Un hermoso articulo

1. A concise review of DNA damage checkpoints and repair in
mammalian cells
Jaco H. Houtgraaf a,b
, Jorie Versmissena
, Wim J. van der Giessenb,4
a
Department of Cell Biology and Genetics, Erasmus MC, PO Box 2040, 3000 CA Rotterdam, The Netherlands
b
Department of Cardiology, Erasmus MC, PO Box 2040, 3000 CA Rotterdam, The Netherlands
Received 12 January 2006; accepted 6 February 2006
Abstract DNA of eukaryotic cells, including vascular cells, is under the constant attack of chemicals, free
radicals, or ionizing radiation that can be caused by environmental exposure, by-products of
intracellular metabolism, or medical therapy. Damage may be either limited to altered DNA bases
and abasic sites or extensive like double-strand breaks (DSBs). Nuclear proteins sense this damage
and initiate the attachment of protein complexes at the site of the lesion. Subsequently, signal
transducers, mediators, and finally, effector proteins phosphorylate targets (e.g., p53) that eventually
results in cell cycle arrest at the G1/S, intra-S, or G2/M checkpoint until the lesion undergoes repair.
Defective cell cycle arrest at the respective checkpoints is associated with genome instability and
oncogenesis. When cell cycle arrest is accomplished, the DNA repair machinery can become
effective. Important pathways in mammalian cells are the following: base excision repair, nucleotide
excision repair, mismatch repair, and DSB repair. When repair is successful, the cell cycle arrest may
be lifted. If repair is unsuccessful (e.g., by high doses of DNA-damaging agents or genetic defects in
the DNA repair machinery), then this may lead to permanent cell cycle arrest (cellular senescence),
apoptosis, or oncogenesis.
D 2006 Elsevier Inc. All rights reserved.
Keywords: DNA damage checkpoints; Repair; Nuclear proteins; Cell cycle
1. Introduction
The genome of eukaryotic cells is under constant attack.
A wide diversity of lesions caused by environmental agents
such as ultraviolet (UV) radiation in sunlight, ionizing
radiation, and numerous genotoxic chemicals can arise in
the DNA. In addition, the genome is also threatened from
within. By-products of normal cellular metabolism, such as
reactive oxygen species (ROS; i.e., superoxide anions,
hydroxyl radicals, and hydrogen peroxide) derived from
oxidative respiration and products of lipid peroxidation, can
cause a variety of damages in the DNA (Fig. 1).
On the other hand, DNA-damaging agents such as
ionizing radiation, UV light (photodynamic therapy), and
most chemotherapeutic agents are increasingly being used to
treat common disorders like arterial (re)stenosis (brachy-
therapy and drug-eluting stents) or cancer.
Whereas DNA damage in terminally differentiated cells
(such as muscle cells) gives rise to DNA damage repair to
ensure the integrity of the transcribed genome, the induction
of DNA damage in dividing cells results in the activation of
cell cycle checkpoints. These checkpoints halt the prolifer-
ating cell in its cell cycle progression in order to give time to
the DNA damage repair machinery to do its work, thereby
avoiding incorrect genetic information from being passed
1553-8389/06/$ – see front matter D 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.carrev.2006.02.002
4 Corresponding author. Tel.: +31 104635245.
E-mail address: w.j.vandergiessen@erasmusmc.nl (W.J. van der Giessen).
Funded by Netherlands Heart Foundation Grant No. 99118.
Cardiovascular Revascularization Medicine 7 (2006) 165–172

2. onto the progeny. Especially when mutations are accumu-
lating, the chance of developing uncontrolled cell growth
(oncogenesis) is substantial. A variety of lesions can occur
in the DNA, including single- and double-strand breaks
(DSBs), mismatches, and chemical adducts. Therefore,
multiple repair pathways have evolved, each directed to a
specific type of lesion. Each pathway consists of numerous
proteins forming a cascade in order to repair the damage as
accurate as possible.
Eventually, when the repair process fails, the cell cycle
can be blocked permanently, leading to a senescent state of
the cell, or alternatively, apoptosis may be induced. Both
mechanisms prevent potentially harmful cells from dividing,
ensuring that no mutations are inherited by the next
generation of cells.
2. Cell cycle checkpoints
The cell cycle in eukaryotic cells consists of four
phases, gap (G)1, synthesis (S), G2, and mitosis (M), and
one phase outside the cell cycle, G0 (Fig. 2). In the G1
phase, directly after mitosis, the cell increases in size and
starts synthesizing RNA (transcription) and proteins (trans-
lation). In the subsequent S phase, DNA is replicated to
produce an exact copy of the genome for the subsequent
daughter cells. During G2, the cell will grow and make
extra proteins to ensure that two viable daughter cells can
be formed. RNA and protein syntheses that started in the
G1 phase are continued during the S and G2 phases.
Finally, the cell will go into the M phase. In this phase, the
chromosomes are organized in such a way that two
genetically identical daughter cells can be produced, after
which the whole cycle can start again. Cells can also stop
dividing and remain in G0. They may stay in this state for
hours, days, weeks, or even years before they start
dividing again or even stay in G0 permanently until the
organism dies.
In a normal cell cycle, the passage from one stage to
another is thoroughly controlled. Although still under
debate, genetic evidence that proteins involved in the
orderly progression through the cell cycle are also involved
in the checkpoint response to DNA damage is accumulating.
This implies that in all cells, these cell cycle checkpoint
DNA REPAIR PROCESS
CC C GT TA A A A G C C GT TAC C CT TA
AG CX GT T TA AG C CGG TA G G TT
GC C GT CA A A C C CT AA
AG C
C
GT T T
G
GA G G
TT
C
G
C C GT TA A A C C CT AA
A CG GT T TG GA G GT G
C
G
C G GT CA A A CT
A CG CT T TG A G
C CA
GT
REACTIVE OXYGEN SPECIES
IONIZING RADIATION
ALKYLATING AGENTS
UV-LIGHT
cis-PLATIN
POLYCYCLIC AROMATIC
HYDROCARBONS
REACTIVE OXYGEN SPECIES
IONIZING RADIATION
REACTIVE OXYGEN SPECIES
ALKYLATING AGENTS
cis-PLATIN
REPLICATION ERRORS
TYPE OF LESION
ALTERATED BASE
ABASIC SITE
SINGLE-STRAND BREAK
intrastrand CROSS-LINKS
BULKY DNA ADDUCTS
BASE MISMATCHES
small INSERTIONS
and DELETIONS
interstrand CROSS-LINKS
DOUBLE-STRAND BREAK
BASE EXCISION REPAIR
(BER)
NUCLEOTIDE EXCISION
REPAIR (NER)
MISMATCH REPAIR
(MMR)
RECOMBINATIONAL
REPAIR (HR, NHEJ)
DNA DAMAGE
Fig. 1. Summary of the most common types of DNA lesions that can be caused by exogenous or endogenous damaging agents. They may affect a single strand
or both strands of the DNA. The assumed repair pathway that operates on the various lesions is also indicated.
Fig. 2. Schematic representation of the cell cycle. DNA damage triggers
activation of these cell cycle checkpoints, which can lead to an arrest at the
G1/S, intra-S, or G2/M phase (indicated in red). During cell cycle arrest, the
DNA damage can be repaired.
J.H. Houtgraaf et al. / Cardiovascular Revascularization Medicine 7 (2006) 165–172166

3. pathways are active, but they are up-regulated when DNA
damage occurs.
The cell cycle checkpoint pathways mentioned above are
operational during the entire cell cycle and, hence, may slow
down the cell cycle at any point during the four phases, but
the term checkpoint is defined more upon the transition
between phases, which is being inhibited by DNA damage
at the G1/S, intra-S, and G2/M checkpoints. Although these
checkpoints are distinct, they all respond to DNA damage
and share many proteins. The intra-S-phase checkpoint
differs from the G1/S and G2/M checkpoints since it also
has to recognize and deal with replication intermediates and
stalled replication forks. Also, in the S phase, another
checkpoint that prevents transmission of unreplicated DNA
is active. This process inhibits mitosis while DNA
replication is ongoing or blocked. The signal for this
checkpoint is unreplicated DNA rather than DNA damage.
The DNA damage response during any phase of the cell
cycle has the same pattern. After the detection of DNA
damage by sensor proteins, signal transducer proteins
transduce the signal to effector proteins. These effector
proteins launch a cascade of events that causes cell cycle
arrest, apoptosis, DNA repair, and/or activation of damage-
induced transcription programs (Fig. 3).
2.1. Damage sensors and signal transducers
Although the sensors required for DNA repair are partly
known, the sensors that eventually lead to cell cycle arrest
are largely unknown. Different types of DNA lesions, as
well as very low levels of damage, have to be recognized.
The lesion itself can be recognized, but it is also possible
that the damage covered by a complex of repair proteins
forms the signal for checkpoint reactions or even inter-
mediates formed during repair. The exact mechanism, as
mentioned before, is still not completely resolved.
The first group that has been found to function as a
sensor consists of proliferating cell nuclear antigen (PCNA)-
like and replication factor C (RFC)-like protein complexes
(Fig. 4). The candidate DNA damage sensors Rad9, Hus1,
and Rad1 form a ring structure (the b9–1–1Q complex) that
can encircle the DNA similar to PCNA. Since it has a ring
structure, this complex has to be loaded on the DNA by a
clamp loader. Whereas, during replication, PCNA is loaded
on the DNA by RFC, which consists of five subunits
(RFC1–5), the 9–1–1 complex is loaded on damaged DNA
by a protein complex that consists of four RFC (RFC2–5)
subunits and Rad17, forming the Rad17–RFC2–5 complex.
As soon as the 9–1–1 complex is bound to the damaged
DNA, it is expected to form a scaffold for downstream
checkpoint and repair proteins.
Another group of important players in the early response
to DNA damage consists of ataxia telangiectasa (AT) mu-
tated (ATM) and ATM and Rad3 related (ATR). AT, caused
by mutated ATM, is an autosomal recessive disorder,
characterized by immunodeficiency, neurological disorders,
and high cancer susceptibility. ATR was identified later on
basis of sequence and functional homology to ATM.
Whereas patients, mice, and cells without active ATM are
viable, the complete absence of ATR leads to embryonic
lethality. This suggests (and evidence is accumulating) that
ATR also functions in essential cellular processes in
undamaged cells like DNA replication and cellular differ-
entiation. The ATM and ATR proteins belong to the
Fig. 3. Flowchart of the cellular response to any kind of DNA damage. In proliferating cells, cell cycle checkpoints will be activated, leading to a cell cycle
arrest and providing time to the activated DNA damage repair machinery to repair the DNA damage. In resting/terminally differentiated cells, DNA repair will
be initiated directly. When repair is complete, the cell may proceed in its cell cycle. If the damage cannot be repaired or if there is too much damage for the
DNA repair machinery to overcome, then the cell cycle can be blocked permanently, leading to a senescent state of the cell, or apoptosis may be induced. If
unrepaired damages remain undetected, then this may lead to mutations and genomic instability that ultimately can lead to oncogenesis.
J.H. Houtgraaf et al. / Cardiovascular Revascularization Medicine 7 (2006) 165–172 167

4. phosphatidylinositol 3-kinase-like (PIKK) family of serine/
threonine protein kinases. This family also includes DNA-
PK, an enzyme that is involved in repair of DSBs during
nonhomologous end joining (NHEJ; Fig. 5D).
ATM appears to be the primary player in response
to ionizing radiation. Recent results suggest that the Rad50/
Mre11/Nijmegen Breakage Syndrome 1 (NBS1) complex
functions as the DSB sensor for ATM but is in turn also
a downstream target. ATR is more important in response
to UV, MMS, and replication inhibitors such as hydroxy-
urea (HU).
The Rad50/Mre11/NBS1 complex, ATM and ATR, the
previously described Rad17/RFC2–5, and the 9–1–1 com-
plex localize to sites of DNA damage independently but
interact to trigger the checkpoint-signaling cascade. For
example, ATR and ATM phosphorylate Rad17. Increased
amounts of 9–1–1 complexes, which are recruited by Rad17
to DNA damage sites, stimulate this phosphorylation. It is
not yet known how Rad17 phosphorylation contributes to
the checkpoint pathway. On the other hand, mammalian
Rad17 and HUS1 are required for the phosphorylation of
Check (Chk)1 by ATR. Therefore, next to the expected
sensor function, Rad17 and the 9–1–1 complex play a role
in the facilitation of signal transduction and, possibly, in the
amplification of the damage signal. ATM and ATR carry out
the actual signal transduction to the effector kinases Chk1
and Chk2.
2.2. Mediators
In addition to damage sensors, signal transducers, and
effector proteins, many other proteins are involved in the
DNA damage response. They are mostly cell cycle specific
and associate with damage sensors, signal transducers, and
effectors at particular phases of the cell cycle and, as a
consequence, help provide signal transduction specificity.
ATM and ATR phosphorylate most of these mediators. Well-
known examples of mediators are p53 binding protein
(53bp), the topoisomerase binding protein TopBP1, and
mediator of DNA damage checkpoint (MDC1). Next to these
mediators, many proteins fulfilling other functions have
additional functions in checkpoint pathways. Examples of
these are BRCA1 and the earlier mentioned Rad50/Mre11/
NBS1 complex, which are also involved in DNA repair.
2.3. Effector proteins: Chk1 and Chk2
Chk1 and Chk2 are, like ATM and ATR, serine/threonine
protein kinases and phosphorylate targets that eventually
result in the cell cycle arrest. In mammalian cells, both
kinases play a role in all checkpoint pathways responsive to
DNA damage. The DSB signal sensed by ATM is
transduced by Chk2, and the UV damage signal sensed by
ATR is transduced by Chk1, although there is some overlap
and redundancy between the functions of these two proteins.
Chk1 and Chk2 transfer the signal of DNA damage to the
phosphotyrosine phosphatases and cell division cycle
proteins Cdc25A, Cdc25B, and Cdc25C. Phosphorylation
of Cdc25A–C by Chk1 or Chk2 inactivates Cdc25A–C,
whereas unphosphorylated Cdc25A–C promotes the G1/S
and G2/M transition by dephosphorylating the cyclin-
dependent kinases (CDKs) directly involved in cell cycle
transition. However, there are some differences between the
different checkpoints, which will be discussed below.
2.4. G1/S checkpoint
Nonphosphorylated Cdc25A protein promotes the G1/S
transition (by dephosphorylation of CDK2, which phos-
phorylates Cdc45 that is involved in initiating replication).
The exact pathway of cell cycle arrest depends on the
kind of damage. DNA DSBs lead to phosphorylation of
ATM that subsequently phosphorylates Chk2. Single-strand
gaps result in the activation of Rad17–RFC, the 9–1–1
Fig. 4. Simplified representation of the DNA-damage-induced checkpoint
response. After the detection of a given damage by sensor proteins, this
signal is transduced to the effector proteins Chk1 and Chk2 via the
transducer proteins ATR and ATM. Depending on the phase of the cell
cycle the cell is in, this can lead to activation of p53 and inactivation of
CDC25, which eventually leads to cell cycle arrest. Mediator proteins
mostly are cell cycle specific and associate with damage sensors, signal
transducers, or effectors at particular phases of the cell cycle and, thus, help
provide signal transduction specificity.
J.H. Houtgraaf et al. / Cardiovascular Revascularization Medicine 7 (2006) 165–172168

5. complex, and ATR, which leads to phosphorylation of
Chk1. Subsequent phosphorylation of Cdc25A by Chk1 or
Chk2 causes inactivation of this protein by nuclear
exclusion and ubiquitin-mediated proteolytic degradation,
leading to G1 arrest. ATM and ATR also phosphorylate p53,
which leads to stabilization and accumulation of the p53
protein and promotes the transcription factor activity of p53.
The target of the transcription factor p53 is p21, which, in
turn, inhibits CDK2 activity, causing maintenance of arrest
of the cell cycle.
2.5. Intra-S-phase checkpoint
Two pathways mediate the intra-S-phase checkpoint.
Firstly, the ATM/ATR–Chk2/Chk1–Cdc25A–CDK2 path-
way is more or less similar to the G1/S checkpoint. In the
S phase, this pathway delays replication (by blocking the
loading of Cdc45 onto chromatin that in turn attracts DNA
polymerase-a into prereplication complexes) and, as a
consequence, extends the DNA replication time, allowing
DNA repair to take place. The second pathway involves
NBS1 (a human disorder characterized by cancer predis-
position, immunodeficiency, hypersensitivity to ionizing
radiation, chromosomal instability, and growth retardation),
which is phosphorylated by ATM together with Chk2,
leading to a cascade involving also Mre11- and Rad50-like
initial DSB recognition, which plays a role not only in cell
cycle arrest but also in activating the repair processes.
2.6. G2/M checkpoint
When cells encounter DNA damage in G2, the G2/M
checkpoint stops the cell cycle in order to prevent the cell
from entering mitosis. As in the G1/S checkpoint, the kind
of DNA damage determines the pathway that will be
activated: ATM–Chk2–Cdc25 for DSBs and ATR–Chk1–
Cdc25 for DNA lesions such as those created by UV light.
Besides down-regulating Cdc25A, both Chk1 and Chk2 up-
regulate WEE1 by phosphorylation, which together control
Cdc2/CyclinB activity. This latter complex promotes G2/M
transition under normal circumstances, and inactivation
blocks the cell cycle when damage occurs in G2. The
maintenance of this arrest seems to be partly p53/p21
independent unlike the G1/S checkpoint, whereas tumor
cells lacking functional p53 still tend to accumulate in G2
after induction of DNA damage.
As the cell cycle checkpoint pathways are predominantly
evolved to prevent transducing DNA damage to daughter
cells, it is obvious that defects in checkpoint responses can
result in genomic instability, leading to the transformation of
normal cells into cancer cells. Indeed, absence of ATM or
p53 causes syndromes featuring cancer susceptibility. In
addition, other genes involved in cell cycle checkpoints are
related to cancer, most of them also having a function in
DNA repair. This combination probably makes mutations or
deletions more severe: less repair and less control.
3. DNA damage repair
DNA damage checkpoints can only prevent the trans-
duction of mutations to daughter cells by means of an
efficient DNA damage repair machinery. As there are many
different lesions possible, different types of repair pathways
have evolved. Important pathways in mammalian cells
include base excision repair (BER), nucleotide excision
repair (NER), mismatch repair (MMR), and DSB repair
(Fig. 5A–D).
3.1. Base excision repair
BER is the main guardian against damage due to cellular
metabolism. Inactivation of the BER core proteins in mice
leads to embryonic lethality, highlighting the importance of
this pathway. Base damages are generated by ROS, ionizing
radiation, and indirectly also by UV radiation (via gen-
eration of ROS) or can be the result of various chemicals
like chemotherapeutic drugs (e.g., adriamycin, mitomycin
C, and psoralen). In BER, the damaged base is removed by
different DNA glycosylases (depending on the damage) and
APE1 endonuclease (Fig. 5A). This results in an abasic site,
from which both ends are trimmed by poly(ADP-ribose)
polymerase and polynucleotide kinase to facilitate repair
synthesis. In mammals, the so-called short-patch repair is
the dominant mode for the remainder of the reaction. The
single-stranded, one-nucleotide gap is filled in by DNA
polymerase-h and subsequently ligated by the Ligase3/
XRCC1 complex. The long-patch repair mode involves
DNA polymerase-h, DNA polymerase-ba¨/a˚,Q and PCNA for
repair synthesis, as well as the FEN1 endonuclease to
remove the displaced DNA flap and DNA ligase1 for
sealing the backbone of the DNA double helix.
3.2. Nucleotide excision repair
NER is the most important repair system to remove
bulky DNA lesions that can be caused by UV radiation
(thymidine dimers), chemicals, or ROS. In NER, there are
two modes of activation of the pathway (Fig. 5B). The
global genome NER pathway scans the genome constantly
and recognizes the damage caused by XPC [named after
one of the seven genes (XPA to XPG) involved in this
pathway, the disruption of which causes the UV-sensitive
and skin-cancer-prone (N1000-fold incidence) disorder
xeroderma pigmentosum in humans]. The second mode
of activation occurs when RNA polymerase II is blocked
by a damage during transcription. This stalled polymerase
must be displaced to make the damage accessible for
repair, which requires at least two transcription-coupled
repair proteins: CS-A and CS-B (for Cockayne syndrome,
another rare UV-sensitive disorder in humans). After the
recognition step, both pathways are identical. The DNA
duplex is unwound by the multi-subunit transcription
factor Transcription Factor IIH that contains the two
J.H. Houtgraaf et al. / Cardiovascular Revascularization Medicine 7 (2006) 165–172 169

6. B.NUCLEOTIDE EXCISION REPAIR
AA
GLOBAL GENOME NER TRANSCRIPTION COUPLED REPAIR
DNA DAMAGE
DAMAGE
RECOGNITION
PROCESSING OF
DAMAGED DNA
REPAIR/
ADHESION of ENDS
XPC
TFIIH
XPA
XPGERCC I/XPF
REPLICATION FACTORS
A. BASE EXCISION REPAIR
X
C
X
C
C
C
PNK
PARP
APE1
GLYCOSYLASES
C
DNA POL-ß
G
DNA pol-β, Pol-δ/ε
PCNA
FEN1
C
XRCC1
Ligase III
G
Ligase I
C
G
SHORT PATCH BER LONG PATCH BER
DNA DAMAGE
PROCESSING OF
DAMAGED DNA
ADHESION
of
ENDS
REPAIR
MISMATCH REPAIRC.
DNA-MISMATCH
T
A
DNA polymerase-α
REPAIR
Exonuclease (3’- 5’)
PROCESSING OF
DAMAGED DNA
T
T
G DNA polymerase-δ/ε
DNA polymerase-α
T
G
MSH2/6
MLH1/PMS2
MISMATCH
RECOGNITION
CSA
CSB
Fig. 5. DNA repair mechanisms. (A) BER: Damage repaired by BER may be caused by ionizing radiation, alkalyting agents, and oxygen radicals. These agents cause single-strand breaks or small alteration of
bases. The mechanism of repair through BER is shown in the figure and explained in the text. (B) NER: Damage that distorts the normal architecture of the DNA helix is repaired through NER. This type of
damage can be caused by UV light, cisplatin, and other chemotherapeutic drugs. Disruption of the DNA helix interferes with base pairing and obstructs transcription and normal replication. The mechanism of
repair through NER is shown in the figure and explained in the text. (C) MMR: Errors occurring during DNA replication can cause base mismatches or small insertions or deletions of nucleotides. These errors are
removed by MMR. A model for MMR is shown in the figure and explained in the text. (D) DSB repair: DNA DSBs can be repaired by at least two mechanically distinct pathways: HR and NHEJ. This figure
shows a simplification of both models for DSB repair. During HR, the damaged DNA (gray) uses the sister chromatid or homologous chromosome (red) as a template to repair the DNA accurately. NHEJ repairs
the DNA by simply joining the DNA ends in a way that is not necessarily error free, since no template is used for the newly synthesized DNA at the damaged sites (red). A number of proteins involved in each
pathway are indicated and discussed in the text.
J.H.Houtgraafetal./CardiovascularRevascularizationMedicine7(2006)165–172170

7. helicases XPB and XPD. During this process, XPA
confirms the presence of DNA damage, and when one is
not detected, it aborts NER. Subsequently, a more stable
preincision complex is formed with the aid of Replication
Protein A. Finally, XPG cleaves the damaged strand 3V of
the damage, and the XPF/ERCC1 complex cleaves the
same strand 5V of the lesion, generating a 24- or 32-base
oligonucleotide fragment containing the injury. The single-
strand gap that remains is filled in by DNA polymerase-y/q
with the aid of the replication accessory protein PCNA
(regular DNA replication machinery).
3.3. Mismatch repair
Replication errors by DNA polymerase-a or -ba¨/a˚Q
(regular replication machinery) can result in mismatched
bases (A–G or C–T). The MMR process begins with the
proteins Msh2–6 that recognize and bind to the mismatched
base pairs (Fig. 5C). Then, Mlh1 and Pms2 are recruited to
the complex. Subsequently, the mismatched strands are
cleaved, and the segment from the cleavage site to the
mismatch is removed by an exonuclease. DNA polymerase-
a fills in the single-strand gap.
DAMAGE RECOGNITION
HOMOLOGOUS RECOMBINATION NON-HOMOLOGOUS END JOINING
END PROCESSING
DNA DOUBLE-STRAND BREAK
Rad51, Rad52, Rad54
Rad50, Mre11, Nbs1
Brca1, Brca2
Ku70, Ku80, DNA-PKcs
Ligase IV, Xrcc4
HOMOLOGY SEARCH
LIGATION
DNA SYNTHESIS
D.
Fig. 5 (continued )
J.H. Houtgraaf et al. / Cardiovascular Revascularization Medicine 7 (2006) 165–172 171

8. 3.4. DSB repair
DSBs are a very genotoxic type of DNA damage.
Because both strands of the DNA double helix are broken,
chromosomal fragmentation, translocation, and deletions
can easily occur and rapid repair is crucial. DNA DSBs
can be caused by ionizing radiation, ROS, and chemo-
therapeutic drugs and can arise during replication of a
single-strand break.
In recent studies, it was shown that one of the first
responses of eukaryotic cells to DSBs is the extensive
phosphorylation of a member of the histone H2A family,
H2AX (g-H2AX), by ATM and other PI3-like kinases.
Phosphorylation already occurs within 1–3 min after
induction of DNA damage. The exact reason of this
phenomenon is not yet clarified, although a role in DNA
damage repair and chromosomal stability is evident.
In order to repair DNA DSBs, two distinct pathways
have evolved: homologous recombination (HR) and NHEJ
(Fig. 5D). The two main differences between these
pathways are the requirement for extensive DNA homol-
ogy on the sister chromatid in HR and the accuracy of
repair. HR is mediated by the Rad52 epistasis group that
includes the Rad51, Rad52, and Rad54 genes. After
introduction of the DSB, Rad51, which is the central
protein in HR, searches the genome for an intact copy of
the broken DNA on the sister chromatid. After this, the
missing information on the broken strand is copied in, and
the damage is repaired without loss of genetic information.
In NHEJ, on the contrary, there is no need for homology.
The two ends of the broken double helix are directly
ligated together by the DNA ligaseIV/Xrcc4 complex.
Other proteins involved in this pathway are the Ku70/80
heterodimer, DNA-PKCS, and the Rad50/Mre11/NBS1
complex. NHEJ is less accurate and might give rise
to deletions.
Although both DSB repair pathways are operational in
mammals, their relative contribution might differ depending
on the stage of the cell cycle or the cell type. For HR to
occur, there is a need for a sister chromatid, which is not
produced until the S phase. For this reason, HR can only
take place in dividing cells that are in the S or G2 phase.
Cells in G0 and G1 or terminally differentiated cells will
have to rely on NHEJ.
A number of the previously mentioned DNA DSB repair
proteins (for instance, Rad51, Rad54, and the MRN
complex) and g-H2AX relocate into bright nuclear foci
after induction of DNA damage (Fig. 6). These foci are
believed to play an important role as DNA damage repair
factories, harboring thousands of repair and cell cycle
checkpoint proteins, although their exact role remains to
be elucidated.
4. Concluding remarks
Over the past 30 years, our knowledge about DNA
damage, DNA damage checkpoints, and DNA repair
has increased dramatically. This brief review should be
regarded as an introduction to the knowledge accumulated
over those years. It should be emphasized that many issues
remain unsolved yet. However, the concept of DNA damage
and repair is very important for our understanding of the
pathogenesis and treatment of many disease processes.
Acknowledgments
We would like to thank Dr L.R. van Veelen for kindly
providing (Figs. 2, 3, and 5) and A.K. Dik for valuable help
with the other figures. Also, we would like to thank V. Smits
and J. Essers for carefully reading the manuscript.
Fig. 6. Ionizing-radiation-induced foci. Immunohistochemical staining for Rad51 (protein involved in HR) in a vascular smooth muscle cell before and 8 h after
irradiation with 12 Gy of g-radiation. Clearly visible are the bright nuclear foci appearing, which are believed to be bDNA damage repair factoriesQ in which
accumulation of many of the proteins discussed in this review (i.e., g-H2AX, Rad54, Rad52, and MRN complex) takes place.
J.H. Houtgraaf et al. / Cardiovascular Revascularization Medicine 7 (2006) 165–172172