Essential role of
of ribonucleotide reductase
at DNA damage sites
in DNA repair during G1 phase
Hiroyuki Niida,1Yuko Katsuno,1
Misuzu Sengoku,1Midori Shimada,1
Megumi Yukawa,1Masae Ikura,2Tsuyoshi Ikura,2
Kazuteru Kohno,3Hiroki Shima,3
Hidekazu Suzuki,3Satoshi Tashiro,3
and Makoto Nakanishi1,4
1Department of Cell Biology, Graduate School of Medical
Sciences, Nagoya City University Medical School, Nagoya
467-8601, Japan;2Radiation Biology Center, Kyoto University,
Kyoto 606-8501, Japan;3Department of Cell Biology, Research
Institute for Radiation Biology and Medicine (RIRBM),
Hiroshima University, Hiroshima 734-8553, Japan
A balanced deoxyribonucleotide (dNTP) supply is essen-
tial for DNA repair. Here, we found that ribonucleotide
reductase (RNR) subunits RRM1 and RRM2 accumu-
lated very rapidly at damage sites. RRM1 bound physi-
cally to Tip60. Chromatin immunoprecipitation analyses
of cells with an I-SceI cassette revealed that RRM1 bound
to a damage site in a Tip60-dependent manner. Active
RRM1 mutants lacking Tip60 binding failed to rescue an
impaired DNA repair in RRM1-depleted G1-phase cells.
Inhibition of RNR recruitment by an RRM1 C-terminal
fragment sensitized cells to DNA damage. We propose
that Tip60-dependent recruitment of RNR plays an es-
sential role in dNTP supply for DNA repair.
Supplemental material is available at http://www.genesdev.org.
Received September 15, 2009; revised version accepted
December 22, 2009.
Maintenance of the optimal intracellular concentrations
of deoxyribonucleotides (dNTPs) is critical not only for
faithful DNA synthesis during DNA replication and
repair, but also for the survival of all organisms. Ribonu-
cleotide reductase (RNR), composed of a tetrameric com-
plex of two large catalytic (RRM1) subunits and two small
subunits (RRM2 or 53R2), catalyzes de novo synthesis of
dNTPs from the corresponding ribonucleotides (Reichard
1993). This reaction is the rate-limiting process in DNA
precursor synthesis and is regulated by multiple complex
mechanisms, including transcriptional and subcellular
localization regulation of RNR (Nordlund and Reichard
2006). In order to duplicate their chromosomal DNA,
mammalian S-phase cells possess 15–20 times more
dNTP pools than resting quiescent cells, whereas whole
dNTP pools were almost unchanged after DNA damage,
suggesting the presence of a unique mechanism that
supplies a sufficient quantity of dNTPs at repair sites
(Hakansson et al. 2006). DNA synthesis must function
properly in both repair and replication (dNTP concentra-
tions in fibroblasts were estimated to be as follows: ;0.5
mM in G0/G1-phase cells, and ;10 mM in S-phase cells,
given that the average volume of a fibroblast is 3.4 pL)
(Imaizumi et al. 1996). Although the amount of dNTPs
required for DNA repair is small, their concentration
during DNA synthesis is critical because DNA poly-
merase involved in DNA repair (Kraynov et al. 2000;
Johnson et al. 2003) has similar kinetic affinities for
dNTPs (;10 mM) to those involved in DNA replication
(;10 mM) (Dong and Wang 1995). Therefore, the dNTPs
might be compartmentalized close to the damage sites
during the DNA repair process. In this study, we show
that, in mammals, both RRM1 and RRM2 rapidly accu-
mulated at double-strand break (DSB) sites in a Tip60-
Results and Discussion
In order to understand the mechanisms by which dNTPs
are sufficiently supplied at DNA damage sites in mam-
mals, we first examined changes in the subcellular
localization of RRM1 and RRM2 subunits after ionizing
irradiation (IR) irradiation. Although both RRM1 and
RRM2 predominantly localized in the cytoplasm as
reported previously (Pontarin et al. 2008), we also
detected trace, but significant, signals of both proteins
in chromatin fraction (see Fig. 1C; Supplemental Fig.
S4A–D). After removing soluble RNR proteins by de-
tergent extraction, we found that RRM1 and RRM2
proteins formed nuclear foci that colocalized with
gH2AX (Fig. 1A). RRM1 nuclear foci were not evident
without DNA damage (Supplemental Fig. S1A) or after
RRM1 depletion by siRNA (Supplemental Fig. S1B).
Ultravioulet A (UVA) microirradiation resulted in the
accumulation of RRM1 and RRM2 along microirradiated
lines as early as 5 min after treatment (Fig. 1B). These
accumulations were also observed when cells were not
subjected to detergent extraction or preincubation with
BrdU (Supplemental Fig. S2A,B), but were significantly
compromised when R1 expression was knockdown by
siRNA (Supplemental Figs. S2C, S4B), excluding the
possibility that accumulated signals at DSB sites were
artifacts during cell-staining processes. These results
indicated that RNR, at least in part, was rapidly recruited
to DSB sites.
In order to determine the molecular basis underlying
RNR recruitment at the sites of DSBs, we performed
of 5 3 106transformants from a HeLa cell cDNA library,
45 positive colonies were confirmed to be lacZ-positive.
They contained overlapping cDNAs derived from three
genes: RRM2 and 53R2 (both encoding a small subunit of
RNR), and another encoding Tip60 histone acetyltransfer-
ase (Tip60). Small C-terminal RRM1 deletion mutants
(D761-C and D781-C) failed to bind Tip60, but retained the
ability to bind to RRM2 (Supplemental Fig. S3A). In
contrast, the N-terminal truncation mutant of Tip60
[Keywords: DNA repair; ribonucleotide reductase; Tip60; dNTPs; geno-
mic instability; DNA double-strand breaks]
E-MAIL firstname.lastname@example.org, FAX 81-52-842-3955.
Article is online at http://www.genesdev.org/cgi/doi/10.1101/gad.1863810.
GENES & DEVELOPMENT 24:333–338 ? 2010 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/10; www.genesdev.org333
(TC2) could interact with RRM1, but no mutant with any
additional truncation of TC2 was able to do so (Supple-
mental Fig. S3B). Full-length Tip60 failed to bind full-
length RRM2 (Supplemental Fig. S3C). We generated the
C-terminal fragment of RRM1 (amino acids 701–792) with
a SV40 nuclear localization signal (NLS-RC1-HA) and
examined its ability to bind Tip60 in vivo and in vitro.
NLS-RC1-HA, but not a control NL-GFP-HA fragment,
was detected in the anti-Myc immunoprecipitates when
transiently coexpressed with Tip60-Myc (Supplemental
Fig. S3D). Purified MBP-fused RC1 produced in Escher-
ichia coli was capable of binding to GST-Tip60 expressed
in insect cells (Supplemental Fig. S3E). Both D761-C and
D781-C failed to bind chromatin, further confirming that
the binding of RRM1 to chromatin required its interaction
with Tip60 (Supplemental Fig. S3F).
Similarly to Chk1 (Niida et al. 2007; Shimada et al.
2008), endogenous RRM1 was present in cytosolic (S1),
nucleoplasmic (S2), and chromatin-bound (P2) fractions
(Supplemental Fig. S4A). Tip60 existed predominantly in
the chromatin-bound fraction (P2). Both RRM1 and Tip60
proteins in this fraction were partly solubilized by treat-
ment with micrococcal nuclease (Mnase), suggesting that
they associated with chromatin. RRM1 knockdown
showed a significant decrease of RRM1 protein levels in
both soluble and chromatin-bound fractions (Supplemen-
tal Fig. S4B). IKKa and Orc2 were detected predominantly
in soluble and chromatin fractions, respectively, indicat-
ing that cell fractionation was done successfully. Ectopic
RRM1-HA present in the chromatin fraction was in-
creased when Tip60-Myc-His was coexpressed, although
a low level of RRM1-HA was detected in the absence of
Tip60-Myc-His, presumably due to the presence of en-
dogenous Tip60 (Supplemental Fig. S4C). The amounts of
RRM1 and Tip60 bound to the chromatin were not af-
fected by DNA damage (Supplemental Fig. S4D). How-
ever, depletion of Tip60 resulted in a reduction in the
amount of RRM1 on chromatin (Fig. 1C). Taken together,
chromatin binding of RRM1 appeared to be Tip60-
dependent. RRM1-HA, but not the RRM2 subunit alone,
formed a complex with GST-His-Tip60 in insect cells
(Fig. 1D, left panels). RRM2 also formed a complex with
GST-His-Tip60 in a manner dependent on the presence of
RRM1-HA. Consistently, accumulation of RRM2 at DSB
sites was compromised when RRM1 was depleted (Sup-
plemental Fig. S2D). Immunoprecipitations using anti-
HA antibodies demonstrated that RRM1-HA bound to
both RRM2 and GST-His-Tip60 (Fig. 1D, right panels).
RRM1 and RRM2 were detected in the precipitates of
anti-Tip60 antibodies from the solubilized chromatin,
even in the absence of DNA damage (Fig. 1E). To further
confirm the interaction between RNR and Tip60, we
purified the Tip60 complex from HeLa cell nuclear
extracts expressing Flag-HATip60 as reported previously
(Ikura et al. 2000, 2007). RRM1 and RRM2, as well as
PAF400/TRRAP as a positive control (Murr et al. 2006),
were detected in Tip60 complex from extracts with or
without DNA damage (Fig. 1F). Tip60 knockdown by
siRNA or shRNA abrogated accumulation of RRM1 along
with microirradiated lines (Fig. 1G; Supplemental Fig.
S2E). These results suggested that RRM1 recruitment at
DSB sites was Tip60-dependent.
To determine precisely whether RRM1 was recruited
at the site of DNA damage, we generated Ku-deficient
mouse embryonic fibroblasts (MEFs) in which a single
DSB was introduced after infection with adenoviruses
expressing I-SceI. This DSB was not rapidly repaired by
nonhomologous end-joining, making it easy to detect pro-
teins accumulating at this DSB site by chromatin immu-
noprecipitation (ChIP) analysis (STEFKu70?/?phprt-DR-
GFP) (Fig. 2A; Pierce et al. 2001). Introduction of the DSB
was confirmed by Southern blotting (Supplemental Fig.
S5). ChIP analyses revealed a substantial increase in the
binding of RRM1 as well as Rad51 and Tip60 to a DNA
break site. An increase in acetylation of histone H4 was
also observed at the damage site (Fig. 2B). These were not
seen on infection with control LacZ. Tip60 depletion by
two independent siRNAs resulted in a loss of RRM1
binding to a DSB site, as well as a reduction in acetylation
of histone H4 (Fig. 2C). A mutant Tip60 lacking histone-
acetylating activity could recruit RRM1 to the DSB site
similarly to wild-type RRM1 (Supplemental Fig. S6A).
Inhibition of ATM, ATR, and DNA-PK by caffeine did
not affect RRM1 recruitment (Supplemental Fig. S6B).
These results further supported the notion that complex
HeLa cells were exposed to IR at 1 Gy, subjected to in situ detergent
extraction after 5 min, and immunostained with the indicated
antibodies. Bars, 5 mm. (B) GM02063 cells were subjected to UVA
microirradiation and immunostained with the indicated anti-
bodies after 5 min. RRM1 or RRM2 and gH2AX signals are shown
in green and red, respectively, in merged images. Bars, 10 mm. (C) IR-
irradiated HeLa cell lysates treated with the indicated siRNAs were
fractionated as described in the Materials and Methods. (Left panels)
The fractions were subjected to immunoblotting using the indicated
antibodies. (Right panel) The RRM1 bands were quantitated, and the
results are presented as percentages of S1 fraction. Data are mean 6
standard deviation (n = 3). (D) Sf9 lysates expressing RRM1-HA,
RRM2, or GST-His-Tip60 were subjected to GST pull-down or HA
pull-down assays using the indicated antibodies. (E) Chromatin frac-
tions from IR- or mock-treated HeLa cells (after 5 min) were solu-
bilized with micrococcal nuclease. The solubilized extracts were
immunoprecipitated with anti-Tip60 antibodies or control IgG. The
resulting precipitates and a 10% input (1/10 Input) were immuno-
blotted with the indicated antibodies. (F) The affinity-purified Tip60
complexes, as described in the Materials and Methods, were sub-
jected to immunoblotting using the indicated antibodies. (G)
GM02063 cells were treated with control, Tip60, or GFP siRNAs
and then subjected to UVA microirradiation as in B.
Tip60-dependent recruitment of RNR at DSB sites. (A)
Niida et al.
334 GENES & DEVELOPMENT
formation between RNR and Tip60 is required for re-
cruitment of RNR to sites of DNA damage.
We then examined if RNR recruitment at damage sites
was required for effective DNA repair. We first generated
RRM1 mutants that lack the ability to bind Tip60 but
retain RNR activity. Given that the C-terminal CXXC
motif of RRM1 is important for RNR function (Zhang
et al. 2007), we constructed RRM1 mutants containing
the CXXC motif but lacking Tip60-binding ability (D761–
786 and A776CD781-C) (Fig. 2D). Wild-type RRM1 or its
mutants were coexpressed with RRM2 in insect cells, and
the resultant complexes were subjected to an in vitro
RNR assay (Fukushima et al. 2001). RNR complexes con-
taining wild-type, D761–786, and A776CD781-C RRM1
retained hydroxyurea (HU)-sensitive RNR activity (HU is
a specific RNR inhibitor), whereas an inactive C429S
mutant or GST protein as a negative control did not show
RNR activity (Fig. 2E). The specific activity of RNR con-
taining wild-type, D761–787, and A776CD781-C RRM1
(;50 nmol/mg per minute) was similar to that reported
previously (Guittet et al. 2001), confirming the reliability
of our results. The A776CD781-C mutant failed to form
a complex with GST-Tip60 (Fig. 2F). ChIP analysis using
RRM1 knockout–knock-in STEFKu70?/?phprt-DR-GFP
cells revealed that the A776CD781-C mutant failed to
accumulate at the DSB site (Fig. 2G). These results in-
dicated that direct interaction of RRM1 to Tip60 is re-
quired for triggering its accumulation at the DSB site.
A comet assay revealed that DNA damage in cells was
repaired efficiently within 1 h in the absence of HU.
However, treatment with HU, and RRM1 or RRM2
depletion, resulted in an impairment of DNA repair
(Fig. 3A,B). RNR activity was thus essential for effective
repair. Ectopic expression of wild-type RRM1 with mu-
tations in a specific sequence targeted by siRNA effec-
tively rescued the impaired DNA repair in cells depleted
of endogenous RRM1 (Fig. 3C). In contrast, ectopic expres-
sion of C429S, D761–786, and A776CD781-C RRM1 failed
manner. (A) Map of the I-SceI cassette construct containing the
I-SceI site, the probe for Southern blotting, and a set of primers for
the ChIP assay. (B) STEFKu70?/?phprt-DR-GFP cells infected with
I-SceI adenoviruses were subjected to ChIP analysis using the in-
dicated antibodies as described in the supporting Materials and
Methods. Data are shown as percentages of increases in PCR prod-
ucts from cells expressing I-SceI (I-SceI) relative to those from cells
expressing Lac Z (LZ). Data are mean 6 standard deviation (n = 3).
(C) STEFKu70?/?phprt-DR-GFP cells were transfected with two
independent Tip60 siRNAs (Tip60-1 and Tip60-2) or control siRNA.
ChIP analysis was performed as in B. (Bottom panels) Aliquots of
cell lysates were subjected to immunoblotting using anti-Tip60
antibodies. (D) The constructs used are schematically represented,
and the specific interaction between RRM1 mutants and Tip60
was assayed using yeast two-hybrid screening. (E) An in vitro RNR
assay of complexes containing wild-type or various RRM1 mutants
was performed as described in the Materials and Methods. (Black
bars) ?HU; (white bars) +HU (10 mM). Data are mean 6 standard
deviation (n = 3). (F) Sf9 lysates expressing GST-His-Tip60 and the
indicated RRM1-HA were subjected to GST pull-down assay using
the indicated antibodies. (G) Knockout–knock-in STEFKu70?/?phprt-
DR-GFP cells expressing wild-type or A776CD781-C RRM1-HA
were generated by transfection with vectors for either wild-type or
A776CD781-C RRM1 and then with RRM1 siRNA. Expression vec-
tors of wild type and A776CD781-C contain mutations in a specific
sequence targeted by siRNA. (Top panel) Cells were subjected to
ChIP analysis using anti-HA antibodies as in B. (Bottom panels)
Aliquots of cell lysates were subjected to immunoblotting using the
RRM1 is recruited at DSB sites in a Tip60-dependent
a prerequisite for effective DNA repair. (A) HeLa cells were treated
with (open bars) or without (filled bars) 2.5 mM HU, exposed to IR (4
Gy), and subjected to a comet assay as described in the Materials and
Methods. The results were obtained by counting at least 50 cells
per sample in three independent experiments. (B) HeLa cells were
transfected with a control (filled bars) or RRM1 or RRM2 siRNA
(open bars), and DNA repair was evaluated as in A. Cell lysates were
subjected to immunoblotting using the indicated antibodies. (C)
HeLa cells were transfected with or without (filled bars) either wild-
type (gray bars), C429S (open bars), A776CD781-C (dotted), or D761–
786 (hatched) RRM1. RRM1-transfected cells were then transfected
with RRM1 siRNA. Expression vectors of wild type and various
RRM1 mutants contain mutations in a specific sequence targeted by
siRNA. DNA repair activity and expression of RRM1 were examined
as in B. (D) Knockout–knock-in HeLa cells expressing wild type or
A776CD781-C RRM1-HA were exposed to IR, and cell lysates were
subjected to immunoblotting as in C.
Recruitment of active RNR at DNA damage sites is
RNR at DNA damage sites
GENES & DEVELOPMENT335
to do so. ATM was activated independently of Tip60
binding to RNR, but this activation was enhanced and
prolonged in cells expressing A776CD781-C, presumably
due to impaired DNA repair (Fig. 3D). It is therefore
conceivable that recruitment of active RNR at DNA
damage sites is a prerequisite for effective DSB repair,
but not for activation of checkpoint signaling. Tip60 is
also known to participate in transcriptional regulation of
several genes. Neither RRM1 nor RRM2 proteins were
affected by Tip60 depletion or overexpression (Supplemen-
tal Fig.S7),indicating that the effectofTip60 did not result
from changes in RRM1 and RRM2 expression.
ChIP analyses revealed that NLS-RC1-HA specifically
inhibited RRM1 binding, but did not affect Rad51 or
Tip60 binding, or increase H4 acetylation at the DSB site
in STEFKu70?/?phprt-DR-GFP cells (Fig. 4A). Expression
of NLS-RC1-HA suppressed accumulation of endogenous
RRM1 at DNA damage sites (Supplemental Fig. S8A,B),
but did not affect the foci formation of 53BP1 at DSB sites
(Fig. 4B), or complex formation and activity (Supplemen-
tal Fig. S9A,B) of endogenous RNR. However, cells
expressing NLS-RC1-HA, but not NLS-GFP-HA, had
unrepaired DNA in the tail at 2 h (Fig. 4C). A quantitative
colony formation assay was used to examine the DNA
damage sensitivity of cells expressing NLS-RC1-HA. In-
duction of NLS-RC1-HA sensitized cells to IR (Fig. 4D).
than during G1 phase (Hakansson et al. 2006), recruitment
of RNR at damage sites may function at a specific phase of
the cell cycle where dNTP pools are low. To address this
issue, we synchronized cells at S phase or G1 phase by
arrest and release of thymidine or nocodazole, respectively.
Recruitment ofwild-typeRRM1 ata DSBsite was observed
at both G1 and S phase (Supplemental Fig. S10). However,
a comet assay revealed that A776CD781-C failed to rescue
the impaired DNA repair in RRM1-depleted cells at G1
phase, but not at S phase (Fig. 4E). Consistently, RRM1
mutation of Tip60 binding slightly sensitizes cells to
Zeocin (Supplemental Fig. S11A), which causes DNA
strand breaks, but not to MMC (Supplemental Fig. S11B),
which can cause interstrand cross-linking repaired mainly
at S–G2 phase. Intriguingly, this G1-phase-specific impair-
ment of DNA repair was restored when excess amounts of
dADP, dGDP, dCDP, and dUMP (250 mM) were supplied in
the culture medium (Supplemental Fig. S12). These results
suggested that recruitment of RNR was required specifi-
cally for effective DNA repair in cells with low levels of
The present study suggests that the RNR recruitment
to DSB sites likely provides mechanistic insights into the
regulatory events that ensure a balanced supply of dNTPs
during mammalian DNA repair. RNR appears to form
a complex with Tip60 independently of DNA damage.
Thus, it is possible that the RNR–Tip60 complex might
have an alternative function, such as regulation of tran-
scription. In response to DNA damage, regulation of the
RNR subunit by Wtm1 and Dif1 in budding yeast is
radically different in terms of cellular localization (Lee
and Elledge 2006; Lee et al. 2008) from that observed in
the present study; however, important changes in the
subcellular localization of RNR might be conserved.
Given that Tip60 is a key regulator of DNA damage
responses, the concomitant recruitment of RNR at dam-
age sites suggests the presence of a synthetic regulatory
mechanism for DNA repair in mammals.
Materials and methods
Antibodies used were as follows: a-Rad51 (Ab-1, Oncogene Research
Products), a-RRM1 (sc-11733 and sc-11731, Santa Cruz Biotechnologies),
a-HA (11 666 606 001, Roche Applied Sciences; and PM002, MBL), a-Myc
(sc-40 and sc-789, Santa Cruz Biotechnologies), a-RRM2(sc-10844, Santa
Cruz Biotechnologies), a-GST (sc-459, Santa Cruz Biotechnologies),
a-Chk1 (sc-8408, Santa Cruz Biotechnologies), a-IKKa (sc-7182, Santa
Cruz Biotechnologies), a-Orc2 (sc-13238, Santa Cruz Biotechnologies),
a-ATM (sc-23921, Santa Cruz Biotechnologies), a-ATMp1981 (no. 4526,
Cell Signaling), a-acetylated histone H4 (no. 06-866, Upstate Biotechnol-
ogies), and a-phospho-histone H2AX (411-pc-020, TREVIGEN; and 05-636,
Upstate Biotechnologies). Anti-Tip60 rabbit polyclonal antibodies were
generated by immunization with recombinant GST-His-Tip60 produced in
insect cells, and the serum obtained was affinity-purified using a GST-His-
Two-hybrid interaction assays
The pGBKT7-RRM1 plasmid was generated by insertion of the full-length
human RRM1-encoding sequence. pGBKT7-RRM1 was transformed into
expression of NLS-RC1-HA abrogates DNA repair and sensitizes
cells to DNA damage. (A) STEFKu70?/?phprt-DR-GFP cells express-
ing NLS-RC1-HA (SV40 NLS-RC1 fragment, 701–792 amino acids)
or NLS-GFP-HA (GFP fragment, 1–93 amino acids) were subjected
to ChIP analysis as in Figure 2B. (Top panels) Cell lysates were
subjected to immunoblotting using the indicated antibodies. (B, left
panels) Tet-on HeLa cells expressing NLS-RC1-HA or NLS-GFP-HA
were treated with or without tetracycline (1 mg/mL), exposed to IR
(4 Gy), and subjected to immunostaining with the indicated anti-
bodies and a comet assay as in Figure 3A. (Right panels) IR-untreated
lysates were subjected to immunoblotting using the indicated
antibodies. (C) Asterisk (*) represents nonspecific bands. (D) These
cells were exposed to the indicated dose of IR, and a quantitative
colony formation assay was performed 8 d after treatment. Data are
mean 6 standard deviation (n = 3). (E) Knockout–knock-in HeLa
cells expressing either wild-type (filled bars) or A776CD781-C (open
bars) RRM1-HAwere synchronized as described in the Materials and
Methods. Synchronized cells were then released into G1 phase or
S phase (time ?3) and exposed to IR (4 Gy) 3 h after release (time 0).
(Right panels) DNA repair was evaluated as in A. (Left panels) Cell
cycle distributions are presented.
Inhibition of recruitment of RNR at DSB sites by ectopic
Niida et al.
336GENES & DEVELOPMENT
the yeast strain AH101 and mated with yeast Y187 pretransformed with
a HeLa cell cDNA library (BD Biosciences). The deletion mutants of
RRM1 and Tip60 were amplified by PCR using specific sets of primers.
Primer sequences are supplied in the Supplemental Material.
Affinity purification of Tip60 complex
Affinity purification of Tip60 complex was performed as described pre-
viously (Ikura et al. 2000, 2007). For the induction of DNA damage, cells
were g-irradiated (12 Gy) after centrifugation.
In situ detergent extraction and immunofluorescence analysis
Immunofluorescence on paraformaldehyde-fixed cells was performed
according to a previous report (Green and Almouzni 2003), using the
Microirradiation was performed as described previously (Ikura et al. 2007).
In brief, GM02063 cells were maintained on the microscope stage in
a Chamlide TC live-cell chamber system (Live Cell Instrument) at 37°C.
Microirradiation was performed using an LSM510 confocal microscope
(Carl Zeiss). Sensitization of cells was performed by incubating the cells
for 20 h in medium containing 2.5 mM deoxyribosylthymine and 0.3 mM
bromodeoxyuridine (Sigma), and then staining with 2 mg/mL Hoechst
33258 (Sigma) for 10 min before UVA microirradiation. The 364-nm line of
the UVA laser was used for microirradiation (three pulses at 30 mW).
Samples were examined with a Zeiss Axioplan 2 equipped with a charge-
coupled device camera AxioCam MRm controlled by Axiovision software
HeLa cells or STEFKu70?/?phprt-DR-GFP cells were transfected with
either control siRNA (Silencer Negative Control #1, Ambion 4611),
siRNAs for human Tip60 (sc-37966, Santa Cruz Biotechnologies), mouse
Tip60-1 (sc-37967, Santa Cruz Biotechnologies), mouse Tip60-2
(D-057795-02-0010, Dharmacon), or RRM1 (GGAUCGCUGUCUCUAA
CUUtt) using Lipofectamine 2000 reagent (Invitrogen).
Subcellular fractionation and Mnase treatment
Subcellular fractionation was performed according to a previous report
(Mendez and Stillman 2000). The isolated chromatin fraction (1 3 106
cells) was treated with Mnase (15 U) for 30 min at 37°C.
Establishment of STEFKu70?/?cells containing
a phprt-DR-GFP cassette
The phprt-DR-GFP vector(10 mg) was linearized with PvuI and transfected
into STEFKu70?/?cells. Cells were selected with 1.25 mg/mL puromycin
for 12 d, and single colonies were screened by Southern blotting using
puromycin cDNA as a probe. Clones having only one copy of the phprt-
DR-GFP cassette were used for experiments.
Establishment of Tet-on HeLa cells expressing NLS-RC1
pcDNA4/TO-NLS-RC1 (10 mg) was linearized with XhoI and transfected
into HeLa T-Rex cells (Invitrogen). Positive clones were selected with
Zeocin (250 mg/mL) and Blastcidin (5 mg/mL) for 12 d and screened by
immunoblotting using anti-HA antibodies for the detection of NLS-RC1
induction in the presence of tetracycline (1 mg/mL).
Generation of adenoviruses expressing I-SceI endonuclease
The full-length I-SceI fragment harboring the CAG promoter and poly
A signal was subcloned into pAd/PL-DEST (Invitrogen). Adenoviruses
expressing I-SceI were generated according to the manufacturer’s protocol
A population of STEFKu70?/?cells (1 3 107) containing phprt-DR-GFP
cells infected with adenoviruses expressing I-SceI was cross-linked with
1% formaldehyde for 10 min at 37°C. ChIP assays were performed
essentially as described (Shimada et al. 2008). Precipitated DNA was
resuspended in 50 mL of water and analyzed by quantitative real-time PCR
with the ABI PRISM7000 system using Power SYBR Green PCR Master
Mix (Applied Biosystems) as described (Katsuno et al. 2009). Primers used
for detection of the I-SceI break site were indicated in Figure 2A. As an
internal control for normalization of the specific fragments amplified,
mouse GAPDH locus was amplified using whole genomic DNAs with
mGAPDH-F and mGAPDH-R. Primer sequences are supplied in the
Alkaline comet assays wereperformedusingaTrevigen’s CometAssay kit
(4250-050-k) according to the manufacturer’s instructions. DNA was
stained with SYBR Green, and slides were photographed digitally (Nikon
Eclipse E800 lens and Fuji CCD camera). Tail moments were analyzed as
reported previously (Park et al. 2006) using TriTek Comet Score Freeware.
Measurement of DNA damage sensitivity
Tet-on HeLa cells expressing NLS-RC1-HA or NLS-GFP-HA were irradi-
ated with varying doses of IR in the presence or absence of doxycycline
(1 mg/mL), and then washed with PBS. Eight days after an additional
incubation, surviving colonies were counted, and their relative numbers
were expressed as percentages of the untreated cells (n = 3).
Insect cells were coinfected with baculoviruses expressing wild-type
RRM1 or its mutants, and with those expressing wild-type RRM2. RNR
complexes were immunopurified, and their activities were determined
according to a method reported previously (Fukushima et al. 2001).
Amounts of wild-type RRM1 protein or its mutant proteins were de-
termined by SDS-PAGE and used for calculating specific activities.
Cell cycle synchronization
For synchronization of cells at S phase, knockout–knock-in HeLa cells
expressing wild-type or A776CD781-C RRM1-HA were first synchronized
at the G1/S boundary by exposure to 2.5 mM thymidine for 16 h, and then
released into S phase by wash-out of thymidine with PBS and the addition
of 20% FBS containing DMEM. Cells were then exposed to IR 3 h after
release. For synchronization of cells at G1 phase, knockout–knock-in
HeLa cells were synchronized at M phase by exposure to 100 ng/mL
nocodazole for 16 h and released into G1 phase by wash-out of nocodazole
with PBS and addition of 20% FBS containing DMEM. Cells were then
exposed to IR 3 h after release.
We thank M. Delhase for critical reading of the manuscript; M. Jasin for
hprt-DR-GFP and pCBASce vectors; M. Fukushima for critical advice on
the RNR assay; A. Kurimasa for STEFKu70?/?MEFs; K. Murata, C.
Namikawa-Yamada, and H. Kojima for technical assistance; and M.
Inagaki and H. Goto for fluorescence microscopy. This work was sup-
ported in part by the Ministry of Education, Science, Sports, and Culture
of Japan through Grants-in-Aid for Scientific Research (B) (to M.N.) and (C)
(to H.N.), the YASUDA Medical Foundation (to M.N.), and the Sagawa
Cancer Foundation (to M.N.).
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