rity by inducing replication fork
stalling and DNA breaks as well
as mutagenesis due to the un-
equal competition between the
dNTPs. Instead, high dNTP lev-
els can impair the polymerase
proofreading activity or pro-
mote polymerase slippage, also
leading to mutagenesis. Hence,
dNTP pool changes can promote
tumorigenesis, framing RNR as
an important target for antican-
cer drugs (6). At high doses, HU
induces replication fork stalling
and activates the DNA dam-
age checkpoint with dramatic
consequences on origin firing.
These include the global inhibition of new origin firing and the
local activation of the so-called
“dormant” origins to enable full
chromosome duplication while
preventing further replication fork stalling
(7). Given that dNTPs are limiting for replication fork progression, treatment with
high HU also indirectly affects replication
fork speed (8, 9).
However, Somyajit et al. show that a low
dose of HU, insufficient to stall replication
forks or activate the DNA damage checkpoint,
was able to modulate replication fork speed
directly, rapidly, and independently of dNTP
levels. This occurs by the reaction catalyzed
by RNR itself. RNR converts ribonucleoside
59-diphosphates (NDPs) into 29-deoxyribo-
nucleoside 59-diphosphates (dNDPs) via an
electron transfer that, when inhibited by
HU, causes a redox imbalance. The observed
replication fork slowdown correlated with a
rapid dissociation of different components
of the replication protection complex, such
as TIMELESS and TIPIN, from replication
forks. Consistently, several studies with the
yeast orthologs of the replication protection
complex have shown that fork rates are reduced in their absence in vivo (10) and accelerated by their presence in vitro (3). Both
the slow replication forks and dissociation
of TIMELESS that were observed with low
doses of HU were rescued by quenching ROS
but not by adding exogenous dNTP precursors, a result that could be recapitulated with
hydrogen peroxide (H2O2) (5), which argues
that it is redox imbalance that triggers TIMELESS dissociation from the replisome and the
resulting replication fork slowdown.
ROS are produced during aerobic metabolism and include H2O2, superoxide (O2–), and
hydroxyl radicals (OH−). In addition to the
pathological consequences of ROS inherent
to their high reactivity, subtle changes in
ROS elicit signaling responses that are key
events in physiological processes such as
cellular differentiation, tissue regeneration,
and prevention of aging (11). Importantly,
the coupling between fork speed and redox
signaling is mediated by the interaction between TIMELESS and one component of
this redox signaling response, peroxiredoxin
2 (PRDX2). This is an antioxidant enzyme
from the peroxiredoxin family, members of
which are highly sensitive to subtle fluctuations in ROS levels. Somyajit et al. propose
that oxidation of PRDX2 oligomers, which
occur on exposure to ROS, induces their
disruption and dissociation from chromatin, dragging out TIMELESS-TIPIN from
ongoing replisomes and thus slowing down
replication fork progression in an oxidizing
environment (see the figure).
Failures in this coupling mechanism (as
in PRDX2-deficient cells) lead to replication-dependent and TIMELESS-mediated genetic
instability, as observed by the increased occurrence of DNA lesions typically derived
from replicative stress, such as ultrafine anaphase DNA bridges and p53 binding protein
1 (53BP1) nuclear bodies (5). The concept of
replicative stress may thus need to be revisited beyond replication fork slowdown and/
or stalling to include also replication fork
acceleration. This indicates that the PRDX2-
TIMELESS interaction prevents the damaging consequences to DNA of metabolite
oscillations. Notably, cancer cells show high
levels of ROS and slow replication forks
and are highly sensitive to PRDX2 inactivation, implying that they exploit this coupling
mechanism to allow tumor cell survival (5).
The necessity of this pathway for tumor cell
survival argues that it is not a frequent oncogenic event and supports that ROS-induced
replicative stress is not a main driver for tumorigenesis (12).
Thus, the coupling of redox signaling
with fork speed emerges as an effective
strategy to prevent the negative conse-
quences of oxidative stress on replication
in strictly aerobic eukaryotic cells, whereas
anaerobic facultative cells such as yeast
can temporarily separate DNA synthesis
from ROS-generating respiration processes
(13). Because cancer cells use this coupling
mechanism to bypass the abnormally high
oxidative stress derived from their altered
metabolism, it seems obvious to see it as a
potential target in chemotherapy. It will be
interesting to investigate how this mecha-
nism affects DNA repair insufficiency–
associated manifestations such as prema-
ture aging and genetic diseases (14) and at
vulnerable DNA regions such as telomeres
or sites of transcription-replication colli-
sions, such as ribosomal DNA or fragile
sites (15). The need for a mechanism of
fork speed regulation to counteract redox
imbalance supposes a new twist in our un-
derstanding of the cellular mechanisms de-
voted to safeguard genome integrity. j
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3. J. T. Yeeles et al., Mol. Cell 65,105(2017).
4. J. T. Yeeles et al., Nature 519,431(2015).
5. K. Somyajit etal. ,Science 358, 797 (2017).
6. Y. Aye et al., Oncogene 34, 2011 (2015).
7. M. Yekezare et al. , J. Cell Sci. 126, 1297 (2013).
8. J. Poli etal.,EMBOJ.31, 883 (2012).
9. H. Técher et al., Cell Rep. 14, 1114 (2016).
10. S. P. Bell, K. Labib,Genetics 203, 1027 (2016).
11. M. Schieber, N. S. Chandel, Curr. Biol. 24, R453 (2014).
12. J. Bartkova et al. , Nature 434, 864 (2005).
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A.A.’s laboratory is funded by the European Research Council,
Spanish Ministry of Economy and Competitiveness, Junta de
Andalucía, Worldwide Cancer Research and European Regional
Funds (FEDER). B.G.-G. is a postdoctoral fellow of the Scientifc
Foundation of the Spanish Association Against Cancer (AECC).
To enable timely duplication of the full genome, the
presence of TIMELESS-TIPIN at the replisome, where
it interacts with PRDX2, speeds up replication.
After subtle changes in ROS, PRDX2 oxidation disrupts its
oligomer conformation and dissociates TIMELESS-TIPIN
from ongoing replisomes, causing fork slowdown.
10 NOVEMBER 2017 • VOL 358 ISSUE 6364 723
Cells adjust replication fork speed to the redox state
Replication forks slow down in the presence of ROS to safeguard genome integrity. Cancer cells, with an abnormally high
oxidative stress derived from their altered metabolism, use this mechanism of fork slowdown for survival.