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DNA repair

DNA DAMAGE RESPONSE


First Printed in R&D Systems 2003 Catalog

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Contents

Introduction
Cell Cycle Checkpoints
G1 Checkpoint
S-phase Checkpoint
G2 Checkpoint
DNA Repair Pathways
References

Introduction
The maintenance of genome integrity and fidelity is essential for the proper
function and survival of all organisms. This task is particularly daunting due to
constant assault on the DNA by genotoxic agents (both endogenous and
exogenous), nucleotide misincorporation during DNA replication, and the intrinsic
biochemical instability of the DNA itself.1
Failure to repair DNA lesions may result in blockages of transcription and
replication, mutagenesis, and/or cellular cytotoxicity.2 In humans, DNA damage
has been shown to be involved in a variety of genetically inherited disorders, in
aging,3 and in carcinogenesis.4,5

Figure 1. DNA Damage Response. DNA damage is caused by a variety of sources. The cellular

DNA repair

response to damage may involve activation of a cell cycle checkpoint, commencement of


transcriptional programs, execution of DNA repair, or when the damage is severe, initiation of
apoptosis.

All eukaryotic cells have evolved a multifaceted response to counteract the


potentially deleterious effects of DNA damage (Figure1).2 Upon sensing DNA
damage or stalls in replication, cell cycle checkpoints are activated to arrest cell
cycle progression to allow time for repair before the damage is passed on to
daughter cells. In addition to checkpoint activation, the DNA damage response
leads to induction of transcriptional programs, enhancement of DNA repair
pathways, and when the level of damage is severe, to initiation of apoptosis. 6 All
of these processes are carefully coordinated so that the genetic material is
faithfully maintained, duplicated, and segregated within the cell.

Cell Cycle Checkpoints


Cell cycle checkpoints are regulatory pathways that govern the order and timing
of cell cycle transitions to ensure completion of one cellular event prior to
commencement of another. The key regulators of the checkpoint pathways in the
mammalian DNA damage response are the ATM (ataxia telangiectasia, mutated)
and ATR (ATM and Rad3-related) protein kinases. Both of these proteins belong to
a structurally unique family of serine-threonine kinases characterized by a Cterminal catalytic motif containing a phosphatidylinositol 3-kinase domain. 7,8
Although ATM and ATR appear to phosphorylate many of the same cellular
substrates,9 they generally respond to distinct types of DNA damage. ATM is the
primary mediator of the response to DNA double strand breaks (DSBs) that can
arise by exposure to ionizing radiation (IR). ATR, on the other hand, plays only a
back-up role in the DSB response, but directs the principle response to UV
damage and stalls in DNA replication.

DNA repair

Figure 2.Mammalian Cell Cycle Checkpoint Pathways. In response to DNA damage, ATM and/or ATR
trigger the activation of a checkpoint that leads to cell cycle arrest or delay. Checkpoint pathways
are characterized by cascades of protein phosphorylation events (indicated with a "P") that alter the
activity, stability, or localization of the modified proteins. A general overview of the G1, S, and G2
cell cycle checkpoint pathways is indicated (left, center, and right panels, respectively). See main
text for additional details.

G1 Checkpoint
The G1 cell cycle checkpoint prevents damaged DNA from being replicated and is
the best understood checkpoint in mammalian cells (Figure 2). 10 Central to this
checkpoint is the accumulation and activation of the p53 protein; two properties
carefully controlled by the ATM and ATR kinases. In normally growing cells, p53
levels are low due to interaction with MDM2, which targets p53 for nuclear export
and proteosome-mediated degradation in the cytoplasm. 11 Following IR damage,
ATM activates downstream kinase Chk2 (by phosphorylation at position T68), 12
which in turn phosphorylates residue S20 of p53. The S20 phosphorylation of p53
blocks p53/MDM2 interaction, resulting in p53 accumulation. ATM exerts a second
control measure on p53 stability by directly phosphorylating the p53 negative
regulator, MDM2, on S395.13 This modification allows MDM2/p53 interaction, but
prevents p53 nuclear export to the cytoplasm where degradation would normally
occur. The role of ATR in p53 S20 phosphorylation (and subsequent stabilization)
is less well established, but implied through in vitro evidence demonstrating S20
phosphorylation by the ATR-dependent kinase, Chk1.14
While phosphorylation of S20 is important to p53 stability, it is the
phosphorylation of S15 that appears crucial in enhancing p53 transcriptional
transactivation activity.15 The S15 residue of p53 can be phosphorylated directly
by ATM or ATR in response to IR (ATM and ATR), UV irradiation (ATR) and stalls of

DNA repair

DNA replication forks (ATR). Activated p53 then up-regulates a number of target
genes, several of which are also involved in the DNA damage response (MDM2,
GADD45a, and p21/Cip). The accumulation of p21, a cyclin-dependent kinase
inhibitor, suppresses Cyclin E/Cdk2 kinase activity thereby resulting in G1 arrest
(see reference 10 and references therein).

S-phase Checkpoint
The S-phase checkpoint monitors cell cycle progression and decreases the rate of
DNA synthesis following DNA damage. Although this pathway is the least
understood of the mammalian checkpoints, recent studies on IR-induced S-phase
checkpoint activation are beginning to provide important insight. Cells from
cancer-prone individuals affected with ataxia telangiectasia (AT) or Nijmegen
breakage syndrome (NBS) fail to slow their rate of DNA replication following IR
exposure; a phenomenon known as radio-resistant DNA synthesis (RDS). This
finding implicates the associated gene products (ATM and NBS1, respectively) in
the S-phase checkpoint pathway. Experimental evidence indicates that IR damage
activates the S-phase checkpoint via at least 2 parallel branches, both of which
are regulated by ATM.16-18 In the first branch, IR damage induces the
phosphorylation of the Chk2 kinase (at T68) by ATM.19 Chk2, once activated,
targets the Cdc25A phosphatase for ubiquitin-dependent degradation by
phosphorylating it on S123.19 The resultant destabilization of Cdc25A prevents it
from performing its normal function of removing inhibitory phosphorylations (T14
and Y15) from Cdk2. The Cdk2/Cyclin E and Cdk2/Cyclin A complexes remain
inactive thus preventing completion of DNA synthesis.
The second branch of the IR-induced S-phase checkpoint pathway is independent
of Cdc25A, but requires the activities of both ATM and NBS1. 16-18 Upon IR damage,
ATM phosphorylates a number of downstream substrates including NBS1 (at
multiple sites including S343), the product of the breast cancer susceptibility gene
1 (BRCA1; at multiple sites including S1387), and SMC1 (structural maintenance
of chromosome protein 1; at S957 and S966). Loss of any of these proteins or
mutation of the indicated phosphorylation sites results in attenuated S-phase
checkpoint activation.16-18,20 Interestingly, the NBS1 and BRCA1 proteins are
required for optimal phosphorylation of SMC1 upon IR, 17,18 perhaps suggesting
that a larger protein complex must be formed before ATM can phosphorylate
SMC1. Indeed, the ATM, NBS1, and BRCA1 proteins have all been shown to be
part of a mega-dalton sized protein complex referred to as BASC (BRCA1associated genome surveillance complex) that also includes numerous other DNA
repair and replication factors.21 The precise roles of these proteins, and
mechanistic details of how activation of this branch of the S-phase checkpoint
leads to reduced DNA synthesis remain to be elucidated.
The involvement of ATR in the S-phase checkpoint also remains relatively obscure.
ATR has been shown to initiate a slow IR- induced S-phase checkpoint response
by phosphorylating its effector kinase, Chk1 (on S317 and S345), which in turn
phosphorylates Cdc25A targeting it for degradation. 22 In addition, SMC1
undergoes S957 and S966 phosphorylation upon UV irradiation or hydroxyurea
treatment in an ATM-independent manner.18 The demonstrated responsiveness of

DNA repair

ATR to UV damage and replication blocks make it the leading suspect in SMC1
phosphorylation under these conditions, however, experimental proof is lacking.
Furthermore, an additional ATR-directed S-phase checkpoint pathway to deal with
UV damage and replication errors has been reported. 7

G2 Checkpoint
The G2 cell cycle checkpoint is an important control measure that allows
suspension of the cell cycle prior to chromosome segregation. Entry into mitosis is
controlled by the activity of the cyclin dependent kinase Cdc2.23 Maintenance of
the inhibitory phosphorylations on Cdc2 (on T14 andY15) is essential for G2
checkpoint activation. ATM and ATR indirectly modulate the phosphorylation status
of these sites in response to DNA damage. Unlike other checkpoints, the response
to IR is mediated primarily by ATR24 with ATM playing a back-up role; the
response to UV damage and replication blocks is controlled by ATR. It should be
noted that the stage of the cell cycle when the DNA damage occurs may influence
whether the response is mediated through ATR or ATM. 7 In any case, upon DNA
damage, the downstream kinases Chk1 and Chk2 (activated by ATR- and ATMdependent phosphorylation, respectively) phosphorylate the dual specificity
phosphatase Cdc25C on position S216.25 Phosphorylation of this residue creates a
binding site for the 14-3-3 proteins. The 14-3-3/Cdc25C protein complexes are
sequestered in the cytoplasm, thereby preventing Cdc25C from activating Cdc2
through removal of the T14 and Y15 inhibitory phosphorylations. This results in
the maintenance of the Cdc2/Cyclin B1 complex in its inactive state and blockage
of entry into mitosis.

DNA Repair Pathways


Direct Reversal The simplest of the human DNA repair pathways involves the
direct reversal of the highly mutagenic alkylation lesion O 6-methylguanine (O6mG) by the product of the MGMT gene (O6-methylguanine DNA
methyltransferase).26 The O6-mG adduct is generated in low levels by the reaction
of cellular catabolites with the guanine residues in the DNA.27 Correction of the
lesion occurs by direct transfer of the alkyl group on guanine to a cysteine residue
in the active site of MGMT in a "suicide" reaction. The inactivated alkyl-MGMT
protein is then degraded in an ATP-dependent ubiquitin proteolytic pathway.28 This
energetically expensive repair mechanism for the correction of a relatively simple
alkyl-adduct implies O6-mG is extremely detrimental to the cell. Accordingly, a
number of chemotherapeutic agents that attack the O6 position of guanine have
been developed and are in clinical use.29
BER Base excision repair (BER) is a multi-step process that corrects non-bulky
damage to bases resulting from oxidation, methylation, deamination, or
spontaneous loss of the DNA base itself.30 These alterations, although simple in
nature, are highly mutagenic and therefore represent a significant threat to
genome fidelity and stability.2
BER has two subpathways (Figure 3), both of which are initiated by the action of a
DNA glycosylase that cleaves the N-glycosidic bond between the damaged base

DNA repair

and the sugar phosphate backbone of the DNA. This cleavage generates an
apyrimidinic/apurinic (AP) or abasic site in the DNA. Eight DNA glycosylases with
partially overlapping base adduct specificity have been identified in humans. 31
Alternatively, AP sites can also arise by the spontaneous hydrolysis of the Nglycosidic bond. In either case, the AP site is subsequently processed by AP
Endonuclease 1 (APE1) which cleaves the phosphodiester backbone immediately
5' to the AP site, resulting in a 3'
hydroxyl group and a transient 5' abasic
deoxyribose phosphate (dRP). Removal
of the dRP can be accomplished by the
action of DNA polymerase beta (Pol b),
which adds one nucleotide to the 3' end
of the nick and removes the dRP moiety
via its associated AP lyase activity.32 The
strand nick is finally sealed by a DNA
ligase, thus restoring the integrity of the
DNA. Replacement of the damaged base
with a single new nucleotide as
described above is referred to as "shortpatch" repair and represents
approximately 80-90% of all BER.
The back-up pathway of BER, termed
"long-patch" repair, is employed when a
modified base resistant to the AP lyase
activity of DNA Pol beta is present in the
DNA.33 Long-patch repair results in the
replacement of approximately 2-10
nucleotides including the damaged base.
This subpathway requires many of the
Figure 3. Base Excision Repair (BER). Shown is
same factors involved in short-patch
a general model of the short patch (left) and
repair, including a DNA glycosylase,
long patch (right) BER pathways. Short patch
APE1 and DNA Pol beta. Unlike shortrepair replaces the lesion with a single
patch repair, however, long-patch repair nucleotide; long patch repair replaces the lesion
with approximately 2 to 10 nucleotides. See
is a PCNA-dependent pathway,34 where
main text for additional details.
the DNA polymerase (beta d, zeta r
epsilon) adds several nucleotides to the repair gap thus displacing the dRP as part
of a "flap" oligonucleotide. The resulting oligonucleotide overhang is excised by
the Flap endonuclease FEN-1 prior to sealing of the nick by a DNA ligase.
NER Nucleotide excision repair (NER) is perhaps the most flexible of the DNA
repair pathways considering the diversity of DNA lesions it acts upon. The most
significant of these lesions are pyrimidine dimers (cyclobutane pyrimidine dimers
and 6-4 photoproducts) caused by the UV component of sunlight. Other NER
substrates include bulky chemical adducts, DNA intrastrand crosslinks, and some
forms of oxidative damage. The common features of lesions recognized by the
NER pathway are that they cause both a helical distortion of the DNA duplex and a
modification of the DNA chemistry.35
Considerable insight into the process of human NER has been gained through the

DNA repair

study of two rare, autosomal recessive, but heterogeneous disorders - xeroderma


pigmentosum (XP) and Cockayne Syndrome (CS). Individuals affected with either
of these diseases display severe
UV hypersensitivity. Both
diseases are genetically
heterogeneous; XP is caused by
mutations in one of seven genes
(XPA to XPG) and CS is caused
by defects in one of two genes
(CSA or CSB). The XP gene
products are now known to
perform various functions during
damage recognition and DNA
incision.36 The CS gene
products, on the other hand, are
required for NER-based repair of
transcriptionally active genes.37
The NER process requires the
action of more than 30 proteins
in a stepwise manner that
includes damage recognition,
local opening of the DNA duplex
around the lesion, dual incision
of the damaged DNA strand,
gap repair synthesis, and strand
ligation (Figure 4).38 As alluded
to above, there two distinct
forms of NER: global genomic
NER (GG-NER), which corrects
damage in transcriptionally
silent areas of the genome, and
transcription coupled NER (TCNER), which repairs lesions on
Figure 4. Nucleotide Excision Repair (NER). A simplified
the actively transcribed strand model of steps in NER is shown. DNA damage recognition
of the DNA. These two
(1) differs between global genomic and transcription
subpathways are fundamentally coupled NER (GG- and TC-NER, respectively). The
subsequent steps of these processes are shared and include
identical except in their
local unwinding of the DNA (2), DNA strand dual incision
mechanism of damage
(3), and DNA repair synthesis and strand ligation (4). See
recognition. In GG-NER, the
main text for additional details.
XPC/HHR23B protein complex is
responsible for the initial detection of damaged DNA. Conversely, damage
recognition during TC-NER does not require XPC, but rather is thought to occur
when the transcription machinery is stalled at the site of injury. The stalled RNA
polymerase complex must then be displaced in order to allow the NER proteins
access to the damaged DNA. This displacement is aided by the action of the CSA
and CSB proteins, as well as other TC-NER-specific factors. The subsequent steps
of GG- and TC-NER proceed in an essentially identical manner. XPA and the
heterotrimeric replication protein A (RPA) then bind at the site of injury and
further aid in damage recognition. Next, the XPB and XPD helicases, components
of the multi-subunit transcription factor TFIIH, unwind the DNA duplex in the

DNA repair

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