AMG2.5 Klein Replication 2018

Replication: The unit of heredity is DNA (or RNA) 
Richard Dawkins: Unit of heredity: DNA (RNA)

Evolution and selection work on replicators = alleles of genes. Individuals (vehicles) come and go – but it is the alleles whose alterations remain.
What is the unit of selection? Individuals or alleles? Alleles are the encoded information that are copied.
Key criterium (of heredity): If a replicator is altered, the alteration will be passed on (forever, or until the next alteration).
Only DNA/RNA fit that definition. Any other cellular component does not have the property, that its alterations will be copied endlessly in the
following generations. (Epigenetics is not generally an exception – its modifications are largely erased in the germline, if not usually fade away
over several generations).
“collapsed” DNA in living cells left handed

right handed right handed role in living cell obscure
role in living cell obscure
Information storage in “the old days” had a big problem. 
Its name was “Oxidative Deamination”
Bases Considering that C should basepair (code for) with G,

but Uracil with Adenine!!
Oxidative
deamination
Hypoxanthine
An engineer would have solved the problem once and for all by
replacing Cytosin with something more stable. But evolution is a
tinkerer …..
It replaced Uracil with something not to be mixed up with the

deamination product of Cytosine.
THUS … the deamination product (Uracil) can be eliminated and
replaced by a repair pathway (Mismatch directed Uracil
degylcosylation)
Klein - AMG 2.5 Replication

Thymine is a Uracil with a Methyl-flag, so it can’t be made  
from Cytosine by Oxidative Deamination
Bases Nukleosides
N N
N N

How did DNA get into the world and how
Thymidine?
By RNR and Thymidilate Synthase

“Frozen accidents” (Francis Crick) – errors “too big to be corrected” help in
reconstruction of early evolution, perhaps even pre-biotic evolution
Billion years
The concept of “Frozen accidents” (Francis Crick) I
It seems that life rarely moves back to correct earlier mistakes. Rather, it repeats the mistakes over and over again, and invents
subsequent patches to make “life easier”…..
We store our genome as DNA – but our pathways make ribonucleotides first – converting each one into deoxynucleotides in a subsequent
step.
An RNA polymerase made from RNA alone
The final evolved ribozyme contains ~200 nucleotides and catalyses extension of an RNA primer on an external RNA template, adding up to 14
successive nucleotides in 24 hours. It is general with respect to the template sequence, yet operates with an average fidelity of ~97% per
nucleotide in copying the template sequence to that of a complementary product.
Johnston, W. K., Unrau, P. J., Lawrence, M. S., Glasner, M. E. & Bartel, D. P. RNA-catalyzed RNA polymerization: accurate and general RNA-templated primer extension. Science
292, 1319–1325 (2001).
Ribo- & Deoxyribonuleotides
Ribonucleotide
Deoxyribonucleotide
RNR, ribonucleotide reductase
Deoxyribonucleosides are made from Ribonucleoside precursors
“Frozen accidents” (Francis Crick) – errors “too big to be corrected” help in
reconstruction of early evolution, perhaps even pre-biotic evolution
Billion years
The concept of “Frozen accidents” (Francis Crick) II
It seems that life rarely moves back to correct earlier mistakes. Rather, it repeats the mistakes over
and over again, and invents subsequent patches to cope with the mistakes …..
Our pathways make ribonucleotides first – converting each one into deoxynucleotides in a subsequent
step.
BUT worse than that ….
In order to make Deoxythymidin we make Deoxyuracil first! By subsequently adding a methyl-tag.

Thymidine is a Uridine modified by a methyl group
dUTP dTTP
Thymidylate Synthase
+ +
N5, N10-Methylen-THF (Tetrahydrofolic acid) DHF (Dihydrofolic acid)

“Molecular fossils” suggest:  
RNA and DNA worlds may have had two stages each
RNA alone can be a polymerase

In vitro evolved ribozyme catalyses extension of an RNA primer
The three-dimensional on an external RNA template, adding 14 successive nucleotides
structure of the original in 24 hours. (Science, 2001)
ribozyme, the self-splicing Collaboration with proteins:
intron of Tetrahymena (13). The large subunit of the
Green and blue ribbons RIBOSOME. Starburst: The
RNA world active site for protein
indicate the path of the
RNA backbone in the two synthesis - consists of RNA
major domains of the RNA, Two ages of the RNA world (white!), not protein (orange)
and the red star marks the invention of translation
active site.
From an RNA/peptide world to an RNA/protein world

(partially single stranded, limited
stability & limited catalytic capacity) (greatly expanded catalytic capacity)
DNA world RNR: invention of ribonucleotide reductase

(greatly expanded stability)
Bacteriophages PBS2 & PBS1 invention of thymidylate synthase (further expanded stability)
(on Bacillus subtilis) have U-DNA.
To survive the T-DNA
environment of their host they
From a U-DNA world to the T-DNA world
developed an inhibitor against
Uracil-deglycosylase.

The free phosphate is on the 5'-C of deoxyribose  
as a remnant from the trinucleotide
Jeder DNA-Einzelstrang
besitzt 5‘- und 3‘- Ende
Normalerweise traegt das freie

5‘- Ende einen Phosphatrest

G-C pairs form three, A-T pairs form only two hydrogen bridges
Watson und Crick Paarung der Basen
G-C Paare bilden 3 Wasserstoffbrücken aus

und sind somit thermodynamisch stabiler als
A-T Paare

DNA polymerases require free 3' ends and a template

DNA replication of the E. coli chromosome

Most DNA replication is bidirectional  
(pulse - chase experiment)

but: E. coli plasmid ColE1 replicates uni-directionally
3 4
1 constant distance
from restriction site
8
5
6 7
one end moves

Replication of the E. coli chromosome,  
Termination sequences (one-sided) avoid fork collisions
Lewin, Genes VII

Phages can switch between θ (τηετα) and "rolling circle" replication
Rolling Circle Replication

Technical application of rolling circle amplification:  
Detection of single RNA molecules in situ (in a fixed cell)
RCA: Rolling Circle Amplification
RCP: Rolling Circle Product
Larsson et al. nature methods | VOL.7 NO.5 | MAY 2010 | 395

Semiconservative Replication:  
Meselson & Stahl
Semikonservative  
Replikation  
sollte dabei
nur 2 Typen 
von Dichten 
DNA ca 1% dichter 
als normal erzeugen
Intermediär (links)  
und
normal (rechts)
Die beiden
gezeigten   Dies ist 
Für eine Strukturen   Um zu   auch 
Generation könnten unterscheiden   der Fall
in 14N
für solch eine ist eine weitere  
wachsen Generation in  
E. coli für mehrere lassen intermediäre 14N nötig Nicht  
Generationen in 15N DNA Dichte
wachsen lassen prozessive 
verantwortlich
Replikation,
sein
Oder DNA  
Rearrangement 
in der 
Interphase würde 
nur einen Typ mit 
ca 25% dichterer 
DNA hervorbringen
Dies ist 
nicht der Fall
Semiconservative Replication  
(because each daughter DNA molecule is one-half "old" and one-half "new".)
Meselson& Stahl grew E. coli in a medium using ammonium ions (NH4+) as the source of nitrogen for DNA
(as well as protein) synthesi s. 14N is the common isotope of nitrogen, but they could also use ammonium
ions that were enriched for a rare heavy isotope of nitrogen, 15N. After growing E. coli for several
generations in a medium containing 15NH4+, they found that the DNA of the cells was heavier than normal
because of the 15N atoms in it.
The difference could be detected by extracting DNA from the E. coli cells and spinning it in an
ultracentrifuge. The density of the DNA determines where it accumulates in the tube.
The DNA in this new generation of cells was exactly intermediate in density between that of the previous
generation and the normal. This tells us that half the nitrogen atoms in the new DNA are 14N and half are
15N.
However, when the bacteria were allowed to divide again in normal ammonium ions (14NH4+), two distinct
densities of DNA were formed:
* half the DNA was normal and * half was intermediate.
Several generations in 15N

Then one generation in 14N
or two generations in 14N

Initiation of DNA-Replication consists of 4 steps
The initiation process can generally be considered to

involve four steps:
•  Ori-Recognition (binding of ORC to the origin)
•  Melting
•  Unwinding (which requires helicase activity)
•  Recruitment of replication factors, such as DNA
polymerases and single-stranded DNA-binding
proteins (SSBs).
The cellular initiator proteins from both eukaryotes and

prokaryotes carry out a subset of these functions.
Some viral initiators, such as E1 and T-antigen (T-
ag), carry out all of these functions.

Simplified overview of bacterial replication, concept of lagging strand

Properties of 3 E. coli DNA polymerases (of 6)
special processive
forces workhorse
E. coli I II III
Polymerization: 5' + + +
Exonuclease activity:
3' + + +
5' + - -
Synthesis from:
Intact DNA - - -
Primed single strands + - -
Primed single strands plus single-strand-binding protein + - +
In vitro chain elongation rate (nucleotides per minute) 600 ? 30,000
Molecules present per cell 400 ? 10-20
Mutation lethal? + - +

Proofreading by DNA polymerases corrects copying errors
E. coli

Schematic model of the proofreading function of a DNA polymerase

Simultaneous synthesis of leading and lagging strand from a single DNA-polymerase
complex
(DnaG)

The processivity of DNA polymerase is increased  
by a β-subunit dimer “β-clamp” or “sliding-clamp”

Prokaryotic replication (2:30...)
Eukaryotic replication
Example (eukaryotic): Lagging strand synthesis, lagging strand Polymerase
handed over when Okazaki fragment detaches with PCNA
Primase
Pol∂ is handed
over to new
primer
PCNA
detaches
new PCNA
loading
(http://www.dnalc.org/resources/3d/04-mechanism-of-replication-advanced.html)

Example (eukaryotic): Lagging strand synthesis,  
emerging new Okazaki fragment loop
Pol∂ is handed
over to new
primer
PCNA
detaches

The hare and the hedgehog: How to be faster, if you are slower
Eukaryotes Prokaryotes
Eukaryotes
Prokaryotes
The E. coli genome contains 4.7 x 106 bp.

DNA replication begins at the replication origin.
Rate: up to 1000 nuc/sec duration ca 40 min.
Error rate: Ca 1/ 109 nucleotides. Thus „normally“ error free.
Eukaryotes
The human genome contains ca 2900 x 106 bp.

The average human chromosome contains 150 x 106 bp.
Rate: 50 nuc/sec duration ca 60 min.
The process would take a month with a single origin. Replication begins at some replication
origins earlier in S phase than at others, but the process is completed for all by the end of S
phase. As replication nears completion, "bubbles" of newly replicated DNA meet and fuse, finally
forming two new molecules.

Early, late and dormant origins can be activated in the emergency case
Zegerman, J.F.X. Diffley / DNA Repair 8 (2009)

Alpha and Delta are the replicative DNA polymerases of Eukaryotes
Additional polymerases serve functions in DNA repair (error-

prone polymerases)

Properties of mammalian DNA polymerases
Mamalian Cells α β γ δ ε
Plymerization: 5'→ 3' + + + + +
Exonuclease proofreading activity: 3 → 5' - - + + +
Synthesis from:
RNA primer + - - + ?
DNA primer + + + + +
Associated DNA primase + - - - -
Sensitive to aphidicolin (inhibitor of cell DNA synthesis) + - - + +
Cell location:
Nuclei + + - + +
Mitochondira - - + - -

How is DNA Replication initiated,  
coordinated with the cell cycle 
and limited to complete rounds of synthesis 
in Eukaryotes ?

Control of Replication in Eukaryotes 
How is a single and complete round of replication achieved?
Question: What happens, if a cell in G2 is fused with a cell in S-phase?
Result: The DNA of the S-phase nucleus replicates normally, but the DNA of the G2 nucleus does not start to
replicate!
Conclusion: A) The regulation is not a diffusible factor, but a state of the nucleus and the chromosomes.
B) Freshly-synthesized DNA requires passage through mitosis to be replicated again.
Cyclins: Cln1-3 Clb5,6 Clb1,2,3,4
CDK: Cyclin Dependent Kinase: Cyclin + Kinase

Kinase: Cdc28(S. cerevisiae) or Cdc2(S. pombe & everywhere else)

Two Ubiquitin-ligases, the SCF and the APC master regulate the cell cycle
G1 S G2

The Problem: Many origins of replication are activated at

different times during S-phase, some are not activated at all. 
If only one of them fires twice, the genome would be unevenly
duplicated leading most likely to death.
The Solution:  
Keep two activities separate: 
 
In G1 - defined by low S-Cdk activity, build up the "preRC" (pre
recombination complex - essentially deposit the helicase). 
 
In S - above a S-Cdk activity threshold, incativate preRC formation. But only
in S the helicase can be activated and start replication. Replication removes  
all inactive preRC when it passes them.

Think of a casino, how do they make sure betting is only once and
comes before rolling the ivory ball?
Rien ne vas plus! - This separates the deposition phase (betting)

from the evaluation phase.
Clb5, Clb6
Prepare, Execute, Allow preparation,
but dont execute  but dont Prepare again
"deposit helicase"
Rien ne vas plus for deposition!

Now fire Origins.
The License to replicate 
 
(ORC) Origin Recognition Complex of proteins. These remain on the DNA throughout replication. 
Licensing factors include CDC-6 and CDT-1, which bind to the ORC during G1 (at low S-phase-CDK
activity) and load MCM2-7. Only DNA coated with the MCM helicase during G1 can be replicated. 
 
After S-phase starts CDC-6 and CDT-1 leave the ORCs (CDT-1 is destroyed) and the MCM proteins leave
in front of the advancing replication fork.

Cdt1 proteolysis pathways.
Two major pathways target Cdt1 for destruction :
Cul4–Ddb1Cdt2 and SCFSkp2 E3 ubiquitin ligases target Cdt1 for

degradation.
The PIP box (PCNA interaction) of Cdt1 is indicated in red.

(A) Cul4–Ddb1Cdt2-dependent Cdt1 destruction
occurs in two steps. First, Cdt1 docks onto PCNA at DNA
replication forks or sites of DNA damage. Second, Cdt2 interacts
with the complex of Cdt1 and PCNA and ubiquitylates Cdt1.
(B) SCFSkp2-dependent Cdt1 destruction also occurs in two steps. First,

CDK phosphorylates Thr 29 (in humans). Second SCFSkp2 binds to the
phoshporylated T29 via Skp2 and ubiquitin tranfer occurs
Think of a casino, how do they make sure betting is only once and
comes before rolling the ivory ball?
Rien ne vas plus! - This separates the deposition phase (betting)

from the evaluation phase.
Clb5, Clb6
Prepare, Execute, Allow preparation,
but dont execute but dont Prepare again
Geminin: Additional negative control of replication  

(only in higher eukaryotes) 
 
In G2  
Geminin prevents re-assembly of MCM proteins on fresh DNA by binding Cdt1
and inhibiting it. Geminin is degraded during the mitotic cell division
upon ubiquitination through the APC to allow response to licensing factors
in the next S phase.
Lee et al Nature 2004

Pre-replicative Complex Formation
( )
A model for pre-replicative complex formation in eukaryotes.

The apparent overabundance of Mcm2–7p relative to other components is illustrated as
additional Mcm2–7p complexes associated with adjacent chromatin;
The fate of Cdc6p after pre-RC formation is distinct in different organisms; expeort and
degradation are illustrated
Klein - AMG 2.5 Replication Bell and Dutta Ann Rev. Biochem. 2002
Licensing factor and ORC are clamp loaders for the Mcm helicase.
Initiation of DNA replication in Saccharomyces cerevisiae.

The first step, licensing, loads the Mcm2-7 helicase onto origin DNA in an inactive form. This is dependent on ATP, the six-subunit
ORC complex and the loading factors Cdc6 and Cdt1. The second step, activation of the helicase, requires CDK and DDK, leading to
the recruitment of the initiation factors Sld2, Sld3, Sld7, Dpb11, Mcm10, Cdc45 and the GINS complex.
Klein - AMG 2.5 Replication Boos, Diffley Current Opinion in Cell Biology 2012
The pre-replication complex - ORC and the MCM helicase
+ATP
Melting
+RPA
Unwinding
Cellular ssDNA
Initiation
B2 element A element
Replication
MCM2-7 complex ORC1-6 complex (x2)

• DNA-helicase • Site specific DNA-
• interaction with binding
Polα • ssDNA binding
Stenlund 03
Klein - AMG 2.5 Replication Boos, Diffley Current Opinion in Cell Biology 2012
Cdc45 functions at the replication fork as a critical regulator of 
the elongation step in replication
Okazaki fragments: RNAse H, Fen1 (flap endonuclease 1), Dna1, Pol δ and DNA ligase 1
ssDNA binding protein

Primase
recruits Primase
Helicase
Topo1 (releases supercoils)
Replication Factor C
(Clamp loader)
Clamp
Helicase activation S-phase Checkpoint
DD (OFF!)
K Cdc2
S-phase (ON) 5A
CDK
CD 2
4-1-2-3 K
Go-Ichi-Ni-San Chk1
Broderick, Nasheuer (2009) Biochemical Society Transactions

Early, late and dormant origins can be activated in the emergency case
Zegerman, J.F.X. Diffley / DNA Repair 8 (2009)

Replication causes torsional stress
Klein - AMG 2.5 Replication Branzei and Foiani, Nature reviews 2010
Positive supercoil in front of the fork, precatenanes in the back
Klein - AMG 2.5 Replication Branzei and Foiani, Nature reviews 2010

AMG2.5 Klein Replication 2018

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Replication: The unit of heredity is DNA (or RNA)

Richard Dawkins: Unit of heredity: DNA (RNA)

“collapsed” DNA in living cells left handed

Bases Considering that C should basepair (code for) with G,

It replaced Uracil with something not to be mixed up with the

Klein - AMG 2.5 Replication

Klein - AMG 2.5 Replication

By RNR and Thymidilate Synthase

Klein - AMG 2.5 Replication

The concept of “Frozen accidents” (Francis Crick) I

An RNA polymerase made from RNA alone

The concept of “Frozen accidents” (Francis Crick) II

BUT worse than that ….

In order to make Deoxythymidin we make Deoxyuracil first! By subsequently adding a methyl-tag.

Klein - AMG 2.5 Replication

Klein - AMG 2.5 Replication

RNA alone can be a polymerase

From an RNA/peptide world to an RNA/protein world

DNA world RNR: invention of ribonucleotide reductase

Klein - AMG 2.5 Replication

Normalerweise traegt das freie

Klein - AMG 2.5 Replication

Watson und Crick Paarung der Basen

G-C Paare bilden 3 Wasserstoffbrücken aus

Klein - AMG 2.5 Replication

Klein - AMG 2.5 Replication

Klein - AMG 2.5 Replication

Klein - AMG 2.5 Replication

Klein - AMG 2.5 Replication

Lewin, Genes VII

Rolling Circle Replication

Klein - AMG 2.5 Replication

RCA: Rolling Circle Amplification

RCP: Rolling Circle Product

Larsson et al. nature methods | VOL.7 NO.5 | MAY 2010 | 395

Klein - AMG 2.5 Replication

Several generations in 15N

Klein - AMG 2.5 Replication

The initiation process can generally be considered to

The cellular initiator proteins from both eukaryotes and

Klein - AMG 2.5 Replication

Klein - AMG 2.5 Replication

Klein - AMG 2.5 Replication

Klein - AMG 2.5 Replication

Klein - AMG 2.5 Replication

Klein - AMG 2.5 Replication

Klein - AMG 2.5 Replication

Klein - AMG 2.5 Replication

Klein - AMG 2.5 Replication

The E. coli genome contains 4.7 x 106 bp.

The human genome contains ca 2900 x 106 bp.

Klein - AMG 2.5 Replication

Zegerman, J.F.X. Diffley / DNA Repair 8 (2009)

Additional polymerases serve functions in DNA repair (error-

Klein - AMG 2.5 Replication

Plymerization: 5'→ 3' + + + + +

Exonuclease proofreading activity: 3 → 5' - - + + +

Associated DNA primase + - - - -

Sensitive to aphidicolin (inhibitor of cell DNA synthesis) + - - + +

Klein - AMG 2.5 Replication

Klein - AMG 2.5 Replication

Question: What happens, if a cell in G2 is fused with a cell in S-phase?

Cyclins: Cln1-3 Clb5,6 Clb1,2,3,4

CDK: Cyclin Dependent Kinase: Cyclin + Kinase

Replication: The unit of heredity is DNA (or RNA) 

Geminin: Additional negative control of replication  