DNA DAMAGE CHECKPOINTS AND THE ROLE OF
PHOSPHORYLATION, PROTEOLYSIS AND INTRA-NUCLEAR
PROTEIN DYNAMICS IN THEIR REGULATION
Jiri Lukas
Danish Cancer Society, Institute of Cancer Biology, Strandboulevarden 49,
DK-2100 Copenhagen, Denmark
[email protected]
INTRODUCTION. DNA lesions severely undermine genome integrity and may
lead to unrestricted cell proliferation, a hallmark of cancer. To protect the
genome, eukaryotic cells evolved surveillance signal transduction pathways, socalled ‘checkpoints’ that delay cell cycle progression and facilitate DNA repair
(1). Among the damage types, DNA double strand breaks (DSB) represent the
most severe (often lethal) lesion. The checkpoint response to DSB is a cascade of
phosphorylation events initiated by ataxia-telangiectasia-mutated (ATM), a
member of the evolutionarily conserved family of phosphatidyl inositol 3-like
kinases (2). Several ATM substrates have been identified (2) and shown to play
pivotal roles in the complex cellular response to DSB including inhibition of cell
cycle progression (p53, Nbs1, Chk1, Chk2), DNA repair (Nbs1, BRCA1),
activation of the damage-induced transcription program (p53, BRCA1),
recombination (Nbs1, Smc1), and apoptosis (p53, Mdm2).
Our laboratory contributed to this field by elucidating the signalling pathways
that lead to a rapid cell cycle arrest in response to DSB-generating insults such as
ionizing radiation (IR). The salient features of this pathway involve sequential
activation of ATM (and/or the related ATR), amplification of the signal by Chk1
and Chk2 kinases, ubiquitin/proteasome-mediated degradation of the Cdc25A
phosphatase, and inhibition of cyclin-dependent kinases (CDKs). Lack of CDK
activity prevents initiation of DNA replication origins thereby blocking S-phase
entry and/or delaying the ongoing S-phase progression (3-5). Interestingly,
ATM-dependent and Nbs1-mediated phosphorylation of the cohesin Smc1 is also
required for a transient cessation of DNA replication in response to ionizing
radiation by a mechanism largely independent of (and parallel to) the DNA
damage-induced CDK inhibition (6-8).
QUESTIONS. The complexity of the signalling network downstream of ATM
poses high demands for coordinating the spatio-temporal distribution of the
ATM-activated effector molecules and raises important conceptual questions.
For instance, why do mammalian cells need two signalling branches to slow
down DNA replication in response to DSB? And how do we reconcile with the
fact that while only a few DSB are able to activate most of the nuclear ATM (9),
inhibition of DNA replication after IR is dose dependent raising up to doses
corresponding to hundreds of DSB per a single cell nucleus? These and related
questions are difficult to address by the available genetic and/or biochemical
analytical approaches. However, combination of these with an insight into how
the checkpoint-associated biochemical reactions and molecular interactions
operate in live cell nuclei can offer surprising solutions.
METHODS. We have combined microlaser-based generation of DBS
restricted to defined subnuclear volumes with a variety of interactive photobleaching analyses. Using these tools and cell lines expressing ATM
effectors tagged with various GFP spectral variants we have been
investigating the dynamics of the checkpoint response in live cells.
RESULTS AND CONCLUSIONS. Although many ATM targets are likely
dedicated solely to the ‘local’ recognition, processing and repair of DSB, their
accumulation does not represent a static interaction or aggregation of the
respective proteins on damaged DNA (10). For instance, Nbs1, one of the earliest
DSB-interacting proteins, undergoes a surprisingly dynamic exchange between
the DSB site and the surrounding undamaged nucleoplasm (Fig. 1a). The
cytologically apparent accumulation of Nbs1 as the IR-induced nuclear foci is
due to only a slight increase in affinity of the Nbs1 protein to the abnormal DNA
structures and/or other DSB-associated proteins such as Mdc1. These
observations are consistent with a model whereby various enzymatic complexes
undergo a rapid exchange on the sites of DNA lesions in a competitive fashion.
This provides an opportunity for a greater flexibility in detecting and repairing
the DNA lesion and allows dynamic monitoring of the DNA and/or chromatin
status by sensor mechanisms. We also have evidence that the ATMphosphorylated Nbs1 is not exported much beyond the actual DSB-containing
nuclear sub-compartments, suggesting that the contribution of the Nbs1-Mre11Rad50 complex to the intra-S-phase checkpoint is restricted to those replicons
that are located within the DSB-surrounding chromosome regions. Real-time
imaging of checkpoint dynamics further revealed that a smaller group of proteins
possess the ability to act in a more ‘global’ fashion in order to spread the signal
from the DSB site(s) to support DNA damage-induced pan-nuclear responses
(10). Thus, the Chk2 kinase appears to be unique among checkpoint transducers
in a sense that it, after a transient interaction with and activation at the DSB sites
rapidly distributes throughout the nucleus (Fig. 1b). It is plausible that spreading
the DSB-activated Chk2 (and likely also Chk1) kinases throughout the nucleus
helps target essential nuclear factors (such as cyclin/CDK complexes) that
themselves do not interact with DSB but that have the potential to control
initiation events of replication-competent origins in the undamaged nuclear
compartments. Finally, the recent upgrade of our imaging workstation allowed us
to measure the dynamics of the checkpoint response within seconds after DSB
generation. We will present evidence that the distribution of ATMphosphorylated (activated) effector proteins in diverse nuclear compartments is
strictly dependent on the orderly recruitment and assembly of the proximal
checkpoint mediators on the sites of DSB lesions.
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Fig. 1. Distinct spatio-temporal modes of ATM-activated checkpoint effectors.