Cyclin /Cdk-Dependent

Initiation of DNA Replication

in a Human Cell-Free System

Torsten Krude, Mark Jackman, Jonathon Pines and Ronald A. Laskey

 Introduction

Initiation of DNA replication:

is a key step of the cell division cycle.

Time for cell to make decision to divide or not.

Cell Fusion Experiments

Yeast: one protein kinase (CDC28) associates

with different cyclin subunits.

Higher eukaryotes the situation is more

complex: different catalytic cyclin-dependent
kinase (Cdk) subunits associate with different
cyclins at specific times.
Existing cell-free DNA replication systems are
insufficient for studying the initiation of chromosomal
DNA replication for two reasons.

Viral cell-free systems depend on essential

viral control elements

Cell-free system derived from Xenopus laevis

can only replicate vertebrate cell nuclei under early
embriyonic cell cycle control. S and M phases
are very rapid and without inverting G1 and G2
phases.
Novel cell-free system from somatic
human cells that obeys somatic cell
cycle control

HeLa Cells

HeLa cells are a human epithelial cervical

cancer, and the first human cells, from which
a permanent (immortal) cell line was
established. On 9 February 1951, the tissue
removed from the patient Henrietta Lacks
(1920-1951), a 31-year-old African American
woman from Baltimore. The tissues were
cultured by George Otto Gey. HeLa cells are
now available in many laboratories of the
world.
Organization of the Presentation

Questions

Experiment

Conclusion of Experiments

Final Conclusions

References
 1º Question
Can G1 phase nuclei initiate DNA synthesis when
coincubated with S phase nuclei and cytosol?

And what happened in G2 phase nuclei?

Analysis of the cell cycle position of nuclei by flow cytometry

 Prelabeled replicated DNA with BrdU “in

vivo”

 The cells synchronized:

 S: blocked with Thymidine.
 G2: cultivate syncronized cells
in S and add Nocodazol
 G1: mitotic cells were
released after 6h.
Confocal Fluorescene microscopy

In vitro Biotin dUTP red initiation

In vivo BrdU green elongation

Early S
phase
Nuclei of nuclei
late S
phase
Percentage of nuclei replicating against ratio of S to G1nuclei

S contaminants S:G1 Ratio 0:1 did not

initiate DNA replication.

S:G1 Ratio 1:1 initiation of

DNA replication.

S phase nuclei induces the

initiation of replication in G1
nuclei.
The equivalent experiment with G2 phase nuclei…

No initiation of DNA synthesis

was observed in true G2 phase
nuclei.

S phase nuclei not induces

the initiation of replication in
G2 nuclei.
The percentage of G1 nuclei initiating replication…

…is depended on the …is depended on the

amount of coreplicating distinct times after
S phase nuclei released from mitosis.
For analysing possible false-positives (S contaminants)…

G1 phase nuclei were incubated with S phase nuclei at a 1:1 ratio in

cytosolic extract
DMAP: the kinase inhibitor.

The elongation is neither depend on which cytosolic

extract used nor kinase activity.
The initiation depended on the cytosolic extract in S
phase and also kinase activity is necessary.
if we consider the time…

In vitro DNA synthesis in S

phase nuclei becomes apparent
after 5 min false-positives
should be detectable after that
time should not continue to
increase entire duration of the
incubation

There are no false positives.

 1º Conclusion
 G1 phase nuclei initiates DNA replication with
S phase nuclei and cytosolic extract.

 The efficiency of initiation depends on,

 The number of S phase nuclei.

 The time G1 phase released from mitosis
 Kinase activity
 Incubation time

 Not occurred same in G2 phase nuclei.

 2º Question

Can S phase nuclear extract

replace with S phase nuclei?
Without nuclear extract only observed elongation

With S phase nuclear extract initiation and elongation

For initiating DNA replication in G1 nuclei
cytosolic and nuclear extracts of S phase were
necessary.
The percentage of G1 nuclei that initiates replication…

…increased with addition of …increased with incubation

different amounts of nuclear time.
extract from S phase.

…and inhibited with DMAP.

and also…

…prepared nuclei from G1

phase at different times after
released from mitosis

The maximum percentage of

G1 phase nuclei initiation in
vitro clearly increased.

…but not in G2 phase nuclei

 2º Conclusion

 Nuclear extract from S phase able to replace

with S phase nuclei initiating DNA synthesis
in G1 but not in G2 phase nuclei.

 The efficiency of initiation somehow less.

 3º Question

Does S phase nuclear extract induce

semiconservative DNA replication in G1 nuclei?
1. αP32-dATP + density gradient centrifugation

In the absence of S nuclear extract

low level of DNA synthesized between
HL and LL

In the presence of S nuclear extract

high level of DNA synthesized HL

Not observed HH in any case

Single round semiconservative

replication.
2. Replication pattern

Early S phase nuclei

Late S phase nuclei

The replication pattern observed

in G1 nuclei that initiate
synthesis “in vitro” is typical of
early S phase nuclei.
3. Measures of the amount of DNA synthesized and the rate of synthesis

G1 nuclei initiate DNA replication in the presence

of nuclear and cytosolic extracts from S phase cells
in vitro and elongate at the same rate as in
preinitiated S phase nuclei.
4. Afidilcoline

Afidilcoline inhibitor of DNA polymerases α and δ.

Afidilcoline treatment inhibits DNA synthesis in

G1 nuclei that have initiated replication.
5. Chromatin

DNA replication in vitro was efficiently assemled into

chromatin, excluding mitochondria as substrates for this
DNA synthesis
 3º Conclusion

 DNA synthesis initiated in G1 nuclei in vitro and

it is semiconservative DNA replication with
dynamic and morphological characteristics of
early S phase.
 4º Question

Can we replace the nuclear extract for

human cyclins and their kinases?
Cyclin A/Cdk2 + Cyclin
E/Cdk2 induces initiation of
replication in G1 nuclei.

Contaminats in S phase
elonagated in the absence of
Cyclin/Cdk. (resistant to
DMAP)

Again observed replication

patterns characteristic for
early S phase nuclei.
Cuantification of fluorecence

B1/Cdc2 and Cdk2

alone do not initiate the
synthesis.

A/Cdk2 and E/Cdk2 are

capable of initiating the
synthesis seperately.
A/Cdk2 + E/Cdk2 synergically induce replication with
the same efficieny observed when using nuclear extracts
from S phase cells.
but not at high concentrations.
also…

Addition of S phase nuclear extracts to the optimal

concentrations of A and E/Cdk2 did not increase the
efficiency of replication

Why?

The cyclin-Cdk2 complexes and the nuclear extract

activated the same targets to trigger initiation of DNA
synthesis in G1 phase nuclei.
Furthermore…

Percentage of nuclei that initiate replication is less if we use the

nuclear extract of cyclin/Cdk instead of S phase nuclei.

S phase nuclei, but not the soluble extracts could inactivate

inhitors (inhibitors of G1 cyclin and their Cdk2, such as p21cip1,
p27kip1) of initiation presents in G1 nuclei.

Addition of excess recombinant cyclins A and/or E/Cdk2 did not

increase the further the efficiency of early G1 nuclei to initiate in
vitro, whereas overexpression of G1 cyclins in vivo could alleviate
inhibitor-mediated G1 arrest.
 4º Conclusion

 Cyclin A and E/Cdk2 are able to replace the nuclear extract and
initiate replication in G1 nuclei with the same efficiency as the
nuclear extract S.

 There is an optimal concentration of both that results in

maximum efficiency. At high concentrations, together act as
inhibitors.
 Final Conclusions

Nuclei, nuclear and cytosolic extracts from HeLa cells

were used to establish cell-free system to initiate
replication under control of somatic cell cycle.
 Final Conclusions

In G1 nuclei, not G2nuclei cytosolic extract from S

phase cells and a signal from the S phase nucleus initiate
nuclear replication. This signal can also be replaced
by cyclin A and E/Cdk2.
 References

Krude, T., Jackman, M., Pines, J., and Laskey, R.A. Cyclin /Cdk-
Dependent Initiation of DNA Replication in a Human Cell-Free
System. Cell, Vol. 88, 109-119, 1997.

Lewin, B. Genes IX. Oxford University Press, 2007.