Kub5-Hera, the human Rtt103 homolog, plays dual functional roles in transcription termination and DNA repair.
ABSTRACT Functions of Kub5-Hera (In Greek Mythology Hera controlled Artemis) (K-H), the human homolog of the yeast transcription termination factor Rtt103, remain undefined. Here, we show that K-H has functions in both transcription termination and DNA double-strand break (DSB) repair. K-H forms distinct protein complexes with factors that repair DSBs (e.g. Ku70, Ku86, Artemis) and terminate transcription (e.g. RNA polymerase II). K-H loss resulted in increased basal R-loop levels, DSBs, activated DNA-damage responses and enhanced genomic instability. Significantly lowered Artemis protein levels were detected in K-H knockdown cells, which were restored with specific K-H cDNA re-expression. K-H deficient cells were hypersensitive to cytotoxic agents that induce DSBs, unable to reseal complex DSB ends, and showed significantly delayed γ-H2AX and 53BP1 repair-related foci regression. Artemis re-expression in K-H-deficient cells restored DNA-repair function and resistance to DSB-inducing agents. However, R loops persisted consistent with dual roles of K-H in transcription termination and DSB repair.
- SourceAvailable from: William S Dynan[Show abstract] [Hide abstract]
ABSTRACT: NONO, SFPQ and PSPC1 make up a family of proteins with diverse roles in transcription, RNA processing and DNA double-strand break (DSB) repair. To understand long-term effects of loss of NONO, we characterized murine embryonic fibroblasts (MEFs) from knockout mice. In the absence of genotoxic stress, wild-type and mutant MEFs showed similar growth rates and cell cycle distributions, and the mutants were only mildly radiosensitive. Further investigation showed that NONO deficiency led to upregulation of PSPC1, which replaced NONO in a stable complex with SFPQ. Knockdown of PSPC1 in a NONO-deficient background led to severe radiosensitivity and delayed resolution of DSB repair foci. The DNA-dependent protein kinase (DNA-PK) inhibitor, NU7741, sensitized wild-type and singly deficient MEFs, but had no additional effect on doubly deficient cells, suggesting that NONO/PSPC1 and DNA-PK function in the same pathway. We tested whether NONO and PSPC1 might also affect repair indirectly by influencing mRNA levels for other DSB repair genes. Of 12 genes tested, none were downregulated, and several were upregulated. Thus, NONO or related proteins are critical for DSB repair, NONO and PSPC1 are functional homologs with partially interchangeable functions and a compensatory response involving PSPC1 blunts the effect of NONO deficiency.Nucleic Acids Research 08/2014; · 8.81 Impact Factor
Kub5-Hera, the human Rtt103 homolog, plays
dual functional roles in transcription termination
and DNA repair
Julio C. Morales1, Patricia Richard2, Amy Rommel3, Farjana J. Fattah1,
Edward A. Motea1, Praveen L. Patidar1, Ling Xiao1, Konstantin Leskov4,
Shwu-Yuan Wu1, Walter N. Hittelman5, Cheng-Ming Chiang1, James L. Manley2and
David A. Boothman1,*
1Simmons Cancer Center, University of Texas Southwestern Medical Center, Dallas, TX 75390-8807, USA,
2Department of Biological Sciences, Columbia University, New York, NY 10027, USA,3Laboratory of Genetics,
Salk Institute of Biological Studies, La Jolla, CA 92037, USA,4Department of Radiation Oncology, Case
Western Reserve University, Cleveland, OH 44106, USA and5Department of Experimental Therapeutics, M.D.
Anderson Cancer Center, Houston, TX 77030, USA
Received November 20, 2013; Revised February 3, 2014; Accepted February 5, 2014
Functions of Kub5-Hera (In Greek Mythology Hera
controlled Artemis) (K-H), the human homolog of the
yeast transcription termination factor Rtt103, remain
undefined. Here, we show that K-H has functions in
both transcription termination and DNA double-
strand break (DSB) repair. K-H forms distinct protein
complexes with factors that repair DSBs (e.g. Ku70,
Ku86, Artemis) and terminate transcription (e.g. RNA
polymerase II). K-H loss resulted in increased basal
responses and enhanced
Significantly lowered Artemis protein levels were
detected in K-H knockdown cells, which were
restored with specific K-H cDNA re-expression. K-H
deficient cells were hypersensitive to cytotoxic
agents that induce DSBs, unable to reseal complex
DSB ends, and showed significantly delayed c-H2AX
and 53BP1 repair-related foci regression. Artemis re-
expression in K-H-deficient cells restored DNA-repair
function and resistance to DSB-inducing agents.
However,R loops persistedconsistent withdual roles
Maintaining genomic stability through progressive cell-
cycle divisions is essential for survival. Genomic instability
can arise by several different mechanisms (1), ultimately
leading to mutations and/or chromosomal rearrangements
that contribute to disease states, such as cancer (2). One
unrepaired DNA double-strand break (DSB) can cause
lethality (3). Mis-repaired DSBs are also a prominent
source of chromosomal rearrangements, resulting in trans-
locations within the genome (4). Thus, DSBs are a
constant threat to genomic stability and can arise natur-
ally during normal metabolic, replication and/or develop-
mental processes (5). Another prominent, yet at this point
understudied, mechanism for genomic instability is
through the formation of persistent RNA:DNA hybrids,
known as R loops (6). R loops are an evolutionarily
conserved consequence of transcription that form under
a variety of conditions, and if not properly resolved lead
to DSBs and genetic instability (6,7). In transient forms,
R-loop formation is an essential process in numerous
normal cellular processes, such as class switch recombin-
ation (8), and may also contributes to normal transcrip-
tion termination by RNA Polymerase II (RNAPII) (9–11).
Transcription termination by RNAPII is a complex
Interestingly, termination factors are linked to several dif-
ferent disease states. Senataxin, a putative DNA:RNA
pathologies, such as amyotrophic lateral sclerosis 4 and
ataxia with oculomotor apraxia 2 (13,14). Polymorphisms
in Xrn2, a 50–30exoribonuclease, are associated with cases
of spontaneous lung cancer in non-smokers (15). PSF,
together with p54(nrb), functions in the recruitment of
Xrn2 (16) and is critical for cellular survival in colon
and prostate cancers (17,18). p54(nrb) is highly expressed
and required for development and progression of malig-
nant melanoma (19).
*To whom correspondence should be addressed. Tel: +1 214 645 6371; Fax: +1 214 645 6347; Email: firstname.lastname@example.org
Nucleic Acids Research, 2014, 1–11
? The Author(s) 2014. Published by Oxford University Press.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0/), which
permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
Nucleic Acids Research Advance Access published March 3, 2014
at UT Southwestern on March 4, 2014
Along with roles in transcription termination, PSF,
p54(nrb) and Senataxin are also implicated in the DNA-
damage response (DDR), particularly in response to
DSBs. PSF and p54(nrb) have direct functional roles in
both the non-homologous end-joining (NHEJ) and hom-
ologous recombination (HR) pathways of DSB repair
(20–22). Loss of either PSF or p54(nrb) abrogates DNA-
repair kinetics and leads to increased chromosomal aber-
rations (21,23). Senataxin is also implicated in the DDR,
and in particular, in response to DSBs created by R loops
formed during various stages of transcriptional pausing
In light of the complex nature of the RNAPII termin-
ation process, it is likely that factors in addition to those
mentioned above are involved. In yeast cells, an additional
factor, Rtt103 contributes to transcription termination
(27). Rtt103 interacts with both RNAPII, via the
C-terminal domain (CTD) of its largest subunit, and
Rat1, the yeast homolog of Xrn2. Rtt103 facilitates the
exonuclease activity of Rat1 in termination (27). The
human homolog of Rtt103 is Ku70 binding protein #5-
Hera (K-H), also known as RPRD1B or CREPT (28,29).
Similar to Rtt103, K-H interacts with RNAPII (29). K-H
also mediates expression of Cyclin D1 and RNAPII occu-
pancy at the 30-end of genes (28). Rtt103 has also been
implicated in the DDR, yet its functions and involvement
in specific DNA-repair pathways remain unknown (30).
While K-H overexpression may also promote cell prolif-
eration and tumorigenesis (28), these functions within the
cell also remain undefined.
Here, we employ genetic and biochemical techniques to
uncover novel functions of K-H. We report that K-H
functions in RNAPII regulation, and aids in stabilizing
interactions between transcription termination factors,
localizing Xrn2 to the 30-end of genes and ultimately sup-
pressing R-loop formation. Importantly, we report that
K-H forms additional complexes and has additional and
separate functions in DSB repair through stabilization of
the DNA-repair factor, Artemis.
MATERIALS AND METHODS
shScr, shk-h and all variations of cells were grown in
DMEM with15% FBS,
hygromycin and 1mg/ml puromycin in a 10% CO2–90%
O2air humidified atmosphere at 37?C. shScr and shk-h
knockdown cells were grown as described above, except
under selection with 1mg/ml puromycin. shScr and shk-h-
MDA231 and all variations of cells were grown in DMEM
with 5% FBS and L-glutamine in a 10% CO2–90% O2air
humidified atmosphere at 37?C.
Mouse embryonic fibroblast production
K-H heterozygote males and females were used for timed
mating. At 12.5p.c., a female mouse was sacrificed and
embryos removed. Individual embryos were then minced
and treated with trypsin at 37?C for 1h to separate cells.
The cell slurry was then plated into a T75 tissue culture
flask and allowed to attach and grow. These cells were
maintained in DMEM with 5% FBS and at 10% CO2.
An antibody recognizing H2AX (a-total H2AX, BL179)
and 53BP1 (A300-272A) was purchased from Bethyl
Laboratories (Montgomery, TX). g-H2AX phospho-
specific antibody (JBW301) was obtained from Millipore
(Billerica, MA). a-Artemis antibodies (E-18, K-14), and
actin (C-11) were obtained from Santa Cruz Biotech
(Santa Cruz, CA). a-Ku70 and a-Ku70/80 heterodimer
specific antibodies (N3H10 and 162, respectively) were
purchased from Genetex (Irvine, CA). K-H monoclonal
University. K-H polyclonal antibody (SAB1102247) was
purchased from Sigma. S9.6, an antibody specific for
R loops (RNA:DNA hybrids) was provided by Dr
Stephen H. Leppla (NIH, Bethesda, MD). PSF (A301-
Laboratories (Montgomery, TX). RNAPII antibodies
(sc-56767) is from Santa Cruz Biotech (Santa Cruz, CA).
shScr, shScr-MDA231, shk-h and shk-h-MDA231 and all
variations of these cells were plated onto 60-mm tissue-
culture plates and allowed to grow for 2 days. Cells were
then exposed to IR (at various doses as indicated), allowed
to grow for 7 days, washed with PBS and stained with
crystal violet solution. Colonies with >50 normal appear-
ing cells were counted and percent survival calculated and
graphed with dose.
Neutral comet assay
Assays were performed as per manufacturer’s instructions
(Trevigen) (Catalog# 4250-050-K). Briefly, cells were
mock or IR treated and allowed to recover for indicated
times. After harvesting, cells were mixed with low melting
agarose. Cells were then exposed to electrophoresis and
stained with SYBR?-green to detect released DNA.
Cells were then imaged and comet tail lengths measured
by NIH Image J.
In order to visualize 53BP1, g-H2AX and Artemis, cells
were plated and grown to 70% confluence on glass cover-
slips and either mock- or IR-treated. Cells were then
washed once with PBS, permeabilized and fixed in
methanol/acetone (70/30, v/v). Cells were then blocked
in PBS containing 5% FBS for 30min at room tempera-
ture. Cells were then washed three times with PBS and
exposed to primary antibody for 1h at room temperature
as indicated. Cells were washed three times with PBS,
exposed to secondary antibody for 30min at room tem-
perature, washed three times with PBS and mounted onto
glass slides. Detection of R loops using the S9.6 antibody
was performed as previously described (31). Visualization
was performed using a 100X oil objective with fluores-
cence on a Nikon microscope.
2 Nucleic AcidsResearch, 2014
at UT Southwestern on March 4, 2014
Plasmid re-ligation assays
The pYes2 plasmid DNA from Invitrogen was digested
with the following restriction enzymes: BamHI, SacI,
PvuII and BamHI+SacI at 37?C for 45min. Cut and
uncut DNAs were purified from Agarose gels using
Qiagen Extraction kits. Linearized and control circular
plasmid DNAs were then used in PEG/LiAc-based yeast
Transformation System 2). Overnight YPDA cultures
(25ml) were grown to an OD600of 0.25, harvested and
re-suspended in 50ml YPDA and incubated for 4h at
30?C until OD600 of 0.4–0.5 were reached. Competent
yeast cells were generated as described (Clontech) and
transformed with equivalent amounts of uncut or restric-
tion enzyme-digested pYes2 plasmid DNA as indicated.
Transformation reactions were performed using plasmid
DNA concentrations of 10mg in 50ml competent rtt103D,
hdf1D, wild-type (WT) and rtt103D yeast stably corrected
with yeast RTT103 and human K-H cDNA (hK-H).
Briefly, after 42?C heat shock to promote DNA trans-
formation, yeast were harvested and resuspended in
0.5ml 0.9% NaCl and plated onto SD–URA selection
plates. Colonies were scored 3 days later and values
graphed as means±standard errors for three separate
Mammalian plasmid re-ligation assays
The pEGFP-Pem1 plasmid was digested with HindIII or
I-SceI for 8–12h to generate free DNA ends. pCherry
plasmid was co-transfected with linearized DNA to
control for transfection efficiency. shScr and shk-h cells
were transfected at roughly 20–25% confluency and
allowed to grow for 3 days. Transfections were performed
using Lipofectamine-2000 using manufacturer’s instruc-
tions. Flow cytometry was performed using a Beckman–
Coulter Cytomic FC 500.
Metaphase spreads and chromosome aberration analyses
Exponentially growing cells were mock- or IR-treated.
Cells were returned to 37?C for 30min to allow for
mitotic exit (particularly IR-exposed cells) and then
incubated with colcemid (1mg/ml) for 2h for mitotic cell
selection before harvest. Harvested cells were fixed in
hypotonic solution containing 75mM KCl and fixed in
methanol:acetic acid (1:1 v/v). Metaphase spreads were
prepared, stained with Giemsa and examined by light mi-
croscopy. Metaphase spreads (>50) were then scored for
chromosome breaks, gaps and aberrations.
HeLa whole-cell extract preparation and gel-filtration
HeLa cells were cultured in two 150mm2dishes (up to
?80% confluency) in DMEM supplemented with 5%
FBS and 1mM L-glutamine in a 5% CO2humidified at-
mosphere at 37?C. Cells were trypsinized, harvested by
centrifugation and washed with cold 1X PBS. Cells were
resuspended in 1ml extraction buffer [25mM Tris–HCl
b-glycerophosphate, 5mM NaF, 0.5mM Na3VO4, 10%
glycerol, 0.1% NP-40, 1X protease inhibitor cocktail
(Sigma), 100 units of turbonuclease (Fisher) and 1mM
DTT]. The cell suspension was incubated on ice for
5min and passed through 1-ml syringe with 27G needle
suspension was incubated on ice for 30min followed by
10min at 37?C. The cell lysate was centrifuged at
14000rpm for 30min at 4?C using a microfuge. The
supernatant was carefully collected as whole cell lysate and
used for gel-filtration chromatography. Chromatography
steps were carried out using AKTA Purifier 10 (GE
Healthcare). For the fractionation of whole cell lysate,
?3.0mg of protein was loaded onto a 24-ml Superose 6
HR 10/30 column (GE Healthcare) pre-equilibrated with
chromatography buffer [25mM Tris–HCl (pH 7.7),
100mM NaCl, 5% glycerol and 1mM DTT] and run in
the same buffer at a flow rate of 0.5ml/min. Molecular
weight standards (Pharmacia Biotech) were used to cali-
brate the column (as indicated in Figure 1A).
Nuclear extract preparation
Cell pellets were re-suspended in Buffer A [10mM Hepes
(pH 7.9), 10mM KCl, 0.1mM EDTA (pH 8.0), 0.1mM
EGTA, 1.0mM DTT, 0.5mM PMSF] and allowed to
swell for 10min, 4?C. NP-40 was then added to cell solu-
tions to a final concentration of 0.5% and vortexed at low
intensity for 30sec. Isolated nuclei were then harvested by
centrifugation (2000? g), the nuclear pellets were resus-
pended in Buffer C [20mM Hepes (pH 7.9), 0.4M NaCl,
1.0mM EDTA, 1.0mM EGTA, 1.0mM DTT, 0.5mM
PMSF] for 15min at 4?C. Nuclear extracts were then
WB: RNA Pol II
669 158 43
WB: Art (High exposure)
#no i t carFECW
22 2120 19187271 262524733236 30 2928353433 32 31
Figure 1. K-H forms a complex with NHEJ factors. (A and B) To
interrogate potential in vivo K-H interacting partners exponentially
growing HeLa cells were collected and lysed for FPLC. Individual frac-
tions were separated by SDS-PAGE and western blot techniques were
used to probe for indicated proteins. (C) To validate K-H and NHEJ
complex member interactions nuclear extracts from shScr cells were
used to perform co-immunoprecipitation (co-IPs) experiments with
and without EtBr (5 ?M), to check for the contribution of DNA to
these interactions. Co-IPs were separated by SDS-PAGE followed by
western blot analysis for indicated NHEJ factor.
Nucleic Acids Research, 20143
at UT Southwestern on March 4, 2014
isolated by centrifugation (25000? g) for 15min, and
assessed for protein concentrations by Bradford assays.
Immunoprecipitation and Chromatin Immunoprecipitation
Of nuclear extract, 0.5 to 1mg was incubated with 5mg
of specified primary antibody conjugated to Protein A/G
with NETN solution [20mM Tris–HCL (pH 8.0), 0.1M
NaCl, 1mMEDTA, 0.05%
each sample was separated on 8% SDS-polyacrylamide gel.
Immunoprecipitation and Chromatin Immunoprecipitation
(ChIP) experiments were performed using previously
described methods (16).
was washed threetimes
All experiments were performed three or more times in
triplicate.Western and immunofluorescence
shown, are representative of these findings. Means and
standard errors were calculated and differences between
treatments were determined by confidence limit calcula-
tions using student’s t-tests. P-values (0.01 and 0.05) for
99% and95% confidence
K-H biochemically interacts with NHEJ factors
Ku70 binding protein #5-Hera (K-H) was isolated in a
screen intendedto identify
involved in NHEJ using a similar yeast two-hybrid
strategy as described (32). The association of K-H with
Ku70 in these assays was relatively strong, comparable to
the association of p53 with SV40 large T as measured by
growth on selection media and beta-galactosidase activity
(Supplementary Table S1). Deletion analyses indicated
that a coiled-coil domain in K-H interacted with a com-
parable coiled-coil domain within Ku70, also known to
associate with nuclear clusterin (33). K-H has two highly
conserved functional domains: an amino-terminal CTD-
interacting domain (CID), that mediates interactions with
the CTD of RNAPII (27,29) and a carboxy-terminal
coiled-coil domain (Supplementary Figure S1A), which
is required for Ku70 interaction. A specific point
mutation, L276A, within the coiled-coil domain of K-H
abolished its binding to Ku70 (Supplementary Table S1).
Human (hK-H) and mouse (mK-H) K-H share significant
regions of homology with yeast Rtt103 as noted by Clustal
Omega analyses (Supplementary Figure 1A).
Due to its association with Ku70, we used a plasmid-
growth on Uracil-deficient medium (UraA cDNAs) were
digested with BamHI (50-overhangs), SacI (30-overhangs),
a combination of BamHI and SacI, (BamHI/SacI, yielding
incompatible 50- and 30-end overhangs) or PvuII (blunt
ends) (Supplementary Figure S1B). Unlike yeast deficient
in Hdf1 (yKu70, the canonical yeast NHEJ protein), that
were unable to repair any type of introduced DSB,
Rtt103D yeast lacked the specific ability to repair DSBs
with incompatible ends (i.e., complex or blunt DNA ends),
which require additional DNA end-processing (Supple-
mentary Figure S1B). Unlike prior reports stating that
Rtt103 is not required for the end-joining process (30),
our data strongly suggest that Rtt103 deficient cells
exhibit a specific defect in the repair of incompatible or
blunt DSB ends. Importantly, re-expression of either yeast
Rtt103 or human K-H (hK-H) cDNAs restored the DNA
repair capabilities of Rtt103D yeast (Supplementary
Figure S1B). Interestingly, the DNA-repair defect noted
in Rtt103D yeast closely resembled the DNA-repair defi-
ciency of yeast lacking Exo1, a 50–30exonuclease that is
required for DNA end-processing during NHEJ (34), a
similar but non-overlapping function performed by the
human Artemis protein (35).
partners, we performed gel filtration on HeLa whole cell
extracts. Interestingly, two separate complexes containing
K-H were readily detectable, suggesting relatively separate
associations with RNAPII (fractions 16–22) (Figure 1A)
and another complex closely associated with p15RS (frac-
tions 27–35) (Figure 1A); an association between K-H and
p15RS was previously reported (29). Additionally, K-H
co-eluted with several known NHEJ factors, including
Ku70, Ku86 and Artemis (fractions 27–29) (Figure 1A
and B), consistent with yeast two-hybrid data suggesting
an association of K-H with Ku70 (Supplementary
Table S1). To visualize Artemis, fractions 27–29 were
concentrated and probed for levels of K-H and Artemis
by western blot analyses (Figure 1B). Importantly,
co-immunoprecipitation (co-IP) analyses using a K-H-
specific antibody generated in our laboratory, which rec-
ognizes K-H but, not the closely related p15RS protein
associated with Ku70 and Artemis. Furthermore, this
association was not due to interactions with DNA, since
co-IPs were noted in the presence or absence of ethidium
bromide (5 ?M) (Figure 1C). DNase treatments also did
not affect the co-IPs (not shown). These data confirm the
initial yeast two-hybrid observation that K-H interacted
with Ku70, and show that Ku86 and Artemis are present
in higher molecular weight protein complexes with K-H,
separate from RNAPII.
revealed that K-H
Increased genomic instability and basal DSB formation
due to K-H loss
To examine possible functions of K-H in DNA repair, we
generated stable shRNA-K-H knockdown cell lines using
human foreskin fibroblast (shk-h) and triple negative,
MDA231) cells. Non-targeted shRNA-Scr fibroblast
(shScr) and MDA-MB-231 (shScr-MDA231) control
cells were generated at the same time. K-H-targeted
shRNA knockdown was directed to a 50-untranslated
region (UTR) sequence to facilitate reconstitution of
cells with human K-H cDNA. We also generated mouse
embryonic fibroblasts (MEFs) from K-H wild type (mk-
h+/+) or heterozygote (het) mice (mk-h+/?). We confirmed
stable loss of K-H in knockdown or mk-h+/?het cells, and
showed that levels of Ku70 remained unchanged (Figure
4 Nucleic AcidsResearch, 2014
at UT Southwestern on March 4, 2014
2A). We used mk-h+/?MEFs in our studies since complete
loss of K-H led to early embryonic lethality (data not
shown) and mk-h+/?cells are haplo-insufficient for pheno-
types examined below. In all three cell systems, loss of K-
H led to increased basal levels of g-H2AX and 53BP1 foci
formed without genomic insult (Figure 2B and C and
Supplementary Figure S2B). Furthermore, K-H deficient
cells showed delayed disappearance of DDR biomarkers
following exposure to IR (Figure 2D and E and
Supplementary Figure 2C). The delay in DDR foci regres-
sion was confirmed by a delay in neutral comet tail regres-
sion after IR treatment in shk-h-MDA231 cells compared
to shScr-MDA231 or K-H reconstituted shk-h-MDA231
cells (Supplementary Figure 2D). As neutral comet
analysis examines DSBs specifically, we concluded that
loss of K-H impaired the ability of affected cells to
repair DSB lesions. We also observed significant increases
in chromosomal aberrations, by metaphase spreads,
before and after exposure to IR (Figure 2F and
Supplementary Figure 3A) in shk-h knockdown fibro-
blasts compared to shScr cells. Elevated levels of chroma-
tid aberrations were also noted in irradiated shk-h
fibroblasts compared to shScr control cells (Figure 2F).
In contrast, levels of other genomic aberrations, such as
di-centric, tri- and tetra-radial chromosomes, were not
statistically different between shScr and shk-h fibroblast
cells (Supplementary Table S2). Collectively, these data
suggested that K-H plays a role in mediating specific
DSB-repair processes. However, direct or indirect role(s)
for K-H in HR repair of DSBs in mammalian cells could
not be ruled out, since increases in chromatid and chromo-
some breaks after IR were observed with loss of either HR
or NHEJ DSB-repair pathways (36). Yet, the lack of
radial chromosomes, commonly seen in cells that have
lost the ability to perform HR; thus reverting to NHEJ
for DSB repair (36,37), in shk-h cells suggest that the
primary DSB repair defect in K-H-lacking cells is in the
NHEJ DSB-repair pathway, and not HR.
K-H loss sensitizes cells to chemotherapeutics that
Cells deficient in specific DSB-repair pathways typically
agents in long-term colony forming (survival) assays
(38,39); for example, NHEJ-deficient Ku70 knockout
cells are hypersensitive to agents that specifically create
DSBs (40). In general, all cells deficient in K-H
(Figure2A) were hypersensitive
compared to mammalian Scr or wild-type (mk-h+/+)
MEF cells (Figure 3A and Supplementary Figure 4A
and B). Furthermore, shk-h-MDA231 cells were also
hypersensitiveto all DSB-inducing
including cisplatin, H2O2, Etoposide, Doxorubicin and
Topotecan compared to genetically matched shScr-
MDA231 cells (Figure 3B and C and Supplementary
Figure 4C–E, respectively). In contrast, shk-h-MDA231
cellswere not sensitive
(Figure 3D). To control for shRNA off-target effects, we
reconstituted shk-h-MDA-231 cells with human K-H
cDNA and observed that such cells were restored for re-
sistance to IR, cisplatin and H2O2treatments to levels
comparable to shScr-MDA231 cells (Figure 3A–D).
These data demonstrate that K-H is intimately involved
in mediating the repair of DSBs and not UV-induced
DNA lesions, such as UV-induced thymine dimers and
6–4 photoproducts (41).
to ultraviolet(UV) light
The CID domain of K-H is required for Artemis protein
As shown above, K-H displays a biochemical association
with NHEJ factors, Ku70, Ku86 and Artemis (Figure 1).
To determine whether loss of K-H had any effect on the
expression levels of NHEJ proteins, we examined their
steady state levels in stable shScr compared to shk-h
knockdown fibroblasts. Significantly, loss of K-H led to
decreased levels of Artemis, but not Ku70 or Ku86.
Importantly, Artemis protein levels were restored upon
re-expression of human K-H cDNA, monitored by both
western blotting and immunofluorescence (IF) (Figure 4A
and B). Since Artemis mRNA levels remained unchanged
after K-H loss (Supplementary Figure S5A) or K-H res-
toration, we suspected that loss of Artemis was due to
protein instability, and that the K-H protein stabilizes
Figure 2. Loss of K-H leads to increased DSBs and genomic instabil-
ity. (A) shScr, shk-h, shScr-MDA231, shk-h-MDA231, mk-h+/+and
mk-h+/–cells were used to interrogate the degree of K-H loss each
cell system. (B and C) Basal levels of the DNA damage indicator
g-H2AX were measured in shScr-MDA231, shk-h-MDA231, mk-h+/+
and mk-h+/–cells by immunofluorescence (IF). (D and E) Rates of
g-H2AX disappearance were measured in shScr-MDA231, shk-h-
indicated times after ionizing radiation (IR) exposure by IF, the first
time point is 0.5h after IR exposure. (F) Amounts of genomic aberra-
tions were quantitated in shScr and shk-h cells with and without
exposure to IR (2Gy). (**P<0.01).
Nucleic Acids Research, 20145
at UT Southwestern on March 4, 2014
Artemis by protein–protein interaction. To determine
regions of K-H required for Artemis stabilization, we
generated various deletion mutants of K-H spanning
specific domains within the protein (Figure 4C) and
overexpressed each of them in 293T cells deficient in en-
dogenous K-H expression by prior exposure to siRNA
specific for the 30-UTR region of K-H mRNA. K-H and
Artemis mRNA and steady state protein levels were con-
firmed by qRT-PCR (data not shown) and western
blotting, respectively. Lamin B protein levels served as
loading controls (Figure 4D). Along with full-length K-
H, fragments F1, F3 and F4 of K-H caused stabilization
of Artemis steady state protein levels (compare lanes 1, 2,
4 and 5 to siRNAk-h+empty vector, lane 6, Figure 4D).
In contrast, overexpression of K-H fragment F2 was not
able to stabilize steady state levels of Artemis (lane 3)
(Figure 4D). These data strongly suggested that the N-
terminal CID domain of K-H (minimally represented by
the region indicated by K-H fragment F3) was required
for Artemis stabilization (Figure 4D).
Artemis overexpression rescues DNA repair defects in
K-H deficient cells
Loss of Artemis impairs DNA repair (particularly in the
very late phase of DSB repair noted by foci regression),
resulting in genomic instability and sensitivity to IR (42).
Because K-H loss led to a concomitant loss of Artemis
protein expression (Figures 4D and 5A), we examined
the effects of Artemis re-expression on the DNA-repair
capacity of K-H deficient fibroblasts. We transfected
shk-h knockdown fibroblasts with Artemis cDNA (shk-
h+Art), which restored Artemis protein expression to
levels found in shScr fibroblast cells transfected with the
empty vector (Figure 5A and Supplementary Figure 5B).
Strikingly,Artemis re-expressionin shk-h cells
compared to non-transfected or vector-alone transfected
shk-h cells (Figure 5A). Artemis re-expression in shk-h
cells also restored resistance to IR and rates of 53BP1
foci regression similar to shScr cells (Figure 5B and C).
We then examined whether loss of K-H expression also
affected NHEJ capacity in shScr, shk-h and shk-h+Art
fibroblasts using a plasmid-based NHEJ assay developed
as described (43). These assays employ an mCherry
expression reporter plasmid linearized by restriction
digestion with HindIII (compatible DNA ends) or I-SceI
(incompatible DNA ends) digestion (43). While cells
knocked down for K-H expression showed only a slight
decrease in the ability to repair compatible ends, there
was a more pronounced deficiency in the ability of these
cells to repair incompatible DNA ends, similar to results
found in Rtt103D yeast (Figure 5D and Supplementary
Intriguingly, we also noted that the DNA-repair
kinetics, monitored by 53BP1 and g-H2AX-foci regres-
sion, in shk-h knockdown cells were significantly and fun-
damentally different from the DNA-repair kinetic-defects
reported in Artemis-deficient cells (42). To explore the
mechanistic basis for this difference, we compared the
DNA-repair kinetics of wild-type mouse (mk-h+/+), het-
erozygote mouse K-H (mk-h+/?) and mouse Artemis?/?
knockout (mart?/?) MEFs. mk-h+/?cells demonstrated a
large increase in basal DSB levels compared to mart?/?
cells (Figure 5E). In contrast, mart?/?cells exhibited basal
DSB levels (averaging 2–4 foci/nucleus) similar to mk-h+/+
loweredbasalDSB levels detected
Relative Art levels
WB: Lamin B
+ + + + + + --
15432 7 86
Figure 4. K-H CID domain is essential for stabilization of Artemis.
(A) shScr, shk-h and shk-h+K-H were monitored for changes in
Ku70, Ku86, Artemis, and K-H expression by western blot. (B)
shScr, shk-h and shk-h+K-H cells were assessed for changes in
Artemis nuclear location monitored by immunofluorescence. (C)
Schematic of K-H deletion mutants tested. (D) K-H domains
required for Artemis expression were monitored in 293T cells trans-
fected with a K-H specific siRNA designed to the 30-UTR, that were
then transfected with wt or mutant K-H plasmid constructs. Cells were
harvested and separated by SDS-PAGE and monitored for Artemis,
c-Myc and Lamin B by western blot. (*P<0.05).
Figure 3. Loss of K-H sensitizes MDA-MB-231 cells to various
chemotherapeutic agents. (A–D) shScr-MDA231, shk-h-MDA231 and
shk-h-MDA231+K-H mock untreated or (A) IR, (B) cisplatin, (C)
H2O2, (D) ultraviolent light (UV) exposed cells were monitored for
survival by colony formation assay. Colonies were determined as accu-
mulations of at least 50 cells. (**P<0.01).
6Nucleic AcidsResearch, 2014
at UT Southwestern on March 4, 2014
cells. After IR treatment (1Gy was an equitoxic dose for
mart?/?and mk-h+/?cells), mk-h+/?cells showed a
dramatic impediment in regression of 53BP1 foci/nucleus
over time compared to mart?/?or wild-type mk-h+/+cells,
which are wild-type for Artemis expression (Figure 5F).
As previously reported, mart?/?cells showed similar
53BP1 foci/nucleus regression as wild-type mk-h+/+cells,
except that mart?/?MEFs exhibited a defect in the slow
phase of DSB repair, noted at >5h post-irradiation
(Figure 5F). This defect is similar to previously published
results using Artemis-deficient human cells (42,44), and
strongly suggested that Artemis loss alone was not suffi-
cient to explain the defective DNA-repair phenotype
(monitored as foci regression) (Figure 5C and F)
observed in mk-h+/?cells (Figure 5F).
Loss of K-H leads to R-loop formation
Given previous observations linking K-H to RNAPII
transcriptional regulation (28,29), we then examined
whether by-products of mis-regulated transcription, in
combination with loss of Artemis expression, may
explain thequalitative and
between the DNA-repair capacities of K-H-deficient and
Artemis knockout cells. One such transcriptional by-
product is an R loop. Since prolonged R-loop formation
leads to increased basal DSBs (45), we monitored R-loop
foci/nuclei formation in shk-h and shScr cells by immuno-
fluorescence (IF) using the S9.6 antibody that specifically
recognizes RNA:DNA hybrids (31). A considerable
increase (?4-fold) in the number of basal R-loop foci/
nucleus was detected in shk-h compared to shScr cells
(Figure 6A and Supplementary Figure S6). We also
found that Artemis reconstitution had no effect on the
level of R loops formed with K-H loss, while R loops
(Figure 6A). Importantly, overexpression of GFP-RNase
H, which alleviated R-loop formation similar to prior
reports (46,47), significantly decreased basal 53BP1 foci/
nuclei in shk-h knockdown fibroblasts to levels detected in
shScr cells, with or without RNase H overexpression
(Figure 6B): similar effects of RNase H overexpression
on R-loop formation and foci representing DSBs were
previously reported (46,47). In contrast, transfection of
shScr and shk-h cells with a GFP-control plasmid did
not alter the increased basal 53BP1 foci/nuclei levels
(Figure 6B) noted in shk-h knockdown compared to
To uncouple the contributions of R-loop formation
versus Artemis loss and its accompanying specific defect
in delayed DSB repair in K-H-deficient cells, we
overexpressed GFP or GFP-RNase H in shScr and shk-
h cells and exposed these cells to IR (1Gy). We then moni-
knockdown cells transfected with GFP alone had similar
defective DNA-repair kinetics associated with K-H loss,
cells transfected with GFP-RNase H had a distinctly dif-
ferent DNA-repair-kinetic profile (Figure 6F), similar to
cells defective in Artemis expression alone (compare repair
kinetics in Figure 6F with Artemis-deficient cells in
Figure 5F). The DNA-repair kinetics in GFP-RNase H
transfected shk-h knockdown cells were similar to GFP-
RNase H shScr cells 3h after IR treatment, but at 6h a
small portion of residual DSBs remained in GFP-RNase
H transfected shk-h cells. This response was very similar
to that observed with Artemis loss in human cells (42,44).
Collectively, our data strongly suggest that R loops
directly affect the rate of 53BP1 and g-H2AX foci regres-
sion in early phases of DNA DSB repair in K-H-deficient
cells, providing an explanation as to why there is a differ-
ence in DNA-repair kinetics in cells with loss of K-H
compared to cells deficient in Artemis expression and
Yeast Rtt103 interacts with Rat1, the yeast homolog of
Xrn2 (27). Rat1 and Xrn2 are 50–30RNA exonucleases
required for efficient RNA-transcription termination
(48). Accordingly, we noted that the Xrn2–RNAPII–
PSF complex observed in shScr fibroblasts was dramatic-
ally disrupted in shk-h knockdown cells as monitored by
directed toward RNAPII or PSF, a known modulator of
Figure 5. Artemis is required for the DNA-repair capability in K-H defi-
cient cells. (A) Basal levels of the DNA-damage repair biomarker 53BP1
were measured in shScr, shk-h and shk-h cells stably over-expressing
Artemis (Art), (shk-h+Art), by immunofluorescence (IF). (B) Sensitivity
to IR in shScr, shk-h and shk-h+Art, mock untreated or IR exposed cells
was monitored by colony forming assay. (C) Disappearance of 53BP1 foci
was measured in shScr, shk-h and shk-h+Art at indicated times after IR,
first time point measured is 0.5h after IR exposure. (D) The ability to
perform NHEJ in shScr, shk-h and shk-h+Art was monitored by a
plasmid-based NHEJ assay with plasmids digested with either HindIII to
study compatible end re-ligation, or I-SceI to study incompatible end re-
K-H heterozygote (mk-h+/–) and Artemis deficient (mart–/–) MEFs by IF.
(F) Rate of 53BP1 foci disappearance after IR at times indicated was
measured in mk-h+/+, mk-h+/–and mart–/–MEFs by IF, first time point is
0.5h after IR. Cells (300) were visualized for 53BP1 foci. Colonies were
determined as ?50 normal-appearing cells in a 7-day period. Events
(10000) were counted by flow cytometry. (**P<0.01, *P<0.05).
Nucleic Acids Research, 20147
at UT Southwestern on March 4, 2014
Xrn2 (16) (Figure 6C). Kaneko et al. (2007) found that
Xrn2 localization to the 30-end of genes is facilitated by
PSF (16). In yeast, Rtt103 and Rat1 interact and
co-localize to the 30-end of genes, mediating transcription
termination (27). K-H localizes to the 30-end of the b-actin
gene, and loss of K-H leads to increased RNAPII occu-
pancy downstream of the poly A cleavage site of the
b-actin gene, suggesting a possible termination defect
(28). Loss of Xrn2 also leads to higher RNAPII occu-
pancy at the 30-end of the b-actin gene (16). We, therefore,
assessed whether loss of K-H affected Xrn2 localization
along the b-actin gene, contributing to this termination
defect. By Chromatin-immunoprecipitation (ChIP) experi-
ments along the b-actin gene we noted that loss of K-H
lowered the level of Xrn2 localizing to the 30-end of the
gene (Figure 6D). Therefore, appropriate K-H levels are
essential for localizing Xrn2 to transcription-termination
regions of DNA. To determine whether loss of other
transcription termination factors led to an accumulation
of basal DSB formation in our cell model system, we
transfected shScr cells with siRNA-targeting p54(nrb),
which functions with PSF in termination (16). Indeed,
loss of p54(nrb) led to increased basal DSB levels
(Figure 6E), similar to previously published results (21).
Consistent with previously published data (30), we dem-
onstrate that Rtt103D yeast cells can not repair incompat-
ible DSBs. In contrast, these cells were competent at
repairing DNA lesions with compatible ends. In human
cells, we demonstrate that K-H-deficient cells are also in-
capable of repairing incompatible DNA ends (Figure 5).
Furthermore, we demonstrate that the repair defect in K-
H-deficient cells is restored by Artemis re-expression. This
suggests that, similar to K-H, the Rtt103 protein might
interact with a corresponding yeast protein important
for incompatible DSB end processing. While we have no
direct evidence for an interaction between these two
proteins, yeast defective in Exo1 display a similar DNA-
repair defect (34) as noted in Rtt103D yeast. That is,
Rtt103D and Exo1D yeast can fully repair plasmid DNA
with compatible DNA ends, but are incapable of repairing
DSBs with incompatible ends (supplementary Figure 1)
(34). Our laboratory is currently investigating whether
Rtt103 and Exo1 interact in a manner similar to the asso-
ciation of Artemis with K-H.
K-H-deficient cells demonstrated increased sensitivity
to genotoxic stresses that specifically induce DSBs,
whereas no hypersensitivity to UV irradiation was noted
(Figure 3). Loss of K-H expression greatly suppressed
DNA-repair foci regression after a genomic insult with
IR, increased genomic instability (Figure 2), and caused
a dramatic decrease in the cell’s ability to specifically
perform NHEJ (Figure 5). Loss of K-H expression was
also accompanied by increased basal DSBs (Figure 2).
RNase H overexpression in shk-h cells suppressed basal
DSBs (Figure 6), indicating a role for RNA metabolism in
the genomic instability observed in these cells.
K-H is known to interact with the CTD of RNAPII
(29), and loss of K-H leads to higher RNAPII occupancy
at the 30-end of genes (28). We showed that K-H mediates
PSF-Xrn2-RNAPII interactions (Figure 6) and that loss
of K-H disrupts these leading to a decrease in Xrn2
recruitment to the 30-end of genes (Figure 6). These data
provide evidence that the RNA PolII termination defect
noted in K-H-deficient cells is most likely due to loss of
Importantly, the function of Xrn2 is not limited to the
30-end of genes. Xrn2 also functions in the premature ter-
mination of elongating RNAPII transcription (49,50).
Thus, it is not known if the accumulation of persistent
and stable R loops is at sites of transcription termination
per se or at mis-regulated premature termination events.
Another interesting possibility is that R loops observed in
K-H-deficient cells may be accumulating in the nucleolus.
Two interesting studies recently demonstrated that loss of
transcriptional regulation of ribosomal DNA leads to R-
IgG + -
Figure 6. Loss of K-H disrupts transcription termination complexes
and leads to R-loop formation. (A) Level of RNA:DNA hybrids
(R loops) in shScr and shk-h cells was measured by immunofluores-
cence (IF). Basal levels of 53BP1 foci in shScr and shk-h cells trans-
fected with either a GFP-expressing plasmid or a GFP-RNase
H-expressing plasmid were measured by IF. (C) Interactions between
transcription termination factors PSF and Xrn2 and RNAPII were
monitored in shScr and shk-h by co-immunoprecipitation (co-IP) ex-
periments. Co-IPs were performed with RNAPII and PSF specific
antibodies and separated by SDS-PAGE. Western blots were then per-
formed with indicated antibodies. (D) ChIP were performed using
Xrn2-specific antibody in HeLa cells treated with K-H-specific siRNA
or control siRNA at the promoter (prom) and designated points
beyond the polyadenylation site. (E) Basal levels of 53BP1-foci forma-
tion were measured in shScr, shk-h and shScr cells transfected with
p54nrb specific siRNA by IF. (F) Rates of 53BP1 foci disappearance
after IR exposure at times indicated were monitored in shScr and shk-h
cell transfected with a GFP or and GFP-RNase H expressing plasmid
by IF, first time point measure is 0.5h after IR exposure. (**P<0.05).
8 Nucleic AcidsResearch, 2014
at UT Southwestern on March 4, 2014
loop formation and subsequent DSB formation (10,24).
Given that Xrn2 plays an integral role in ribosomal
RNA trimming (51), it is highly conceivable that the R
loops we observe in K-H-deficient cells are accumulating
in the nucleolus.
Our data demonstrate that expression of the NHEJ
protein, Artemis, was reduced after K-H depletion by
transient or stable siRNA knockdown, or through
somatic haplo-insufficiency. Since Artemis mRNA levels
were not altered in these cells and re-expression of K-H
cDNAs containing specific wild-type CIDs restored
normal Artemis level, we concluded that K-H is necessary
to stabilize Artemis. Interestingly, overexpression of
Artemisto over-ride normal
caused by K-H loss, restored the DNA-repair capabilities
and sensitivities to DNA-damaging agents in K-H-defi-
cient cells to levels comparable to wild-type cells. Thus,
Artemis expression can repair DSBs created from persist-
ent R-loop formation in K-H deficient cells. This provides
insight into the nature of DSB ends created due to
R loops,since Artemis
small subset of DSBs with known substrate specificity,
including stem and heterologous loops, flap and gapped
DSBs, but did not affect persistent R loops (Figure 6) in
K-H-deficient cells. We conclude that Artemis acts down-
stream of RNA:DNA hybrid formation, on DSBs pre-
sumably created by DNA or RNA polymerase collisions
with R-loop roadblocks. Interestingly, persistent R-loop
formation correlates with Histone H3 S10 phosphoryl-
ation and chromatin condensation (52), suggesting that
the localized chromatin compartment surrounding the
R loop is more heterochromatic than euchromatic.
Given that Artemis is required for the repair of DSBs in
heterochromatic regions of DNA (44), these findings
support our data that DSBs created in response to persist-
ent R-loop formation would require Artemis for repair.
Furthermore, RNase H overexpression restored DNA-
repair kinetics in shk-h knockdown cells to relatively
normal rates similar to those found in Artemis-deficient
cells after IR. These data suggest that removal of R loops
results only in a defect in the slow phase of DSB repair,
similar to that of Artemis-deficient cells. It is known that
DSBs in heterochromatic regions of DNA are repaired at
a slower rate than DSBs in euchromatin after IR (53). K-
H-deficient cells also lose Artemis expression, which is
required for efficient slow phase repair of DSBs in hetero-
chromatin (44). R-loop-mediated chromatin condensation
would also increase in K-H-deficient cells (52). Couple this
with the fact that IR does not adversely affect global tran-
scription rates (54), one would anticipate that cells defi-
cient in K-H expression will have a higher proportion of
DSBs with slower DNA-repair kinetics.
Our data provide evidence that K-H exists in multiple
protein complexesand with
properties. K-H associates with several different DNA-
repair proteins, such as Ku70, Ku86 and Artemis. In
separate complexes, K-H associates with RNAPII and
factors that function in transcription termination. While
the amino acid sequence of K-H does not appear to
two clear functional
contain any obvious functional enzymatic domains, it
domains, suggesting that it may function as a molecular
matchmaker, or a protein important for functional scaf-
folding. The coiled-coil region of K-H appears to be dis-
pensable for Artemis stabilization and mediates an
interaction with Ku70. Importantly, the CID region of
K-H was necessary and sufficient for stabilization of the
Artemis protein. CID-domain-containing proteins primar-
ily interact with the CTD of RNAPII (55). Indeed, the CID
domain of K-H can associate with the CTD of RNAPII
(29). Yet, our data suggest that CID domains may mediate
protein–protein interactions independent of RNAPII. The
nature of how the CID domain of K-H stabilizes Artemis
through protein–protein interactions is unknown and is
currently under investigation in our laboratory.
Taken together, our data strongly suggest that loss of
K-H copy number and corresponding steady state protein
level leads to simultaneous R-loop-derived DSBs and loss
of DNA-repair capacity of specific DSBs with complex
ends. We theorize that this combination confers a chromo-
somal ‘mutator’ phenotype on cells and likely contributes
to carcinogenesis, since K-H knockdown led to chromatin
aberrations. Our data demonstrate that K-H is important
in mediating NHEJ, through stabilization of Artemis. In
mouse models, Artemis loss was linked to the formation of
several different tumor types ranging from pro-B cell
lymphomas to sarcomas and malignant gliomas (56,57).
Consistent with these mouse data, loss of NHEJ capability
and chromosomal translocations
associated with the formation of many different tumor
types in humans (58). Interestingly, overexpression of
K-H has also been observed in multiple tumor types,
such as liver, lung, prostate and colon (28), although
current data on K-H deficiency in cancers are extremely
limited. In tumors with overexpressed K-H, then, one
might expect heightened resistance to genotoxic agents
and possibly enhanced cancer progression, particularly if
complexes needed in RNA transcription or DNA repair
(shown in Figure 1) are functionally altered. Data pre-
sented in this article suggest that K-H expression must
be critically controlled in the cell for balanced and
normal cell growth. Altering its expression may alter its
roles as a potential scaffolding protein mediating a
balance between transcription termination, genetic in-
stability and Artemis-dependent DSB repair. Loss or
gain of expression of K-H may then lead to a chromo-
somal-level ‘mutator’ phenotype.
Supplementary Data are available at NAR Online.
We thank Dr Randy J. Legerski and Dr Robert J. Crouch
forproviding us with
overexpression plasmids respectively. We also thank Dr
Stephen H. Leppla for providing us with antibodies
directed against RNA:DNA hybrids (R loops) (S9.6).
Artemis and RNaseH
Nucleic Acids Research, 20149
at UT Southwestern on March 4, 2014
We also thank Dr. Hongtao Yu and his lab members for
help with optimizing gel filtration chromatography. We
also thank the members of the Boothman lab and Drs
David Guis, Jessica Tyler and Shigeki Miyamoto for
critical reading of the article.
National Institutes of Health [grants CA139217 to D.A.B,
GM28983 to J.L.M. and CA103867 to C.-M.C.]; CPRIT
[grant RP110471 to C,-M.C.]; Welch Foundation [grant I-
1805 to C.-M.C.]; Cancer Biology T32 training grant
Funding for open access charges: NIH [CA139217 to
PI: Dr.Jerry Shay.
Conflict of interest statement. None declared.
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