DNA Damage Detection and its Impact on the Cell Cycle

Monika Kurpas, Katarzyna Jonak and Krzysztof Puszynski

Faculty of Automatic Control, Electronics and Computer Science, Silesian University of Technology,

Akademicka 16, 44-102 Gliwice, Poland

1 STAGE OF THE RESEARCH

1.1 Biological Background

Thousands of DNA lesions are formed daily in each

cell of the human body. They can be induced either

endogenously, as well as exogenously - by physical

and chemical agents from outside of the body. There

are several types of DNA damage, from small chem-

ical modiﬁcations of single-stranded DNA through

the photoproducts and adducts caused by UV irradia-

tion, to the potentially most dangerous double-strand

breaks. The existence of such a large number of ab-

normalities in many cells may cause the death of the

body after a very short time. During evolution num-

ber of mechanisms that protect cell from damages

evolved to prevent cell death and lesions transforma-

tion to future generations. There are several path-

ways of DNA repair depending on nature of dam-

age, their review can be found in (Ciccia and Elledge,

2010). For all of these complex mechanisms of re-

Figure 1: DNA damage detection and signal ampliﬁcation.

pair, it is necessary to detect DNA damage just af-

ter it arises and spread the information about it to the

proper regulatory units. This process takes place in a

manner speciﬁc to the type of lesion. ATR (ataxia

telangiectasia mutated and Rad3-related) module is

activated by presence of single stranded DNA areas

in the cell, which are caused by resection of various

types of lesions or by stalled replication forks. Dou-

ble strand breaks (DSBs) are detected indirectly by

ataxia telangiectasia mutated (ATM). Stages of DNA

damage detection by ATR and ATM and its further

ampliﬁcation are presented in ﬁg. 1.

In case of ATR subpathway, the signal strength

of the checkpoint cascade is dependent on the

length of RPA-ssDNA regions and possibility of ATR

molecules located closely to each other. Other ATR

phosphorylation targets are RPA subunits (RPA70 and

RPA32), ATRIP, TopBP1, Chk2, p53 and histone

H2AX. Double strand breaks are detected by repair

complexes like MRN complex. MRN binds to DNA

damage site and recruits ATM kinase, that after its

autophosphorylation interacts with several protein in

pathway (Chk1, Chk2, p53, Mdm, CREB, Wip1) and

leads to p53 stabilization.

1.2 ATR-p53 Model

Our existing stochastic mathematical model of ATR

signaling pathway is based on the Haseltine-Rawlings

postulate (Haseltine and Rawlings, 2002) and is an

extension of our previous model of the p53 signaling

pathway (Puszynski et al., 2008). The model is

activated by UV irradiation which results in SSBs

lesions occurrence. The output of the model is the

level of p53 protein which determines cell fate: DNA

damage repair or cell apoptosis. Spontaneous DNA

damage formation implemented in presented model

results in basic ATR pathway activation. The core

of the model are states of ATR: inactive protein, its

phosphorylated state, and fully activated form. In this

model, there are two feedback loops: positive, with

the participation of PTEN protein, and negative, con-

taining MDM - p53 suppressor (ﬁg. 2). Details about

Kurpas M., Jonak K. and Puszy

nski K..

DNA Damage Detection and its Impact on the Cell Cycle.

 2014 SCITEPRESS (Science and Technology Publications, Lda.)

Figure 2: DNA damage detection model vizualization. Solid lines represent change of protein form; dashed lines describe the

interactions that occur in the path. Components of ATR module are colored in blue.

p53 signaling pathway are available in (Puszynski

et al., 2008). The model distinguish the nucleus and

cytoplasm. It was assumed that each gene has two

copies. None of them can be active, one of them or

both can be active. For some proteins production

and degradation was not modeled directly assuming

that they are equal and protein only change the form

(from active to inactive and vice versa). In presented

model a simpliﬁed DNA repair was implementing

depending on the number of p53 tetramers, repair rate

and the amount of repair complexes, which is limited.

Apoptosis condition is recognized as a permanently

elevated level of the p53 protein (over 6 hours). Then

the cell dies and all of its elements are degraded, thus

further protein levels and the number of lesions are

not taken into account.

1.2.1 Simulation Analysis of ATR Module

In the simulation analysis we examined cell response

to different doses of radiation, we set the threshold

of detection and apoptosis, as well as showed sponta-

neous activation of the ATR/p53 pathway. Determin-

istic and stochastic (for 100 cells) experiments were

performed. At t=24 hours after start, simulated cells

were irradiated by a speciﬁc dose of UVC, and then

observed over the next 48 hours. The correctness of

the model was veriﬁed based on the results of biolog-

ical experiments from the literature.

3A. Basic Activation

According to (Kohn, 2002) in every cell of the human

body in a day are formed approximately 55 000

single-strand breaks, which are responsible for base

activation of the path (ﬁg. 3A).

3B. Damage Detection Threshold

We examined response of the model (ﬁg. 3B) to ra-

diation dose causing one single strand break (0.0665

mJ/m

4A. Apoptotic Death Threshold

Based on the observation of simulation results, the

dose of 18 J/m

in which more than half of the cell

becomes apoptotic cells was taken as the threshold

for apoptosis (ﬁg. 4A). . For comparison, the dose

17 J/m

causing death of 44/100 cells. Assumed that

apoptosis occurs when the level of p53 is increased

by more than 6 hours (in simulation exceeding the

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Figure 3: A. Spontaneous DNA damage formation and p53 activation. Result of 10 stochastic simulations; B. Response to

one lesion occurrence. Red - number of SSBs, green - p53 total level.

threshold 2.1·10

). Further experiments were run for

the radiation dose of 18 J/m

4B-D. Disabling Selected Effects

Inhibition of the p53 phosphorylation caused by Chk1

and Chk2 kinases results in the reduction of apoptotic

fraction size (ﬁg. 4B: 17/100 cells for the Chk1 and

14/100 cells to Chk2, for which plot appears almost

identical), and a signiﬁcant prolongation of DNA re-

pair time. In the case of the inhibition of the ATR-

dependent p53 activation, the apoptotic fraction de-

cline was smaller (size of fraction was 46/100 cells).

A stronger effect was obtained excluding Chk1

(ﬁg. 4C) and Chk2 dependent degradation of Mdm.

None of the cells entered the state of apoptosis. As in

the previous case, less system response change was

observed for the ATR. Apoptotic fraction size was

20/100.

When total Chk1 (ﬁg. 4D) or Chk2 protein kinase

activity is blocked, none of the cells reach the state of

apoptosis. Simulation analysis show the correct func-

tioning of the model stress response which effect is

known from the literature.

1.2.2 Conclusions from Recent Work

ATR module is able to detect a single strand break

caused by UVC appearing in the cell and enhance the

signal so as to cause an increase in p53 protein level.

The threshold of apoptosis in the healthy cell is 18

J/m

. However, if the pathway is defective, apoptotic

threshold shifts. Despite extensive damage, the cells

may not die, but transfer incorrect genetic material to

daughter cells (because DNA damage repair takes a

lot longer). This state could potentially be a cause of

cancer and other genetic diseases. Developed model

can be used to study the behavior of cells with spe-

ciﬁc mutations without the need for costly and time-

consuming experiments in the laboratory.

1.3 ATM-p53 Model

Independently, the ATM model was created. ATM-

p53 model is based on information from literature re-

garding DSBs detection pathways and role of Wip1

protein (Shimada and Nakanishi, 2013). ATM detec-

tion module regulates p53 phosphorylation via posi-

tive loop. Wip1 creates negative feedbacks for all of

the proteins except Mdm2 in nucleus, where it acti-

vates inactive Mdm2 by dephosphorylation. Activa-

tion of the model takes place by the application of ion-

izing irradiation. The signaling pathway is also stim-

ulated continuously by the small number of damages

that occur spontaneously. Most of the assumptions

about the structure of the model is the same as in ATR

part. ATM model is also an extension of the p53 sig-

naling pathway model (Puszynski et al., 2008). This

model shows that cells with blocked transcription of

Wip1 are more affected to apoptosis. The absence of

PTEN and Chk2 in the model signiﬁcantly affect the

results of the simulation.

2 OUTLINE OF OBJECTIVES

First of all we planned to combine models ATR-p53

and ATM-p53. This approach is caused by interac-

tions between these paths. However, a more interst-

ing goal is modelling dependence of described above

path on cell cycle phase. We plan to investigate how

the speciﬁc cell cycle phase affects the ATM-ATR-

p53 path and the other hand, how the path inﬂuences

the cell cycle.

3 RESEARCH PROBLEM

In our study we plan to examine how cell cycle pro-

gression inﬂuences DNA damage detection pathway.

DNADamageDetectionanditsImpactontheCellCycle

Figure 4: A. Apoptotic death threshold; B-D. Disabling selected effects. Results for 100 stochastic simulations. Solid line -

median; dashed line - upper and lower quartile of results.

The speed of reactions occuring in the cell depends

on the concentration of molecules participating in it.

This concentration differs over time. It is caused e.g.

by the variable volume of the cell depending on its

cell cycle phase. It must be taken into account for

modeling of the ATM-ATR-p53 pathway. Expression

of some proteins and its nucleus or cytoplasm loca-

tion may be different depending on the phase of the

cycle, what will have to be reﬂected in the proposed

model.

In another look at the model, we plan to examine

how the damage formation caused by stressful factor

in various forms (ionizing radiation, UV light) affects

the cell cycle and for which radiation doses cell cy-

cle will be stopped for DNA damage repair. We plan

to determine which dose will cause apoptosis of cells.

Another important issue is to check how the disabling

of selected interactions in the pathway will affect the

response of the cell.

The purpose of the construction of such a model

is to illustrate the processes occurring in the cell af-

ter a lesion is detected in the different phases of the

cycle, depending on the given force. With this model

we can revise how mutations of the genes encoding

the individual elements of the path and causing inhi-

bition of their activity may inﬂuence the behavior of

cells. The model can be used to verify the hypotheses

without the need for costly and long lasting biological

experiments.

4 STATE OF THE ART

4.1 Cell Cycle

The cell cycle consists of two main stages: interphase,

which prepares the cell to the next division and the

division (M phase): mitosis (somatic animal cells) or

meiosis (generative animal cells). Mitosis (which we

will deal more in our model) includes laryokinesis

(division of the cell nucleus) and cytokinesis (divi-

sion of cytoplasm). It results in the separation of the

one cell into two daughter cells (Cooper, 2000). The

major stages of interphase are:

• G

Phase - growth phase; biosynthetic processes

in the cell, which were signiﬁcantly slowed in the

M phase, will be taken up again. In this phase the

synthesis of various enzymes required for DNA

replication in S phase takes place. Length of the

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phase differs even between cells of the same

species.

• S Phase - begins with DNA synthesis, and lasts

for a similar period of time in all cells. The pur-

pose of the processes taking place in the S phase is

to double the amount of DNA present in the cell.

Each chromosome has been replicated. RNA and

protein synthesis in this phase of the cycle is very

slow (with the exception of histone protein syn-

thesis).

• G

phase - in this phase synthesis of proteins is

increased again, mainly those responsible for the

formation of the mitotic spindle (tubulin), which

are necessary for the occurrence of a subsequent

process of mitosis.

• G

Phase - eukaryotic cells (especially those fully

differentiated) may move from the G1 phase to the

G0 phase, where they do not undergo divisions

and can remain for a long period of time. The

aging of cells in response to damage is a process

that prevents (without causing apoptosis) transfer

of incorrect genetic material to progeny cells.

4.2 Cell Cycle Checkpoints

Cell cycle checkpoints (ﬁg. 5) are mechanisms which

verify correctness of the DNA. If genetic material is

damaged or not all cellular processes speciﬁc for each

phase have been completed, cell cycle progression is

stopped until all will be ﬁnished and repaired. If the

damage is too big that could have been repaired, cell

is directed to apoptosis (Cooper, 2000; Shapiro and

Harper, 1999). The main checkpoints in the eukary-

otic cells are:

• G

/S checkpoint - at the end of G

phase (before

synthesis phase) decision whether cell should di-

vide, delay division or enter a G

phase is taken.

• G

/M checkpoint - occurs at the end of G

phase

(before mitosis) and checks if cell is ready to mi-

tosis (whether mitotic apparatus is fully formed

and whether DNA lesions occur).

4.3 Cyclins, Cyclin-Dependent Kinases,

Cyclin-Dependent Kinase

Phosphatases and

Cyclin-Dependent Kinase Inhibitors

To move to next phases of cell cycle is needed co-

operation of two types of molecules: cyclin and

cyclin-dependent kinases. Cyclins and the cyclin-

dependent kinases (CDKs) form together the active

Figure 5: Cell cycle checkpoints and DNA damage detec-

tion.

heterodimer, where cyclins represent a regulatory unit

and are synthesized in speciﬁc phases of the cell cy-

cle in response to various molecular signals. CDKs

play a catalytic function and their expression is inde-

pendent of phase of cell cycle. CDKs upon binding to

cyclins are activated and performs the target protein

phosphorylation reactions, which thus become acti-

vated or inactivated, what coordinate entry into the

next phase of the cell cycle. CDKs are often activated

by cyclin-dependent kinase phosphatases (for exam-

ple Cdc25) - tyrosine phosphatases, which acts by

removing the blocking the CDKs activity phosphate

residues (Orlando et al., 2008). Regulation of CDKs

activity might be performed by CDKs inihibitors (like

p21 encoded by a gene CDKN1) which nhibits the

CDK-cyclin complexes activity. p21 binds to cyclin

E/Cdk2 and cyclin D/Cdk4 complexes and inhibiting

their activity acts as a regulator of the cell cycle in the

G1 phase. p21 gene expression is tightly controlled

by the p53 protein (Gartel and Radhakrishnan, 2005).

4.4 Role of Chk1 and Chk2 in

Checkpoint Mechanism

Between G1 and S phase DNA damage results in the

activation of ATM and ATR and following Chk2 and

Chk1 phosphorylation and next phosphorylation of

p53 and Mdm2, which results in activation and sta-

bilization of p53. Active tetramers act as a transcrip-

tion factor of (among others) p21 protein, which is a

potent inhibitor of cyclin-dependent kinases and pre-

vents cells before the entry to the S phase. In addition,

Chk1 phosphorylates and inactivates Cdc25A phos-

phatase that is essential in CDKs activation.

Blocking of the G2-phase is performed with the

signal transducers ATM or ATR (depending on the

type of damage). They activate Chk1 and Chk2 ki-

nases which phosphorylate Cdc25 phosphatase, caus-

ing its inactivation. This avoids the activation of Cdk1

kinase (encoded by the gene CDC2) necessary to ini-

tiate mitosis (Shapiro and Harper, 1999).

DNADamageDetectionanditsImpactontheCellCycle

Figure 6: Schema of new ATM-ATR-p52 and cell cycle pathway.

5 METHODOLOGY

We created scheme od combined ATM-ATR-p53

pathways model with taking into account the key ele-

ments of cell cycle regulation in checkpoints (ﬁg. 6).

Both the scheme and the model are a simpliﬁcation

of reality what is necessary to enable the modeling of

the pathway.

In signaling pathways modelling we use the basic

laws known from biochemistry: the law of mass ac-

tion and Michaelis-Menten kinetics. The kinetic pa-

rameters for our model we obtain from the results of

biological experiments performed by us and from lit-

erature. Unknown parameters are estimated by ﬁtting

the model to the known data.

As described in caption 1 proposed model will

be based on Haseltine-Rawlings postulate (Haseltine

and Rawlings, 2002) which binds deterministic and

stochastic approach. In our models we use ODE

to simulate fast reactions (in example protein-protein

interactions) and direct Gillespie method (Gillespie,

1977) to simulate slow reactions (enabling genes

and DNA lesions number). In near future we will

have computational system which enables modeling

of variable terms for each of cell cycle phase. We

plan to perform stochastic simulation for population

of cells (in example 1000 cells).

6 EXPECTED OUTCOME

Expected outcome of this research will be to develop

a model illustrating the processes occurring in the cell

associated with the detection of damage and cell cy-

cle progression. The resulting model will allow for

the imaging of various kinds of extortions acting on

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the cells (ioinizing irradiation, UV-light). The result

of model action will be a system response which will

be adequate to the applied force. For healthy cells

at too high dose of damaging agents cells should be

directed to apoptosis, whereas at lower doses cell cy-

cle arrest and repair of damages should occur. For

the population (in example, 1000) modeled stochasti-

cally, desynchronized cells after forcing will show a

division into fractions of cells retained in the appro-

priate phase of the cycle. Constructed model will let

investigate the effect of disable selected interactions

at overall pathway answer. This allows to investigate

how mutation emerged in a cell can inﬂuence the cell

cycle and damage detection. Effect will show inﬂu-

ence of abnormalities appearing for example in tumor

cells which are known in the literature.

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DNADamageDetectionanditsImpactontheCellCycle