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Summary Molecular Genetics (WBBY008-05)
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Lecture 1 Genome ReplicationSemiconservative is how it actually works.
But: 2 topological problems:
➢ How to pull the two polynucleotides of the parent helix apart? Helicases
➢ How to unwind the helix (without pulling it into a knot)? → Topoisomerases
Meselson-Stahl experiment: proved that replication is semiconservative.
They grew E. coli in a 15NH4Cl medium → transferred part of the culture to a 14NH4Cl medium
➔ After 20 min → 1 cell division → extract DNA → density gradient centrifugation → 1 DNA band, this DNA
contains equal amounts of newly synthesized (14) and parental DNA (15).
➔ After 40 min → 2 cell divisions → extract DNA → density gradient centrifugation → 2 DNA bands, this DNA
contains equal amounts of newly synthesized (14) and parental DNA (15).
Topoisomerases: prevent DNA overwinding during replication.
➢ Topoisomerase I: cuts one strand of the double helix, so that strand rotate automatically, because of the
stress. The rotation energy originates from the tension in the DNA itself. The energy of the phosphodiester
bond is maintained.
Summary: single-strand break and doesn’t need ATP.
- Topoisomerase IA: uses the single-strand break as a gate through which it passes the uncut
polynucleotide. The 2 ends of the broken strand are then religated. This mode of action results in the
Linking number (the number of times one strand crosses the other in a circular molecule): down by 1
- Topoisomerase IB: acts as a molecular rotor, relaxing the torsional stress in an overwound helix by
,Displacement replication: a variance of DNA copying via a replication fork, it’s used by some smaller circular
molecules. The point at which replication begins is marked by a D-loop: a region of ~500 bp where the double helix is
disrupted by the presence of an RNA molecule base paired to one of the DNA strands.
This RNA molecule acts as the starting point for synthesis.
After completion of first-strand synthesis, a second RNA primer attaches to the displaced strand and initiates
replication of this molecule.
Rolling-circle replication: a special type of displacement replication, used by certain bacteriophages and plasmids.
It’s an efficient mechanism for rapid synthesis of multiple copies of a circular genome.
It initiates at a nick made in one of the parent polynucleotides. The free 3’-end that results is extended, displacing
the 5’-end of the polynucleotide. Continued DNA synthesis rolls off a complete copy of the genome.
INITIATION OF DNA REPLICATION
Initiation of bidirectional genome replication:
Replication ‘’machine is assembled at ‘’replication forks’’.
Starting position is NOT random but at ‘’origin of replication’’.
➢ Circular bacterial genome: 1 origin of replication, 2 replication forks.
➢ Eukaryotic genome: multiple origins of replication.
INITIATION OF REPLICATION IN E. COLI:
oriC: origin of replication in E. coli. ~245 bp.
Red: high affinity DnaA binding sites of oriC → are always occupied.
Blue: other (low affinity) DnaA sites of oriC → are filled just prior to replication.
Green: DNA unwinding element (DUE): AT-rich part of oriC.
The high-affinity sites of the replication origin are permanently occupied by DnaA proteins, while the other sites are
filled immediately before replication begins.
DnaA binds and forms a barrel → opening (melting) of the double helix of the AT-rich DUE with the help of HU
proteins → allows the attachment of prepriming complex BC (= DnaB + DnaC)
➢ DnaC ‘’helps’’ DnaB to attach to DUE
,2 limitations of DNA synthesis by DNA polymerase:
1) It can only synthesize DNA in the 5’ → 3’ direction.
2) It needs a primer to initiate the synthesis of a new polynucleotide.
In eukaryotes, the primase forms a complex with DNA polymerase α, which is shown synthesizing the RNA
primer followed by the first few nucleotides of DNA. Then it’s taken over by DNA polymerase ε or δ.
Primase makes an RNA primer of ~10 nt.
NB: primase is NOT involved in transcription, it’s just involved in making RNA primers needed for replication.
Okazaki fragments:
Lagging strand: strand with Okazaki fragments (discontinuous copying).
Leading strand: other strand (continuous copying).
Single-strand binding protein (SSB): bind to phosphate backbone forming a channel
to:
➢ Prevent ssDNA from re-associating.
➢ Protect against degradation by e.g. single-strand-specific nucleases.
They’re obtained by helicase.
Parallel synthesis of DNA leading- and lagging-strands:
The E. coli model:
DNA synthesis is done by 2 DNA polymerase III molecules linked together (dimer).
One DNA polymerase III copies leading strand, the other copies lagging strand.
Lagging strand spools through its DNA polymerase III.
→ 1 dimer can replicate both strands at the same time.
A few proteins are involved in attaching and detaching of the DNA polymerase from the
template. (Primarily during lagging strands replication):
➢ ‘’Clamp loader’’
➢ ‘’Sliding clamp’’ (binds the DNA polymerase to the DNA)
, TERMINATION OF REPLICATION IN E. COLI:
Genomes are replicated bidirectional, so the fork meets the other side at some point. This should be exactly
opposite to the origin of replication, but this doesn’t always happen.
Replication is faster than transcription. So the replication fork pauses behind a RNA polymerase that is transcribing a
gene, until it’s done transcribing. So if at one side there are more genes being transcribed than at the other side, the
fork doesn’t meet the other side exactly in the middle.
This is not allowed to happen because of the presence of terminator sequences (they prevent fork ‘’overshoot’’).
These act as recognition sites for a sequence-specific DNA-binding protein called the terminator utilization
substance (Tus).
Tus protein: is a sequence-specific DNA binding protein. When Tus is bound to a terminator sequence, it allows a
replication fork to pass if the fork is moving in one direction, but blocks if the fork is moving in the opposite
direction. So it allows passage of replication fork only in 1 direction.
Magnetic tweezers: they use magnetic tweezers to examine the role of the Tus protein. The magnetic tweezers
enable the magnetic bead to be manipulated so that the 2 polynucleotides are pulled apart.
The resulting replication fork is able to pass a bound Tus protein only when approaching from the permissive
direction. As none of the proteins of the replisome are present, the conclusion is that interactions between Tus and
the DNA being replicated are at least partly responsible for the ability of Tus to block progression of the replication
fork.
TERMINATION OF REPLICATION IN EUKARYOTES:
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