
3
, Two reasons why linear DNA could become shorter after replication…
Primer removal:
requires extension of
3’-end of
adjacent Okazaki
fragment
Telomeres; Table 15.3
▪ Consist of multiple copies of a short repeat motif
▪ Several 100 copies in tandem repeats (total length: 2-15
kilobases in humans)
▪ A telomere is a region of repetitive nucleotide sequences at each end of a
chromosome, which protects the end of the chromosome from deterioration
or from fusion with neighboring chromosomes.
▪ Telomeres are extended by Telomerase
T-loop formation in mammalian cells
▪ Through invasion of free G-rich 3ʹ-end in double helix
▪ Basepairs with complementary sequence on C-rich strand
▪ Might provide additional stabilization of chromosome end
▪ At the very distal end of the telomere is a 300 base pair single-stranded portion, which forms the T-loop. This loop is
analogous to a knot, which stabilizes the telomere, preventing the telomere ends from being recognized as break
points by the DNA repair machinery.
Shelterin complexes are essential for formation of T-loop
Protein complex of telomere-binding proteins that:
▪ Binds to telomere repeat sequence
▪ Regulates telomerase activity
▪ Protects against degradation by nucleases
▪ Protects telomeres in many eukaryotes from DNA repair mechanisms
Absence of shelterin (telosome):
• Causes telomere uncapping
• Activates damage-signaling pathways
• May lead to non-homologous end joining (NHEJ; prevented by t-loop)
• Homology directed repair (HDR)
• End-to-end fusions
• Genomic instability
• Senescence, or apoptosis
Telomere length is implicated in cell senescence and cancer
Inhibition of telomerase as an anticancer treatment
Antibody against protein part of telomerase
Oligonucleotide complementary to RNA of telomerase
When telomeres are short, cells stop dividing and
undergo a growth arrest (called replicative
senescence).
4
