Short summary of the information given in the Molecular Genetics course. Together with my longer summary I was able to get a 9.0 for my exam. This summary mainly contains the pathways and steps that are important.
Most Important information
Meselson-Stahl experiment
N15: heavier and thus this DNA strand will have a higher
density when incorporated. It was indeed incorporated
in the DNA strand
Then this DNA strand was put in another medium with
N14, these mixed and created an ‘intermediate’ density
gradient.
Initiation of replication in e.coli:
- oriC
- fully methylated by Dam methylase
- 6 proteins:
- DnaA: this is the initiator protein. It is an ATP-binding protein, and it is activated
when it is bound to ATP. DnaA-ATP binds in the fully methylated oriC. First in the
high affinity sites, then the DNA wraps around DnaA and it binds to the low affinity
sites that are AT-rich (easy to separate). It twists and melts the helix with the help of
HU
- Two DnaB/DnaC complexes. DnaB is an ATP-hydrolysis-dependent 5’-3’ helicase. It
unwinds the DNA by breaking the hydrogen bonds between the nucleotides. DnaC is
a chaperone. They form the two replication forks.
- Gyrase (Type II topoisomerase) relaxes DNA supercoils
- SSB (single-strand binding proteins) stabilize DNA, keep the replication bubble open,
protect against degradation of ssDNA from ss-specific-nucleases.
Joining of okazaki fragments
e.coli
DNA polymerase III has only 3’-5’ exonuclease activity. It stops synthesizing DNA when
it finds a primer
DNA polymerase I has both exonuclease activities. It degrades the primer with the 5’-3’
exonuclease activity and synthesizes new complementary DNA simultaneously
DNA ligase ligates the adjacent fragments
Eukaryotes
Two-step process:
DNA polymerase delta and helicase displace the primer, creating a 5’ flap.
Simultaneously the DNA polymerase fills the gap
Flap endonuclease I (FEN1) cleaves the flap, removing the primer
DNA ligase ligates the adjacent fragments
Replisome:
- 2x DNA polymerase (for lagging and leading strand)
- 2x dimerizing subunit T that link DNA pol. together
- 2x sliding clamp from B-rings that encircles the DNA and assures contact between
DNA and DNA polymerase
- Clamp loader (group of proteins) places the clamp on DNA, keeping all of the
structure together
The end problem
1
, a. Final Okazaki fragment cannot be primed, because primase doesn’t have space to
add a primer
b. The primer of the last Okazaki fragment is at the very last 3’ extreme, so it cannot be
removed and synthesized again by DNA polymerase
Spontaneous mutations:
Point mutations
- Transitions
- transversions
Insertions/deletions
- Can lead to frameshift mutation
Replication slippage
- Micro/minisatellites
Tautomerization
Induced mutations
Chemicals
- Base analogs
5-bromouracil is an analog of thymine
- Deaminating agents
Nitrous acid removes the amine of C, so it becomes a U
- Alkylating agents
Methylation of G makes it bp with T not C
- Intercalating agents
Ethidium bromide intercalates which makes DNA polymerase to add or pass
over some nucleotides
Physical agents
- Ionizing radiation
- UV
Causes pyrimidine dimers
- Heat
Causes break of glycosidic bonds, so there is an AP-site
BER
Glycosylase > AP-site
Long-patch pathway
- Endonuclease APE1 cleaves on the 5’ side of the AP site
- Replication complex with DNA polymerase synthesizes 2-10 nt in the 5’-3’ direction
creating a 5’ flap
- FEN1 (flap endonuclease) removes the displaced DNA
- Ligase seals the nick
Short-patch pathway
- Lyase breaks the sugar ring creating a nick on the 3’ side of the AP site
- APE1 and DNA polymerase replace a single nucleotide
- Ligase seals the nick
NER
2
, UvrABC system
1. Recognition step: the UvrAB complex recognizes the damage and binds to DNA
2. Incision step: then, UvrA dissociates and UrvC joins, creating the UvrCB complex
that cleaves on each side of the damage
3. Excision step: helicase UvrD removes the damaged DNA sequence
4. Gap-filling step: DNA polymerase synthesizes the replacement DNA and DNA
ligase seals the nick
Recognition step in eukaryotes
1. Global Genome Repair: XPC protein recognizes the damage
2. Transcription-coupled repair: RNA polymerase recognizes the damage during
transcription
MMR – Mut system e.coli
1. MutS dimer recognizes and binds to the mismatch
2. MutL dimer binds to MutS
3. MutS translocates the DNA until a GATC site is encountered, creating a loop in
the DNA
4. MutH endonuclease joins MutSL and cleaves the unmethylated strand
5. Cleaved DNA is excised by an exonuclease or/and helicase from the GATC to the
mismatch site
6. DNA polymerase synthesizes a new strand and DNA ligase seals the nick
SOS response E.coli
- LexA is a repressor of many genes involved in DNA repair
- When severe damage in the cell induces RecA, RecA triggers the cleavage of LexA
and several genes involved in DNA repair systems are expressed and repair the
damage
Homologous recombination
1. DSB initiates recombination (DSB can be spontaneous or induced)
2. Exonuclease (5’-3’ activity): degradation of the 5’ ends. It creates 3’ overhangs
3. Single strand invasion of one 3’ end into the homologous chromosome, it forms a D-loop
structure
4. Heteroduplex is formed. The recombinant joint moves with Branch migration (energetically
neutral)
5. Extension of the 3’ end by DNA polymerase
6. Displaced D-loop pairs with the other strand and DNA polymerase fills the gap
7. The free 5’ end performs a second single strand invasion, creating a second recombinant
joint
8. DNA ligase seals the nicks generating 4 complete DNA strands and 2 Holliday junctions
9. Resolution of the Holliday junctions is a critical step in HR
Two possible outcomes:
- Non-crossover DNA: both cut in the same axis
- Crossover recombinant DNA: cut in different axis
RecBCD in E.coli
3
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