Cyclin Cdk Dependent Initiation of Recombination

8/7/2019 Cyclin Cdk Dependent Initiation of Recombination

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Cyclin /Cdk-Dependent

Initiation of DNA Replication

in a Human Cell-Free System

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


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Introduction

Initiation of DNA replication:

is a key step of the cell division cycle.

Time for cell to make decision to divide or not.


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Yeast: one protein kinase (CDC28) associates

with different cyclin subunits.

Higher eukaryotes the situation is morecomplex: different catalytic cyclin-dependent

kinase (Cdk) subunits associate with different

cyclins at specific times.

Cell Fusion Experiments


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Existing cell-free DNA replication systems are

insufficient for studying the initiation of chromosomalDNA 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.


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Novel cell-free system from somatic

human cells that obeys somatic cell

cycle control

HeLa Cells

HeLa cells are a human epithelial cervicalcancer, 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 werecultured by George Otto Gey. HeLa cells are

now available in many laboratories of the

world.


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Organization of the Presentation

Questions

Experiment

Conclusion of Experiments

Final Conclusions

References


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1 Question

Can G1 phase nuclei initiate DNA synthesis when

coincubated with S phase nuclei and cytosol?

And what happened in G2 phase nuclei?


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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.


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Nuclei of

late Sphase

Early Sphase

nuclei

In vitro Biotin dUTP red initiation

In vivo BrdU green elongation

Confocal Fluorescene microscopy


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S:G1 Ratio 0:1 did notinitiate DNA replication.

S:G1 Ratio 1:1 initiation ofDNA replication.

S phase nuclei induces the

initiation of replication in G1nuclei.

S contaminants

Percentage of nuclei replicating against ratio of S to G1nuclei


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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.


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The percentage of G1 nuclei initiating replication

is depended on theamount of coreplicating

S phase nuclei

is depended on thedistinct times after

released from mitosis.


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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 cytosolicextract used nor kinase activity.

The initiation depended on the cytosolic extract in S

phase and also kinase activity is necessary.


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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.


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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.

1 Conclusion


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Can S phase nuclear extractreplace with S phase nuclei?

2 Question


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Without nuclear extract only observed elongation

With S phase nuclear extract initiation and elongation


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For initiating DNA replication in G1 nuclei

cytosolic and nuclear extracts of S phase werenecessary.


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The percentage of G1 nuclei that initiates replication

increased with addition of

different amounts of nuclear

extract from S phase.

and inhibited with DMAP.

increased with incubation

time.


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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


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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.

2 Conclusion


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Does S phase nuclear extract induce

semiconservative DNA replication in G1 nuclei?

3 Question


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1. P32-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.


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2. Replication pattern

The replication pattern observed

in G1 nuclei that initiatesynthesis in vitro is typical of

early S phase nuclei.

Early S phase nuclei

Late S phase nuclei


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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 inpreinitiated S phase nuclei.


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4. Afidilcoline

Afidilcoline inhibitor of DNA polymerases and .

Afidilcoline treatment inhibits DNA synthesis inG1 nuclei that have initiated replication.


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5. Chromatin

DNA replication in vitro was efficiently assemled into

chromatin, excluding mitochondria as substrates for this

DNA synthesis


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DNA synthesis initiated in G1 nuclei in vitro and

it is semiconservative DNA replication with

dynamic and morphological characteristics of

early S phase.

3 Conclusion


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Can we replace the nuclear extract for

human cyclins and their kinases?

4 Question


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Cyclin A/Cdk2 + Cyclin

E/Cdk2 induces initiation ofreplication in G1 nuclei.

Contaminats in S phaseelonagated in the absence of

Cyclin/Cdk. (resistant to

DMAP)

Again observed replicationpatterns characteristic for

early S phase nuclei.


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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.


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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.


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also

Addition of S phase nuclear extracts to the optimal

concentrations of A and E/Cdk2 did not increase the

efficiency of replication

The cyclin-Cdk2 complexes and the nuclear extract

activated the same targets to trigger initiation of DNA

synthesis in G1 phase nuclei.

Why?


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Furthermore

Percentage of nuclei that initiate replication is less if we use thenuclear 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.


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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.

4 Conclusion


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Final Conclusions

Nuclei, nuclear and cytosolic extracts from HeLa cells

were used to establish cell-free system to initiatereplication under control of somatic cell cycle.


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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 replacedby cyclin A and E/Cdk2.

Final Conclusions


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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.

Documents

Cyclin Cdk Dependent Initiation of Recombination