Results Probl Cell Differ (42)
P. Kaldis: Cell Cycle Regulation
DOI 10.1007/b136684/Published online: 14 July 2005
© Springer-Verlag Berlin Heidelberg 2005
Checkpoint and Coordinated Cellular Responses
to DNA Damage
Xiaohong H. Yang
1
·LeeZou
1,2
(✉)
1
MGH Cancer Center, Harvard Medical School, Charlestown, MA 02129, USA

2
Department of Pathology, Harvard Medical School, Charlestown, MA 02129, USA

Abstract The DNA damage and replication checkpoints are signaling mechanisms that
regulate and coordinate cellular responses to genotoxic conditions. The activation of
checkpoints not only attenuates cell cycle progression, but also facilitates DNA repair
and recovery of faulty replication forks, thereby preventing DNA lesions from being con-
verted to inheritable mutations. It has become increasingly clear that the activation and
signaling of the checkpoint are intimately linked to the cellular processes directly in-
volved in chromosomal metabolism, such as DNA replication and DNA repair. Thus, the
checkpoint pathway is not just a surveillance system that monitors genomic integrity and
regulates cell proliferation, but also an integral part of the processes that work directly
on chromosomes to maintain genomic stability. In this article, we discuss the current
models of DNA damage and replication checkpoints, and highlight recent advances in the
field.
1
Introduction

pathway, as well as the mechanisms by which the checkpoint regulates key
downstream processes such as DNA replication, DNA repair, and chromatin
modulation. In this review, we will discuss an updated model of checkpoint
signaling that begins to explain how different processes involved in the main-
tenance of genomic stability are integrated and coordinated by the checkpoint
pathway.
2
Sensing DNA Damage and DNA Replication Stress
ATM ( ataxia telangiectasia mutated)andATR(ATM- an d R a d3 - related)are
two large PI3K-like protein kinases that play central roles in the checkpoint-
signaling pathway (Abraham 2001). In response to DNA damage, ATM and
ATR phosphorylate Chk1 and Chk2, two downstream effector kinases, and
numerous substrates involved in various cellular processes (e.g. p53, Brca1,
Nbs1). The DNA damage specificities of ATM and ATR are distinct from
each other. While ATM primarily responds to double-strand DNA breaks
(DSBs), ATR is involved in the responses to DSBs as well as a broad spec-
trum of DNA damage caused by DNA replication interference. Although ATM
is important for genomic stability, patients, mice, and cells lacking ATM are
viable (Kastan and Bartek 2004), suggesting that ATM is not essential for nor-
mal cell proliferation in the absence of significant DSBs. On the other hand,
ATR is indispensable for the proliferation of human and mouse cells (Brown
and Baltimore 2000; Cortez et al. 2001). These findings indicate that ATR
has a critical role even in normal cell cycles, and that the function of ATR
might be regulated by certain DNA structures generated by intrinsic DNA
metabolism.
Checkpoint and Coordinated Cellular Responses to DNA Damage 67
2.1
Recruitment of ATR to DNA
What is the DNA structure sensed by the ATR kinase? Several important clues
came from the studies of Mec1, the budding yeast homologue of ATR (see

and other Brca1/53BP1/Mdc1 Rad9
signaling molecules TopBP1 Dpb11
hTim1 Tof1
hTipin Csm3
Effector kinase Chk1 Chk1
Chk2 Rad53
68 X.H. Yang · L. Zou
recruit the ATR-ATRIP kinase complex. Consistent with this idea, increased
amountsofssDNAwasalsoobservedatDNAreplicationforkshaltedbyhy-
droxyurea (HU) treatment (Sogo et al. 2002), suggesting that ssDNA might
also be important for the activation of Mec1 by replication fork stalling.
Studies using Xenopus egg extracts have also revealed important clues
of how ATR is recruited to DNA. In Xenopus extracts, ATR associates with
chromatin during S-phase in a replication-dependent manner (Hekmat-Nejad
et al. 2000). Depletion of RPA, an ssDNA-binding protein complex essential
for DNA replication, abolished the chromatin association of ATR (You et al.
2002), suggesting that RPA is either directly or indirectly required for the re-
cruitment of ATR to chromatin. Similarly, in Xenopus extracts RPA is also
needed for the recruitment of ATR to DNA lesions generated by etoposide
(Costanzo et al. 2003), a DNA topoisomerase II inhibitor. This finding indi-
catesthatRPAitselfortheDNArepairprocessinvolvingRPAisrequiredfor
ATR recr uit ment.
In human cells, RPA is required for the localization of ATR to DNA
damage-induced nuclear foci and the efficient phosphorylation of Chk1 by
ATR (Zou and Elledge 2003). In yeast, depletion of RPA in the cells arrested
in G2 abolished the localization of Ddc2 to the HO-induced DSBs (Zou and
Elledge 2003), suggesting that RPA is required for the recruitment of Ddc2
to DNA damage in vivo and its function is independent of its role in DNA
replication. Indeed, rfa1-t11, a mutant of RPA that is proficient for DNA repli-
cation but partially defective for checkpoint responses (Umezu et al. 1998;

DNA at stalled forks. Although the checkpoint can be activated through different mech-
anisms, cell cycle arrest and inhibition of DNA replication are common results. The
activated checkpoint may play important roles at DSBs and stalled replication forks to
facilitate DNA repair and fork recovery
other proteins cannot be ruled out. Furthermore, it is also possible that
the interaction between ATR-ATRIP and RPA-ssDNA is regulated by other
proteins in vivo (see below). It was recently reported that ATR-ATRIP can
associate with proteins such as Claspin, Msh2 and Mcm7 (Chini and Chen
2003; Wang and Qin 2003; Cortez et al. 2004). It is possible that the interac-
tions of ATR-ATRIP with additional proteins on DNA also contribute to the
localization of ATR-ATRIP to specific types of DNA damage.
2.2
DNA Damage Recognition by the RFC- and PCNA-like Checkpoint Complexes
Although ssDNA plays a crucial role in the recruitment of ATR-ATRIP, ss-
DNA alone is not sufficient to elicit the checkpoint responses, suggesting that
additional DNA structures induced by DNA damage are also necessary for
the activation of ATR-ATRIP. In addition to ATR-ATRIP itself, several other
checkpoint proteins are also required for the initiation of checkpoint signal-
ing. In human cells, these proteins include Rad17, Rad9, Rad1, and Hus1.
Rad17 is a homologue of all five subunits of RFC, and its forms an RFC-like
protein complex with the four small subunits of RFC (Lindsey-Boltz et al.
2001; Shiomi et al. 2002; Ellison and Stillman 2003; Zou et al. 2003). Rad9,
Rad1, and Hus1, on the other hand, are all structurally related to PCNA
(Venclovas and Thelen 2000), and they assemble into a hetero-trimeric ring-
shaped complex (termed the 9-1-1 complex) resembling PCNA (Volkmer and
Karnitz 1999). Ablation of Rad17 or the 9-1-1 complex in human or mouse
cells resulted in severe chromosomal instability and failures in activating the
ATR-mediate checkpoint (Weiss et al. 2002; Zou et al. 2002; Roos-Mattjus et al.
2003; Wang et al. 2003; Bao et al. 2004; Loegering et al. 2004). During DNA
replication, RFC specifically recognizes the 3

servations, purified Rad17 complexes recruit 9-1-1 complexes onto primed
ssDNA or gapped DNA structures in an RPA-dependent manner (Ellison and
Stillman 2003; Zou et al. 2003) (Fig. 1a, b). Unlike RFC, which only uses the
3

dsDNA/ssDNA junctions to load PCNA, the Rad17 complex can apparently
use the 5

dsDNA/ssDNA junctions to recruit 9-1-1 complexes, providing
a possible explanation of the DNA damage specificity of the Rad17 complex.
Together, the studies described above have revealed that the ATR-ATRIP,
Rad17, and 9-1-1 complexes can independently recognize damage-induced
DNA structures such as ssDNA and junctions of dsDNA/ssDNA. Moreover,
RPA appears to play important roles in the damage recognition by both the
ATR-ATRIP and the Rad17 complexes. Once recruited to the sites of DNA
damage, the Rad17 and 9-1-1 complexes might facilitate the recognition and
phosphorylation of ATR substrates by interacting with ATR-ATRIP and/or
other proteins at the damage sites. Alternatively, the Rad17 and 9-1-1 com-
plexes might stimulate the kinase activity of ATR on DNA. Although studies
using Xenopus extracts suggested that ATR can be activated on DNA (Ku-
magai et al. 2004), how ATR is stimulated by DNA or its regulators on DNA
remains to be elucidated.
2.3
Processing of DNA Lesions
Single-stranded DNA is a common DNA structure generated by DNA repli-
cation and many types of DNA repair processes. The discovery that ssDNA
plays a crucial role in the activation of ATR provides a plausible explanation
for why ATR can respond to different types of DNA damage. Furthermore, it
clearly suggests that the processing of DNA lesions plays an important role in
the activation of ATR. Many cellular processes involved in the maintenance

2000; Lupardus et al. 2002), suggesting that junctions of dsDNA/ssDNA might
also accumulate at the stressed forks and they might facilitate the functions of
Rad17 and 9-1-1 complexes.
DNA repair also plays important roles in the processing of DNA lesions
and the activation of checkpoint. In yeast, the DSBs generated by the HO en-
donuclease are recessed by exonucleases in the 5

-to-3

direction. The reces-
sion of DSBs requires Xrs2, the yeast homologue of the human Nbs1 protein,
and Exo1 (exonuclease I) (Nakada et al. 2004). The generation of ssDNA at
the ends of DSBs not only presents a signal for Mec1-Ddc2 activation, but
also creates an important DNA intermediate for homologous recombination
(Wang and Haber 2004). Furthermore, the recession of DSBs is controlled
by the cyclin-dependent kinase Cdk1 (Ira et al. 2004). Hence, the processing
of DSBs in yeast cells has presented a clear example how the activation of
checkpoint by a particular type of DNA damage is coupled to a specific DNA
repair pathway as well as the cell cycle regulatory apparatus. Certain types of
Checkpoint and Coordinated Cellular Responses to DNA Damage 73
DNA damage might be sensed by both replication-dependent and replication-
independent mechanisms. For example, in the cells arrested in G0, G1 or
G2, UV-induced DNA damage can activate the checkpoint in a NER (nu-
cleotide excision repair)-dependent manner (Neecke et al. 1999; O’Driscoll
et al. 2003). Moreover, the endonuclease and helicase involved in NER are
needed to process the DNA lesions into structures that can elicit checkpoint
responses (Giannattasio et al. 2004). In addition to processing DNA lesions,
many DNA repair proteins also physically interact with the checkpoint sen-
sors. For example, the NER protein Rad14 associates with the 9-1-1 complex
in yeast (Giannattasio et al. 2004), and the mismatch repair protein Msh2

ciates from its multimeric form to become monomers (Bakkenist and Kastan
2003). The monomerization of ATM appears to be an important step for its
74 X.H. Yang · L. Zou
activation. Intriguingly, the autophosphorylation of ATM can be induced by
very low doses of DNA damage or treatments disrupting chromatin structures
in the absence of detectable DSBs, leading to the hypothesis that ATM might
be activated by changes of chromatin structures (Bakkenist and Kastan 2003).
Although monomeric ATM is capable of phosphorylating non-DNA-bound
substrates such as p53, the phosphorylation of other substrates at the sites of
DSBs requires the MRN complex and Brca1 (Kitagawa et al. 2004). The mech-
anisms by which the autophosphorylation of ATM is regulated are yet to be
elucidated. Proteins including Nbs1, 53BP1, Mdc1, PP5, PP2A, and p18 might
be involved in the regulation of ATM autophosphorylation at various stages
of signaling (Mochan et al. 2003; Uziel et al. 2003; Ali et al. 2004; Goodarzi
et al. 2004; Park et al. 2005). Elevated kinase activity of ATM can be detected
in vitro after DNA damage (Canman et al. 1998). A study by Paull’s laboratory
demonstrated that the MRN complex stimulates the phosphorylation of ATM
substrates in vitro even in the absence of DNA (Lee and Paull 2004). Although
this study did not reveal the contribution of DSBs in the activation of ATM,
it clearly shows a direct role of the MRN complex in the stimulation of ATM.
Together these recent findings suggest that the activation of ATM is a multi-
step process that involves the autophosphorylation of ATM, the interactions
of ATM with other regulatory factors, and the localization of ATM to DSBs.
The MRN complex is not only important for the localization of ATM to DSBs,
but also critical for the activation of the kinase activity of ATM.
The MRN complex might also be involved in the activation of ATR (Car-
son et al. 2003; Pichierri and Rosselli 2004; Stiff et al. 2005). The budding and
fission yeast mutants lacking the MRN complex display defective checkpoint
responses after HU or MMS treatments (D’Amours and Jackson 2001; Chah-
wan et al. 2003). In human cells, like ATR, the MRN complex is implicated in

addition to the Rad17 and 9-1-1 complexes, the phosphorylation of Chk1 re-
quires Claspin, Mcm7, Brca1, CtIP, and TopBP1 (Kumagai and Dunphy 2000;
Yarden et al. 2002; Yamane et al. 2003; Cortez et al. 2004; Lin et al. 2004; Tsao
et al. 2004; Yu and Chen 2004). Because these groups of proteins function to
mediate the DNA damage signals between ATM/ATR and C h k 1 /Chk2, they
were appropriately termed “mediators”. However, the exact biochemical ac-
tivities of these proteins in checkpoint signaling are not known. The mediator
proteins do share several features that might be important for their func-
tions in signaling. First, most if not all mediators are present at the sites of
DNA damage or stalled replication forks. Some mediators, such as Claspin
and Mcm7, are probably components of DNA replication forks (Chini and
Chen 2003; Lee et al. 2003), whereas others, such as Mdc1, 53BP1, and Brca1,
are recruited to sites of DNA damage (Scully et al. 1997; Schultz et al. 2000;
Goldberg et al. 2003; Lou et al. 2003; Peng and Chen 2003; Stewart et al. 2003;
Xu and Stern 2003). Second, most mediator are themselves substrates of ATM
or ATR after DNA damage (Tibbetts et al. 2000; Chini and Chen 2003; Stew-
art et al. 2003). Third, many mediators possess phospho-serine/threonine-
binding motifs, such as the BRCT and the FHA motifs (Durocher et al. 1999;
Manke et al. 2003; Rodriguez et al. 2003; Yu et al. 2003). It has been shown that
certain mediators can associate with each other or with downstream kinase
in a phosphorylation-dependent manner (Kumagai and Dunphy 2003; Yu and
Chen 2004), suggesting that the damage-induced phosphorylation of these
mediators and their phospho-Ser/Thr-binding motifs might be involved in
organizing the protein complex at the sites of DNA damage and/or recruiting
downstream kinases. Consistent with the idea that mediators are important
for the recruitment of downstream kinases to ATM/AT R, cer tain mediators
in budding and fission yeast can be partially bypassed by fusing the down-