Lecture 1
Genome replication
Genome → contains all genetic information of a organism
- DNA → made up of deoxyribose (sugar group), phosphate and a nitrogen base (A, T,
C, G)
Transcriptome → set of all RNA transcripts from a genome
- RNA → made up of ribose (sugar group), phosphate and a nitrogen base (A, U, C,
G)
- Ribose differs from deoxyribose since it lacks the OH group at position 2
(ribose only contains a H)
Proteome → set of all proteins expressed by an organism
Metabolome → the complete set of small-molecule chemicals found within a biological
sample
How does DNA replicate
→ DNA replication is semiconservative and not conservative or dispersive
- Semiconservative → DNA strands will separate and each strand
serves as a template for a new DNA strand
- New DNA contains 1 parental strand and 1 new DNA strand
- Conservative → each DNA molecule serves as a template for a
new DNA molecuul
- Dispersive → DNA replication results in two DNA molecules that
are mixtures of parental and daughter DNA
→ semiconservative replication is proved with E. coli an experiment (Meselson-Stahl
experiment)
1. E. coli was grown in N15 medium which makes DNA ‘heavy’ after it is incorporated in
the DNA
2. E. coli was cultured in N14 medium for 1 replication → DNA appeared to be ‘light’
a. Both DNA molecules were light (so contained N 15 and N14)
3. After a second replication in N14 medium → DNA appeared to be ‘lighter’ and ‘light’
a. 2 DNA molecules were light (N 15 and N14) and 2 molecules were lighter (2x
N14)
→ DNA replication is bidirectional
Topological problem
Topology of DNA → how the two DNA strands are intertwined (met elkaar verweven)
- Relaxed state → +/- 10.5 bp per turn
- Overwound → < 10.5 bp per turn which can cause positive supercoils
- Underwound → > 10.5 bp per turn which can cause negative
supercoils
- This state makes DNA more pre-relaxt
→ normal DNA contains both positive and negative supercoils
1
,How does the DNA unwind without generating a big knot?
Topoisomerase → enzyme that cuts in DNA strands to makes changes in the topological
state of DNA
- Topoisomerase 1 → makes single strand breaks
- Cuts DNA, DNA opens and passes the other strand to make the
DNA more relaxt
- Topoisomerase 2 (gyrase) → makes double strand breaks
- Gyrase → plays an essential role in DNA replication since it relaxes positive
supercoils by introducing negative supercoils
- Binds a positive supercoil, makes a double strand break and
introduces a negative supercoil
Initiation of DNA replication
- Origin of replication (ORI) → the place where DNA replication starts and DNA
strands are separated generating a replication bubble (contains two replication forks)
- Bacterial circular chromosomes contain a single ORI
- Eukaryotic linear chromosomes have multiple ORI
- Replicon → the entire region of DNA that is independently replicated
from a single ORI (so bacteria have 1 replicon and eukaryotes have
multiple)
1. Methylation regulates initiation (E. coli example)
- Dam methylase enzyme methylates the A on the OriC, if both strands are
methylated, the OriC is active and replication starts
- After replication, only 1 strand is methylated and the other will not be
methylated for a period of time
- Hemimethylated → 1 strand is methylated and the other is
demethylated
- Inhibits initiation of replication
2. Initiation of replication (E. coli example)
a. 6 proteins are involved in the initiation of the replication:
i. DnaA → active when bound to ATP (so ATP dependent)
1. DnaA-ATP binds fully methylated OriC at the high affinity site
and causes DNA to wrap around DnaA
2. DnaA is now also bound to the low affinity site which is AT rich
3. Gets help from HU to melt the helix
ii. DnaB/DnaC complex → unwinds DNA further (forms replication forks)
1. DnaB → ATP dependent 5’ → 3’ helicase (separates DNA
strands by breaking the hydrogen bonds between nucleotides)
2. DnaC → chaperone
iii. Gyrase (type 2 topoisomerase) → relaxes positive DNA supercoils
iv. Single strand binding protein (SSB) → stabilizes DNA, keeps the
replication bubble open and
protects against degradation
of ssDNA nucleases
→ other proteins can now bind
2
,DNA polymerases
→ there are a lot of different DNA polymerases and they can have a lot of different functions.
All DNA polymerases have 3 things in common:
1. DNA synthesis direction from 5’ → 3’ direction
2. Cannot start from scratch, they need a primer (RNA or DNA) with a free 3’-OH end
3. Exonuclease activity in 3’ → 5’ direction (proofreading error-control mechanisms)
a. DNA polymerase I also has 5’ → 3’ exonuclease activity (to remove the RNA
primers of the Okazaki fragments)
i. Note: not all DNA polymerases can do this
→ DNA polymerases differ between prokaryotes and eukaryotes
Priming and semi-discontinuous replication
- DNA polymerase cannot synthesize DNA from scratch → it needs a starting point
(DNA or RNA)
- Primase → DNA-dependent RNA polymerase that synthesizes RNA primers
- DNA polymerase can elongate this primer
- Genome replication is semi-discontinuous
- Leading strand is synthesized continuously → one primer needed
- Lagging strand is is synthesized discontinuously → multiple primers needed
- A new primer will be added close to the replication fork and DNA
polymerase can synthesize DNA, this happens over and over again as
the DNA gets single stranded by helicase
- Forms Okazaki fragments*
*Joining of Okazaki fragments in E. coli (prokaryotes)
- DNA polymerase I has 3’ → 5’ as well as 5’ → 3’ exonuclease activity (remember:
DNA polymerase III only had 3’ → 5’ exonuclease activity for proofreading)
- It can degrade the RNA primer and synthesize DNA at the same time
- DNA ligase (enzyme) ligates the Okazaki fragments together completing the lagging
strand
*Joining of Okazaki fragments in eukaryotes
- DNA polymerase δ and helicase displace (verdringen) the primer
creating a 5’ flap and DNA polymerase fills the gap with DNA
- Flap endonuclease I (FEN1) cleaves the flap, removing the
primer
- DNA ligase ligates the fragments completing the lagging strand
How is the synthesis of the leading and lagging strand parallel? → replisome
The replisome → multiprotein structure at the replication fork to perform synthesis of DNA
- 2x DNA polymerase (one for the leading and one for the lagging strand)
- DNA polymerase synthesizing the lagging strand repetitively associate and
dissociate from the DNA
- 2x dimerizing subunit T → links DNA polymerases together
- 2x sliding clamps → connects the DNA polymerases to the DNA
- Clamp loader (group of 5 proteins) → places the sliding clamp on the DNA and keeps
the structure together
3
, Movement of the replisome
Trombone model of DNA replication → suggests the lagging strand forms a loop such that
the leading and lagging strand replisome proteins contact one another
→ https://www.youtube.com/watch?v=IjVLhoyfGAM&t=143s (from 2:25)
Termination of replication
→ DNA replication is bidirectional (and semiconservative and semi-discontinuous)
- In prokaryotes, two replication forks will meet halfway from the ORI → the replication
fork trap
- Tus proteins recognize Ter sites and can bind in a specific orientation to let the
replication fork stop or continue
- Binding in the same direction allows the replication fork to continue
- Binding in the opposite direction stops the replication fork
The end problem of eukaryotic chromosomes
→ linear chromosomes could become shorter after replication, how?
1. There is no space to generate a primer for the last Okazaki fragment
2. The primer of the last Okazaki fragment is at the very last 3’ extreme, it cannot be
removed and synthesized again by DNA polymerase
→ this only applies for the lagging strand
The solution → telomeres
→ short, repetitive 3’ G-rich sequences that seals the end of a chromosome
- Telomeres have different functions
1. The cell can distinguishes between the end of a chromosome and a DNA
break
2. Telomeres can be extended to prevent shortening of chromosomes
3. Telomeres stabilize the end of a chromosome by forming a T-loop (the G-rich
region of the telomere is complement to an earlier part of the telomere)
→ shelterin (complex of 6 proteins) is important for telomeres
- Protects telomeres from DNA repair mechanisms
- Regulates telomeres length control
- Catalyzes the T-loop formation
→ telomeres can be extended by telomerase (enzyme)
- Telomerase is a reverse transcriptase enzyme (RNA-dependent DNA polymerase)
- Synthesizes DNA from an RNA template
- RNA template is the reverse complement of the 3’ G-rich region
- Consists of protein + RNA template
- Telomerase in only active in dividing cells (early embryo, reproduction and stem cells)
- If not active → chromosomes shorten → senescence → cell death
The fidelity problem
→ DNA replication is not flawless, mutations are produced (despite the proofreading system)
- Mutations can cause cancer and other diseases but also benefit to evolution
4
Genome replication
Genome → contains all genetic information of a organism
- DNA → made up of deoxyribose (sugar group), phosphate and a nitrogen base (A, T,
C, G)
Transcriptome → set of all RNA transcripts from a genome
- RNA → made up of ribose (sugar group), phosphate and a nitrogen base (A, U, C,
G)
- Ribose differs from deoxyribose since it lacks the OH group at position 2
(ribose only contains a H)
Proteome → set of all proteins expressed by an organism
Metabolome → the complete set of small-molecule chemicals found within a biological
sample
How does DNA replicate
→ DNA replication is semiconservative and not conservative or dispersive
- Semiconservative → DNA strands will separate and each strand
serves as a template for a new DNA strand
- New DNA contains 1 parental strand and 1 new DNA strand
- Conservative → each DNA molecule serves as a template for a
new DNA molecuul
- Dispersive → DNA replication results in two DNA molecules that
are mixtures of parental and daughter DNA
→ semiconservative replication is proved with E. coli an experiment (Meselson-Stahl
experiment)
1. E. coli was grown in N15 medium which makes DNA ‘heavy’ after it is incorporated in
the DNA
2. E. coli was cultured in N14 medium for 1 replication → DNA appeared to be ‘light’
a. Both DNA molecules were light (so contained N 15 and N14)
3. After a second replication in N14 medium → DNA appeared to be ‘lighter’ and ‘light’
a. 2 DNA molecules were light (N 15 and N14) and 2 molecules were lighter (2x
N14)
→ DNA replication is bidirectional
Topological problem
Topology of DNA → how the two DNA strands are intertwined (met elkaar verweven)
- Relaxed state → +/- 10.5 bp per turn
- Overwound → < 10.5 bp per turn which can cause positive supercoils
- Underwound → > 10.5 bp per turn which can cause negative
supercoils
- This state makes DNA more pre-relaxt
→ normal DNA contains both positive and negative supercoils
1
,How does the DNA unwind without generating a big knot?
Topoisomerase → enzyme that cuts in DNA strands to makes changes in the topological
state of DNA
- Topoisomerase 1 → makes single strand breaks
- Cuts DNA, DNA opens and passes the other strand to make the
DNA more relaxt
- Topoisomerase 2 (gyrase) → makes double strand breaks
- Gyrase → plays an essential role in DNA replication since it relaxes positive
supercoils by introducing negative supercoils
- Binds a positive supercoil, makes a double strand break and
introduces a negative supercoil
Initiation of DNA replication
- Origin of replication (ORI) → the place where DNA replication starts and DNA
strands are separated generating a replication bubble (contains two replication forks)
- Bacterial circular chromosomes contain a single ORI
- Eukaryotic linear chromosomes have multiple ORI
- Replicon → the entire region of DNA that is independently replicated
from a single ORI (so bacteria have 1 replicon and eukaryotes have
multiple)
1. Methylation regulates initiation (E. coli example)
- Dam methylase enzyme methylates the A on the OriC, if both strands are
methylated, the OriC is active and replication starts
- After replication, only 1 strand is methylated and the other will not be
methylated for a period of time
- Hemimethylated → 1 strand is methylated and the other is
demethylated
- Inhibits initiation of replication
2. Initiation of replication (E. coli example)
a. 6 proteins are involved in the initiation of the replication:
i. DnaA → active when bound to ATP (so ATP dependent)
1. DnaA-ATP binds fully methylated OriC at the high affinity site
and causes DNA to wrap around DnaA
2. DnaA is now also bound to the low affinity site which is AT rich
3. Gets help from HU to melt the helix
ii. DnaB/DnaC complex → unwinds DNA further (forms replication forks)
1. DnaB → ATP dependent 5’ → 3’ helicase (separates DNA
strands by breaking the hydrogen bonds between nucleotides)
2. DnaC → chaperone
iii. Gyrase (type 2 topoisomerase) → relaxes positive DNA supercoils
iv. Single strand binding protein (SSB) → stabilizes DNA, keeps the
replication bubble open and
protects against degradation
of ssDNA nucleases
→ other proteins can now bind
2
,DNA polymerases
→ there are a lot of different DNA polymerases and they can have a lot of different functions.
All DNA polymerases have 3 things in common:
1. DNA synthesis direction from 5’ → 3’ direction
2. Cannot start from scratch, they need a primer (RNA or DNA) with a free 3’-OH end
3. Exonuclease activity in 3’ → 5’ direction (proofreading error-control mechanisms)
a. DNA polymerase I also has 5’ → 3’ exonuclease activity (to remove the RNA
primers of the Okazaki fragments)
i. Note: not all DNA polymerases can do this
→ DNA polymerases differ between prokaryotes and eukaryotes
Priming and semi-discontinuous replication
- DNA polymerase cannot synthesize DNA from scratch → it needs a starting point
(DNA or RNA)
- Primase → DNA-dependent RNA polymerase that synthesizes RNA primers
- DNA polymerase can elongate this primer
- Genome replication is semi-discontinuous
- Leading strand is synthesized continuously → one primer needed
- Lagging strand is is synthesized discontinuously → multiple primers needed
- A new primer will be added close to the replication fork and DNA
polymerase can synthesize DNA, this happens over and over again as
the DNA gets single stranded by helicase
- Forms Okazaki fragments*
*Joining of Okazaki fragments in E. coli (prokaryotes)
- DNA polymerase I has 3’ → 5’ as well as 5’ → 3’ exonuclease activity (remember:
DNA polymerase III only had 3’ → 5’ exonuclease activity for proofreading)
- It can degrade the RNA primer and synthesize DNA at the same time
- DNA ligase (enzyme) ligates the Okazaki fragments together completing the lagging
strand
*Joining of Okazaki fragments in eukaryotes
- DNA polymerase δ and helicase displace (verdringen) the primer
creating a 5’ flap and DNA polymerase fills the gap with DNA
- Flap endonuclease I (FEN1) cleaves the flap, removing the
primer
- DNA ligase ligates the fragments completing the lagging strand
How is the synthesis of the leading and lagging strand parallel? → replisome
The replisome → multiprotein structure at the replication fork to perform synthesis of DNA
- 2x DNA polymerase (one for the leading and one for the lagging strand)
- DNA polymerase synthesizing the lagging strand repetitively associate and
dissociate from the DNA
- 2x dimerizing subunit T → links DNA polymerases together
- 2x sliding clamps → connects the DNA polymerases to the DNA
- Clamp loader (group of 5 proteins) → places the sliding clamp on the DNA and keeps
the structure together
3
, Movement of the replisome
Trombone model of DNA replication → suggests the lagging strand forms a loop such that
the leading and lagging strand replisome proteins contact one another
→ https://www.youtube.com/watch?v=IjVLhoyfGAM&t=143s (from 2:25)
Termination of replication
→ DNA replication is bidirectional (and semiconservative and semi-discontinuous)
- In prokaryotes, two replication forks will meet halfway from the ORI → the replication
fork trap
- Tus proteins recognize Ter sites and can bind in a specific orientation to let the
replication fork stop or continue
- Binding in the same direction allows the replication fork to continue
- Binding in the opposite direction stops the replication fork
The end problem of eukaryotic chromosomes
→ linear chromosomes could become shorter after replication, how?
1. There is no space to generate a primer for the last Okazaki fragment
2. The primer of the last Okazaki fragment is at the very last 3’ extreme, it cannot be
removed and synthesized again by DNA polymerase
→ this only applies for the lagging strand
The solution → telomeres
→ short, repetitive 3’ G-rich sequences that seals the end of a chromosome
- Telomeres have different functions
1. The cell can distinguishes between the end of a chromosome and a DNA
break
2. Telomeres can be extended to prevent shortening of chromosomes
3. Telomeres stabilize the end of a chromosome by forming a T-loop (the G-rich
region of the telomere is complement to an earlier part of the telomere)
→ shelterin (complex of 6 proteins) is important for telomeres
- Protects telomeres from DNA repair mechanisms
- Regulates telomeres length control
- Catalyzes the T-loop formation
→ telomeres can be extended by telomerase (enzyme)
- Telomerase is a reverse transcriptase enzyme (RNA-dependent DNA polymerase)
- Synthesizes DNA from an RNA template
- RNA template is the reverse complement of the 3’ G-rich region
- Consists of protein + RNA template
- Telomerase in only active in dividing cells (early embryo, reproduction and stem cells)
- If not active → chromosomes shorten → senescence → cell death
The fidelity problem
→ DNA replication is not flawless, mutations are produced (despite the proofreading system)
- Mutations can cause cancer and other diseases but also benefit to evolution
4
