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3. DNA REPLICATION
see CHAPTER 12 of GENOMES by T.A. Brown
or
CHAPTER 13 of GENOMES 2nd edition by T.A. Brown
General aspects :
Concepts
- duplication of entire genome, each time the cell divides
- double-helix model of Watson & Crick suggested a mode of DNA replication
=> subsequently confirmed by the experiment of Meselsohn & Stahl
- topological problem: solved by the discovery of topoisomerases :
=> removal of torsional stress ahead of the replication fork
- well-defined origins of replication in bacteria, plasmids, yeast ; less so in
higher organisms
- distinction between leading and lagging strand during replication
=> leading strand is copied continuously
=> lagging strand is copied discontinuously => Okazaki fragments
- strand synthesis requires a primer
- multiple enzymes involved at the replication fork
- termination only partially understood ;
special mechanisms required with linear molecules
- cell cycle checkpoints to coordinate DNA replication with cell division
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DNA replication
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The topological problem
Unzipping the helix?
leading and lagging strand synthesis
=> replication fork
=> uni-directional progress
=> bi-directional progress
unzipping creates superhelicity ahead of the fork : removal required
=> enzymes found more than 25 years later : cleavage and resealing
Topoisomerases (IA, IB, II)
- IA : breakage in one strand : one rotation of the broken strand : L = 1
- IB : similar as IA but mechanisms differ by details (probably separate evolution)
- II : break in both strands: entire helix passes through the ‘gate’ : L = 2
E. coli topoisomerases I and III :
type IA
E. coli topoisomerases II (DNA gyrase) :
II
E. coli topoisomerases IV :
II
Eukaryotic topoisomerase I :
IB
Yeast and human topoisomerase III :
IA
Archaeal reverse gyrase :
IA
Eukaryotic topoisomerases II and IV :
II
- involved in many cellular processes (a.o. transcription, replication, recombination)
- at the fork: removal of positive superhelicity that is produced by rotation
front of the fork
- resolving catenanes that may otherwise remain entangled at the end of
replication of circular molecules
G. Volckaert
DNA replication
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The Meselsohn-Stahl experiment (1958)
- dispersive (Delbrück) versus semi-conservative (Watson-Crick) versus
conservative replication
-
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N versus 14N
- E. coli cell culture fully grown in “heavy” NH4Cl
- start by switching to “normal” growth medium (time = 0)
- samples at 20 min, 40 min, etc. (cell divisions)
- DNA analysis by CsCl density gradient centrifugation
- after one cell division: all DNA having the same density
=> no conservative replication
- after second division: two specific bands
=> only consistent with semi-conservative mechanism
Variations
Displacement replication
- e.g. human mitochondrial DNA
- one strand replicated, the second strand (circle) remains uncopied and is
copied after completion of the first strand
Rolling-circle replication
- many bacteriophages
- nicking the ds and copying one strand by elongation of the 3’-end
- the second strand stepwise converted to ds, and unit-length circularized
- may lead to ss products if the second strand remains protected
by single-stranded DNA binding protein
G. Volckaert
DNA replication
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The replication process :
Basic aspects : initiation, elongation, termination, regulation
two complications :
=> different polarity of the single strands :
=> lagging strand to be replicated discontinuously
=> requirement of a primer : no DNA polymerase is able to start de novo
Initiation
the ‘origin’
- E. coli genome : oriC
- about 245 bp
- three copies of 13-nt motif (direct repeats) + five copies of 9-nt motif
- DnaA binding sites in 9-nt motifs: non-stoechiometrically
(up to 30 molecules bound)
- DnaA complex leads to melting of 13-nt repeat region
- negative superhelicity is required
- next, DnaBC attaches to the melted region => prepriming complex
DnaC is soon released
DnaB is a helicase that starts unzipping the helix
- S. cerevisiae : ARS (autonomously replicating sequence) (one ori per 40 kb?)
- multiple ARS per chromosome (CEN is not ori !!!)
- ARS less than 200 bp : 4 subdomains are recognized
=> A + B1 : origin recognition sequence
=> B2 : melted region (as the 13-nt repeat in E.coli)
=> B3 : attachment of ABF1 (ARS binding factor 1)
=> causes torsional stress to induce melting
- then binding of helicase
- looks a bit similar to E. coli oriC but nevertheless quite different
G. Volckaert
DNA replication
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- replication origins in higher eukaryotes
- apparently difficult to isolate, if any specific origins exist
- probably an 8 kb region involved with primary (high frequency sites) and
secondary (low frequency sites) involved
- still much to be discovered
- ori‘s spaced about 100-200 kb ?
- plasmids:
- ColE1 and analogues : ori about 600 bp
- RNA primer (RNAII) by transcription from a regular promoter
- regulation of priming events by RNAI and Rop protein: copy number
- processing of transcript by RNaseH
- pSC101 (and many broad host range plasmids have similar ori’s)
- about 300 bp
- DnaA binding site, 3 iteron sequences of about 27 bp
- RepA is the only plasmid-encoded protein required for replication
- RepA binds to the iterons and initiates DNA synthesis
- copy number control
- autoregulation of RepA synthesis (RepA binds to its own promoter)
- handcuffing: RepA binds inverted repeats (IR1 & IR2) of 2 plasmid molecules
- par site for partitioning of the plasmid copies over the daugther cells
- viruses
- Ff-bacteriophage
- transcription initiation for copying of ssDNA genomic DNA
- gene II product for nicking of the ds RF DNA (ds => ds)
- phage lambda
- early & late replication :  mechanism (early),  mechanism (late)
G. Volckaert
DNA replication
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The fork :
- elongation : leading and lagging strand
- melting (see above : initiation)
- + helicase => pre-priming complex (DnaB on lagging strand)
(Rep & PriA on leading strand, or only in  replication)
- + primase => primosome
- + SSB proteins : 1000-2000 nt replication on leading strand
then: start of discontinuous strand synthesis on lagging strand
- DNA polymerase III : 2 copies except  complex ( +  + ’ +  + )
forms replisome
 complex interacts with  subunit (attaches & detaches repeatedly in
discontinuous synthesis)
in eukaryotes, PCNA is functional equivalent of  subunit in E. coli
(proliferating cell nuclear antigen)
- after passage of the replisome : finalization by joining of Okazaki
fragments: removal of RNA primers / fill-in / ligate
for this purpose, pol III is released and replaced by pol I
- leading strand : synthesized continuously after priming
lagging strand : repeated process with start of every new Okazaki fragment
- Okazaki fragments
- in prokaryotes : size of about 1000 bp
- in eukaryotes : size of about 200 bp
- growth & division
- prokaryotes grow and divide at rates depending on culturing conditions.
rate of chromosome replication is relatively constant
=> fast growth : initiation of new rounds before the first doubling is complete
=> slow growth : intervals between doublings
- eukaryotes : DNA synthesis in a distinct portion of the cell cycle : S phase
- primers : prokaryotes : primase (DnaG) provides starter of 4-15 nt in length
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eukaryotes :
primase is tightly bound to polymerase 
primase makes starter of 8-12 nt,
then polymerase  continues by adding 20 deoxynucleotides
then polymerase  continues
- enzymes and proteins
- DNA polymerases
E. coli : pol III , pol I, (pol II, pol IV, pol V in repair of damage)
Eukaryotes : polymerase , , ,  (detection DNA damage),
               , , , , , 
n repair
mitochondrion : polymerase , encoded in nuclear DNA
polymerase  : detection of damaged DNA (compare to pol I)
- exonuclease activities :
pol I : proofreading + 5’-3’exo
pol III : only proofreading
polymerase ,  : none
polymerase , ,  : proofreading
- helicases : DnaB is main (5’=>3’) helicase (lagging strand template) in
replication but there are at least 10 other helicases in E. coli
(reflecting diversity of functions where DNA unwinding is necessary?)
they - bind ssDNA
- migrate along the strand in either 5’ or 3’ direction
- require energy : hydrolysis of ATP
Rep : 3’=>5’ helicase involved in  replication (only?)
- ssDNA binding proteins (SSB)
binds ssDNA following unwinding by helicase
- inhibits base-pairing to reform
- protects against nuclease action
- genes : see table
G. Volckaert
DNA replication
12/02/2016
8
Termination
- in E. coli
circular DNA : replication bidirectionally
termination within a defined region : 7 sequences defined
interaction with Tus (DNA-binding protein)
Tus operates (blocks helicase DnaB from progressing) direction-specific
- in eukaryotes
linear DNA’s : unknown how mechanism of termination functions
no Tus equivalent found
forks of replication loops meet and strands ligate?
- terminations in lagging strand (polymerases have no 5’=>3’ exo-activity)
flap endonuclease (FEN1) : two models
- displacement activity and cleavage by FEN1 at the branch point
- RNase H removes RNA primer ; FEN1 removes last ribonucleotide
and some deoxynucleotides
- in both models : ligase seals the ends
- telomerase
how the size of the (repetitive) telomeric DNA is maintained?
Regulation
The cell cycle
M phase : nucleus and cell divide
G1 phase (gap 1) : interval between M and S (transcription, translation, etc)
S phase : DNA synthesis ; genome is replicated
G2 phase (gap 2) : interval between S and M
Checkpoints
G. Volckaert
DNA replication
12/02/2016