Molecular Biology of the Cell
summary (exam 1+2)
1
,DNA Replication & Sequencing & PCR 3
Genomes & Omics & Bioinformatics 6
RNA Gene Expression 8
Translation & Protein Degradation 11
Gene Cloning & Manipulation 13
Transgenesis 16
Epigenetics & Chromatin 18
DNA Methylation, Gene Regulation & Genome Stability 20
Histone Modi cations 25
Histone Variants 28
Non-coding RNAs 31
Protein Synthesis, Structure & Puri cation 35
Membranes & Protein Targeting 39
Vesicular Transport 42
Cellular (Stress) Signaling & Cell Division 45
2
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,DNA Replication & Sequencing & PCR
THE CENTRAL DOGMA
- Going from the gene to function: DNA -> RNA -> protein -> metabolite -> phenotype
- Replication (DNA synthesis): DNA -> DNA
- ~ PCR, sequencing
- Transcription (RNA synthesis): DNA -> RNA
- ~ promoters, regulatory factors, splicing
- Translation (protein synthesis): RNA -> protein (made from amino acids)
- ~ RNA structure and stability, ribosomes, post-translational modi cations (=decoration of
proteins)
DNA SYNTHESIS / REPLICATION
- DNA replication is semi-conservative: every new double-stranded molecule consists of 1 old
and 1 new strand
- The old strand is the template (i.e., example) for the new strand
- 1 parental DNA double helix results in 2 identical daughter DNA double helices
- DNA replication always occurs 5’->3’; the template strand is anti-parallel (3’->5’)
- DNA synthesis is the process of the formation of phosphodiester bonds while hydrolyzing
the matching dNTP (deoxyribonucleoside triphosphate) molecule
- The energy comes from the DNA backbone: as the phosphodiester bond forms between
the 5’ phosphate group of the new nucleotide and the 3’ OH of the last nucleotide, 2
phosphates are removed providing energy for bonding
- DNA polymerase synthesizes DNA from a double-stranded ‘primer’
- DNA polymerase requires a stable primer already bound to the template strand to
synthesize DNA, since it needs a pre-existing 3’ OH to elongate the strand
- The replisome is a molecular machine with essential components for DNA replication:
- Topoisomerase: enzyme that regulates the over-/underwinding of DNA by making a nick
in the DNA, thereby releasing the tension of the coiled DNA helix
- Helicase: breaks hydrogen bonds between DNA strands, thereby separating the strands
using ATP
- Primase: RNA polymerase that synthesizes a short RNA primer that is complementary to
a single-stranded DNA template, which is needed by DNA polymerase
- After elongation of the strand, the primer is removed by a 5’ to 3’ exonuclease and
replaced by DNA
- 2 DNA polymerases: one synthesizes leading strand, while the other synthesizes the
lagging strand
- Leading strand: template (antisense) strand that goes 3’->5’
- Replication occurs continuously 5’->3’
- 1 RNA primer is made at the origin
- Lagging strand: anti-parallel template strand (coding/sense) that goes 5’->3’
- Replication occurs discontinuously 5’->3’
- Many RNA primers are required to synthesize Okazaki fragments (=small 5’->3’
DNA fragments)
- Ligase: covalently links adjacent Okazaki fragments together
MISTAKES IN DNA SYNTHESIS
- Mistakes in DNA synthesis can be restored through ‘proofreading’ activity of the DNA
polymerase complex
- Without proofreading (5’->3’ polymerization): 1 error per 105 nucleotides
- With proofreading: 1 error per 102 nucleotides
- Proofreading: DNA polymerase recognizes a mismatched nucleotide by 3’->5’
proofreading, removes it, replaces it with the correct nucleotide, and allows synthesis
to continue 5’->3’
3
fi
, - With strand-directed mismatch repair: 1 error per 103 nucleotides
- Strand-directed mismatch repair: mismatch proofreading proteins (MutS and MutL)
recognize and bind to an error in newly-made strand -> DNA scanning detects a nick
in new DNA strand -> strand removal containing the error -> repair DNA synthesis
using template
- Occurs after DNA replication
- These 3 mechanisms combined give 1 error per 1010 nucleotides
- If the error would be 0, there would be no evolution: allowing some errors is a good
thing
DNA MUTATIONS
- Chemical changes in DNA bases can cause mutation in the DNA
- Depurination: involves the removal of a purine (G/A) from the DNA, leaving only the sugar
phosphate of the nucleotide
- Results in 1 mutated daughter helix with an A-T/G-C deletion, and 1 unchanged daughter
helix
- Is the most common DNA mutation
- Deamination: involves the removal of an amino group
- Results in 1 mutated daughter helix with a change in base pair, and 1 unchanged daughter
helix
- C -> T
- Deamination of C results in a U -> the mutated daughter strand will have a G to
A conversion in the new strand while U will be repaired into a T -> C to T
conversion at the position of the deaminated C
- A -> G
- G stops replication (stutter/deletion)
DNA SEQUENCING
- DNA sequencing (whole genome) is used to determine the order of the bases A, C, G, and T
- Sanger sequencing / dideoxy sequencing: DNA synthesis while incorporating chain
terminators in separate reaction for the bases A, C, G, and T
- A chain terminator (dideoxynucleotide) lacks the 3’ OH necessary for strand elongation,
thereby preventing extension of the DNA strand, resulting in DNA fragments
- With a high concentration of all dNTPs and low concentrations of ddATP, ddCTP, ddGTP
and ddTTP, in separate reactions DNA synthesis will continue, but occasionally synthesis
will stop when a dideoxynucleotide is incorporated
- The oligonucleotide primer (needed by DNA polymerase) determines the start of the
reaction
- With 4 separate reactions (ddATP/ddCTP/ddGTP/ddTTP + DNA polymerase) and a
labelled (radioactive or uorescent) primer, fragments of di erent lengths are generated:
these fragments are separated alongside using electrophoresis, and every visible fragment
represents a termination in the DNA synthesis
- The DNA sequence is read from the bottom of the gel upward
- E ciency of manual sequencing methods:
- Radioactive nucleotides in 4 lanes: ~8000 bases per day
- Fluorescent nucleotides in 4 lanes: ~10,000 bases per day
- Fluorescent nucleotides in 1 lane: ~30,000 bases per day
- Nucleotides in capillary gel (1-96 wells): ~150,000 bases per day
- Genome sequencing / hierarchical shotgun sequencing: multiple copies of the genome are
randomly fragmented -> the nucleotide sequence of each clone is stitched together, using the
overlaps between clones as a guide
- Works well for small genomes that lack repetitive DNA
- Uses contigs: assembly of smaller DNA sequences into 1 continuous strand
- Needs markers such as restriction enzymes
4
ffi
fl ff
summary (exam 1+2)
1
,DNA Replication & Sequencing & PCR 3
Genomes & Omics & Bioinformatics 6
RNA Gene Expression 8
Translation & Protein Degradation 11
Gene Cloning & Manipulation 13
Transgenesis 16
Epigenetics & Chromatin 18
DNA Methylation, Gene Regulation & Genome Stability 20
Histone Modi cations 25
Histone Variants 28
Non-coding RNAs 31
Protein Synthesis, Structure & Puri cation 35
Membranes & Protein Targeting 39
Vesicular Transport 42
Cellular (Stress) Signaling & Cell Division 45
2
fi fi
,DNA Replication & Sequencing & PCR
THE CENTRAL DOGMA
- Going from the gene to function: DNA -> RNA -> protein -> metabolite -> phenotype
- Replication (DNA synthesis): DNA -> DNA
- ~ PCR, sequencing
- Transcription (RNA synthesis): DNA -> RNA
- ~ promoters, regulatory factors, splicing
- Translation (protein synthesis): RNA -> protein (made from amino acids)
- ~ RNA structure and stability, ribosomes, post-translational modi cations (=decoration of
proteins)
DNA SYNTHESIS / REPLICATION
- DNA replication is semi-conservative: every new double-stranded molecule consists of 1 old
and 1 new strand
- The old strand is the template (i.e., example) for the new strand
- 1 parental DNA double helix results in 2 identical daughter DNA double helices
- DNA replication always occurs 5’->3’; the template strand is anti-parallel (3’->5’)
- DNA synthesis is the process of the formation of phosphodiester bonds while hydrolyzing
the matching dNTP (deoxyribonucleoside triphosphate) molecule
- The energy comes from the DNA backbone: as the phosphodiester bond forms between
the 5’ phosphate group of the new nucleotide and the 3’ OH of the last nucleotide, 2
phosphates are removed providing energy for bonding
- DNA polymerase synthesizes DNA from a double-stranded ‘primer’
- DNA polymerase requires a stable primer already bound to the template strand to
synthesize DNA, since it needs a pre-existing 3’ OH to elongate the strand
- The replisome is a molecular machine with essential components for DNA replication:
- Topoisomerase: enzyme that regulates the over-/underwinding of DNA by making a nick
in the DNA, thereby releasing the tension of the coiled DNA helix
- Helicase: breaks hydrogen bonds between DNA strands, thereby separating the strands
using ATP
- Primase: RNA polymerase that synthesizes a short RNA primer that is complementary to
a single-stranded DNA template, which is needed by DNA polymerase
- After elongation of the strand, the primer is removed by a 5’ to 3’ exonuclease and
replaced by DNA
- 2 DNA polymerases: one synthesizes leading strand, while the other synthesizes the
lagging strand
- Leading strand: template (antisense) strand that goes 3’->5’
- Replication occurs continuously 5’->3’
- 1 RNA primer is made at the origin
- Lagging strand: anti-parallel template strand (coding/sense) that goes 5’->3’
- Replication occurs discontinuously 5’->3’
- Many RNA primers are required to synthesize Okazaki fragments (=small 5’->3’
DNA fragments)
- Ligase: covalently links adjacent Okazaki fragments together
MISTAKES IN DNA SYNTHESIS
- Mistakes in DNA synthesis can be restored through ‘proofreading’ activity of the DNA
polymerase complex
- Without proofreading (5’->3’ polymerization): 1 error per 105 nucleotides
- With proofreading: 1 error per 102 nucleotides
- Proofreading: DNA polymerase recognizes a mismatched nucleotide by 3’->5’
proofreading, removes it, replaces it with the correct nucleotide, and allows synthesis
to continue 5’->3’
3
fi
, - With strand-directed mismatch repair: 1 error per 103 nucleotides
- Strand-directed mismatch repair: mismatch proofreading proteins (MutS and MutL)
recognize and bind to an error in newly-made strand -> DNA scanning detects a nick
in new DNA strand -> strand removal containing the error -> repair DNA synthesis
using template
- Occurs after DNA replication
- These 3 mechanisms combined give 1 error per 1010 nucleotides
- If the error would be 0, there would be no evolution: allowing some errors is a good
thing
DNA MUTATIONS
- Chemical changes in DNA bases can cause mutation in the DNA
- Depurination: involves the removal of a purine (G/A) from the DNA, leaving only the sugar
phosphate of the nucleotide
- Results in 1 mutated daughter helix with an A-T/G-C deletion, and 1 unchanged daughter
helix
- Is the most common DNA mutation
- Deamination: involves the removal of an amino group
- Results in 1 mutated daughter helix with a change in base pair, and 1 unchanged daughter
helix
- C -> T
- Deamination of C results in a U -> the mutated daughter strand will have a G to
A conversion in the new strand while U will be repaired into a T -> C to T
conversion at the position of the deaminated C
- A -> G
- G stops replication (stutter/deletion)
DNA SEQUENCING
- DNA sequencing (whole genome) is used to determine the order of the bases A, C, G, and T
- Sanger sequencing / dideoxy sequencing: DNA synthesis while incorporating chain
terminators in separate reaction for the bases A, C, G, and T
- A chain terminator (dideoxynucleotide) lacks the 3’ OH necessary for strand elongation,
thereby preventing extension of the DNA strand, resulting in DNA fragments
- With a high concentration of all dNTPs and low concentrations of ddATP, ddCTP, ddGTP
and ddTTP, in separate reactions DNA synthesis will continue, but occasionally synthesis
will stop when a dideoxynucleotide is incorporated
- The oligonucleotide primer (needed by DNA polymerase) determines the start of the
reaction
- With 4 separate reactions (ddATP/ddCTP/ddGTP/ddTTP + DNA polymerase) and a
labelled (radioactive or uorescent) primer, fragments of di erent lengths are generated:
these fragments are separated alongside using electrophoresis, and every visible fragment
represents a termination in the DNA synthesis
- The DNA sequence is read from the bottom of the gel upward
- E ciency of manual sequencing methods:
- Radioactive nucleotides in 4 lanes: ~8000 bases per day
- Fluorescent nucleotides in 4 lanes: ~10,000 bases per day
- Fluorescent nucleotides in 1 lane: ~30,000 bases per day
- Nucleotides in capillary gel (1-96 wells): ~150,000 bases per day
- Genome sequencing / hierarchical shotgun sequencing: multiple copies of the genome are
randomly fragmented -> the nucleotide sequence of each clone is stitched together, using the
overlaps between clones as a guide
- Works well for small genomes that lack repetitive DNA
- Uses contigs: assembly of smaller DNA sequences into 1 continuous strand
- Needs markers such as restriction enzymes
4
ffi
fl ff
