HAL Id: hal-03027155
https://hal.archives-ouvertes.fr/hal-03027155
Submitted on 27 Nov 2020HAL is a multi-disciplinary open access archive for the deposit and dissemination of sci-entific research documents, whether they are pub-lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.
L’archive ouverte pluridisciplinaire HAL, est destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.
A disease-causing single amino-acid deletion in the
coiled-coil domain of RAD50 impairs MRE11 complex
functions in yeast and humans
Marie Chansel-da Cruz, Marcel Hohl, Ilaria Ceppi, Laëtitia Kermasson,
Laurence Maggiorella, Mauro Modesti, Jean-Pierre de Villartay, Talia Ileri,
Petr Cejka, John Petrini, et al.
To cite this version:
Marie Chansel-da Cruz, Marcel Hohl, Ilaria Ceppi, Laëtitia Kermasson, Laurence Maggiorella, et al.. A disease-causing single amino-acid deletion in the coiled-coil domain of RAD50 impairs MRE11 complex functions in yeast and humans. Cell Reports, Elsevier Inc, In press. �hal-03027155�
1 2 3 4
A disease-causing single amino-acid deletion in the coiled-coil domain of RAD50
5
impairs MRE11 complex functions in yeast and humans
6 7 8 9
Marie Chansel-Da Cruz1, 2, 3, Marcel Hohl4, †, Ilaria Ceppi 5,6, †, Laëtitia Kermasson1,2,
10
Laurence Maggiorella3, Mauro Modesti7, Jean-Pierre de Villartay1, 2, Talia Ileri8,
11
Petr Cejka5, 6, ‡, John. H. J. Petrini4, ‡, Patrick Revy1, 2, *, #
12 13 14 15
1
INSERM UMR 1163, Laboratory of Genome Dynamics in the Immune System, Equipe Labellisée la
16
Ligue contre le cancer, Paris, France
17
2
University of Paris-Sorbonne Paris Cité University, Imagine Institute, Paris, France
18
3
Genomic Vision, R&D innovation department, Bagneux, France
19
4
Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA
20
5
Institute for Research in Biomedicine, Università della Svizzera italiana (USI), Faculty of Biomedical
21
Sciences, Via Vincenzo Vela 6, 6500 Bellinzona, Switzerland.
22
6
Department of Biology, Institute of Biochemistry, Eidgenössische Technische Hochschule (ETH), 8093
23
Zürich, Switzerland.
24
7
Cancer Research Center of Marseille; CNRS UMR7258; INSERM U1068; Institut Paoli-Calmettes;
25
Aix-Marseille Université, Marseille, France
26
8
Ankara University School of Medicine, Pediatric Hematology and Oncology, Ankara, Turkey
27
† These authors equally contributed to the study
28
‡ These authors equally contributed to the study
29 30 * Lead contact 31 #
Correspondance: Patrick Revy, Imagine Institute, 24 bd du Montparnasse, 75015 Paris, France.
32 Email: [email protected] . 33 Tel. (33) +1 427 542 92 34 35
36
Summary
37 38
The MRE11-RAD50-NBS1 complex plays a central role in response to DNA double-strand breaks. Here,
39
we identified a patient with bone marrow failure and developmental defects caused by biallelic RAD50
40
mutations. One of the mutations created a null allele while the other (noted RAD50E1035Δled to the loss
41
of a single residue in the heptad repeats within RAD50 coiled-coil domain. This mutation represents a
42
human RAD50 separation-of-function mutation that impairs DNA repair, DNA replication, and DNA end
43
resection without affecting ATM-dependent DNA damage response. Purified recombinant proteins
44
further indicated that RAD50E1035Δ impaired MRE11 nuclease activity. The corresponding mutation in
45
Saccharomyces cerevisiae caused severe thermosensitive defects in both DNA repair and Tel1ATM
-46
dependent signaling. These findings demonstrate that a minor heptad break in the RAD50 coiled-coil
47
suffices to impede MRE11 complex functions in human and yeast. Furthermore, these results emphasize
48
the importance of the RAD50 coiled-coil to regulate MRE11-dependent DNA end resection in humans.
49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71
Introduction
72 73
DNA double-strand breaks (DSBs) are considered the most toxic form of DNA damage. While unrepaired
74
DSBs can cause cell death, improperly repaired DSBs represent a source of mutation and translocation
75
that challenges genome stability and can ultimately result to cancer development (Scully et al., 2019).
76
MRE11-RAD50-NBS1 (MRN; MRX in yeast for Mre11-Rad50-Xrs2) is a conserved complex that plays
77
a central role in the sensing of and response to DSBs (Lisby et al., 2004; Mirzoeva and Petrini, 2001).
78
MRN is one of the first molecular entities detected at DSBs where it activates the kinase ATM to trigger
79
DNA damage response (DDR) signaling to induce cell cycle checkpoints and apoptosis (Lisby et al.,
80
2004; Stracker and Petrini, 2011). The MRN complex also influences DSB repair via the endo- and exo-
81
nuclease activities of MRE11, initiates DNA end resection to generate, in concert with other nucleases,
82
single stranded DNA ends that drive homology-directed repair (HDR) (Scully et al., 2019; Syed and
83
Tainer, 2018).
84
RAD50 belongs to the highly conserved structural maintenance of chromosome (SMC) family of
85
proteins (de Jager et al., 2004; Hopfner and Tainer, 2003). RAD50 possesses an extended coiled-coil
86
domain that folds back upon itself to form an intramolecular anti-parallel coiled‐ coil structure of about
87
500 angstroms that leads to juxtaposition of the N‐ and C‐ terminal Walker A and B domains and
88
generates a functional ATPase that directly interacts with MRE11. The coiled‐ coil structure, which is
89
conserved across the SMC proteins and all the known Rad50 orthologs (de Jager et al., 2004; Paull, 2018;
90
Stracker and Petrini, 2011; Syed and Tainer, 2018), comprises a heptad repeat pattern wherein the first
91
and the fourth residue are hydrophobic to allow the association of the anti-parallel helices via their
92
hydrophobic faces (de Jager et al., 2001; Truebestein and Leonard, 2016; van Noort et al., 2003). The
93
apex of the RAD50 coiled-coil consists of a zinc hook domain by which two MRE11-RAD50 complexes
94
can dimerize to form intra- and intermolecular complexes that have been proposed to bridge DNA ends
95
(Hohl et al., 2015; Hopfner, 2014; Park et al., 2017; Paull, 2018; Stracker and Petrini, 2011; Syed and
96
Tainer, 2018; Tatebe et al., 2020). It has also been recently suggested that MRE11-RAD50 complexes
could promote DNA tethering of sister chromatids at stalled forks by facilitating Cohesin loading
98
(Delamarre et al., 2020). Although the flexibility of the RAD50 coiled-coil domain has prevented its
99
whole structure determination at the atomic resolution (Kashammer et al., 2019; Park et al., 2017), several
100
studies suggested that this domain participates in the regulation of MRN functions by propagating
101
information from the Zn-hook domain to the globular domain (Hohl et al., 2015; Hohl et al., 2011; Park et
102
al., 2017). Recent analyses using crystallographic, cryo-electron, and high-throughput single-molecule
103
microscopy suggested that dimers of ATP-bound MRE11-RAD50 intracomplex (M2R2) mostly adopt a
104
ring conformation able to interact and scan DNA homoduplex to recognize DNA end and trigger
105
ATM/Tel1-dependent DDR (Deshpande et al., 2014; Hopfner, 2014; Kashammer et al., 2019; Myler et al.,
106
2017; Tatebe et al., 2020). A recent model suggested that, upon ATP and DNA binding, the
MRE11-107
RAD50 intracomplex undergoes a conformational switch leading to the interaction of intermolecular
108
coiled-coil domains resulting in a rod shaped structure. This structural modification would allow the
109
exposure of the catalytic domain of MRE11 to DNA end and stimulates nuclease activity and DNA end
110
processing (Deshpande et al., 2014; Hopfner, 2014; Kashammer et al., 2019; Lammens et al., 2011; Lim
111
et al., 2011; Liu et al., 2016; Rojowska et al., 2014). Several studies conducted in yeast and bacteria
112
suggested that the RAD50 coiled-coil domain was particularly important during these conformational
113
transitions by propagating spatial information from the Zn hook to the globular domain (Hohl et al., 2015;
114
Hohl et al., 2011; Hopfner et al., 2002; Kashammer et al., 2019; Lee et al., 2013; Park et al., 2017).
115
Nonetheless, despite the recent progress in the structure determination of all or part of the MRN complex,
116
our knowledge of the RAD50 coiled-coil functional role in the regulation of the multiple MRE11
117
complex functions remains limited, especially in mammals (Paull, 2018).
118
Here, we identified an individual, P1, presenting with immunodeficiency and developmental
119
defects and has compound heterozygous mutations in RAD50. One of the mutations creates a null allele
120
while the other appears to be hypomorphic due to the loss of a single amino-acid residue in the coiled-coil
121
domain of RAD50 (noted RAD50E1035Δ). The analysis of the human RAD50E1035Δ mutant as well as its
yeast counterpart provided unprecedented information, emphasizing the fundamental role of the RAD50
123
coiled-coil domain in modulating MRE11 complex functions. Furthermore, this study reports the first
124
RAD50 separation-of-function mutation that does not affect ATM-dependent DNA damage response but
125
severely impairs DSB repair. These findings demonstrate the crucial role of the RAD50 coiled-coil
126
domain to regulate MRE11-dependent DNA end resection and repair in humans.
Results
128 129
Clinical features of individual P1
130
This study was initiated by the analysis of an individual P1 who was diagnosed at 7-years of age
131
with bone marrow failure and developmental defects. In the family, the two other children and the parents
132
were healthy (Figure 1A). The P1's cell blood count revealed thrombocytopenia (platelet count of 113
133
G/liter) associated with anemia (hemoglobin level of 10 g/dL), and neutropenia (neutrophil count of 1
134
G/L) (Table S1). Bone marrow smears and biopsies showed reduced cellularity (15%) without
135
myelodysplastic features, consistent with aplastic anemia. Immunophenotyping highlighted a virtual
136
absence of circulating B lymphocytes (CD19+) while T lymphocytes (CD3+) that were normal in number
137
exhibited an increased proportion of central memory T cells (CD3+ CCR7- CD45A-) (Table S1).
138
Conversely, the proportions of both naive CD4+ T cells (evaluated by the CD45RA marker) and recent
139
thymus emigrant (characterized by the CD45RA+CD31+markers) were markedly reduced, suggesting an
140
impaired T lymphopoiesis (Table S1). Moreover, physical examination of P1 revealed failure to thrive
141
(height 108.5cm, <3rd percentile; weight 17.2 kg, <3rd percentile), skin pigmentation, nail dysplasia,
142
leukoplakia, dental loss, microcephaly (cranial perimeter 48cm, <3rd percentile), and dysmorphia. P1 also
143
developed cataract at 10 years of age that necessitated surgical intervention. Collectively, P1's clinical
144
features were reminiscent of a defect in DNA repair and/or telomere maintenance (Glousker et al., 2015;
145
Rivera-Munoz et al., 2007). However, telomere restriction fragment (TRF) analysis indicated that
146
telomere length in P1's peripheral blood cells was not reduced as compared to his parents and his healthy
147
sister (Figure S1).
148 149
Individual P1 carries biallelic RAD50 mutations
150
To determine the molecular etiology of the disease, we performed whole exome sequencing
151
analysis in P1 byconsidering coding sequences carrying rare biallelic variants (frequency less than 0.1% 152
in 1000 genomes, EVS, dbSNP, and our in-house database (8319 individuals)) predicted to be deleterious 153
(Figure 1A). This approach highlighted two mutations in RAD50 that were confirmed by Sanger 154
sequencing and present in a compound heterozygous configuration (Figure 1B). One consisted in a
nucleotide insertion leading to a frameshift and a premature stop codon (c.2165dup; p.Glu723Glyfs*5)
156
(Figures 1B, 1C) inherited from the asymptomatic mother and present in both healthy sister and brother
157
(Figure S2A). The other mutation was an in-frame deletion of three nucleotides resulting to the loss of a
158
single glutamic acid residue at position 1035 (c.3109_3111del; p.Glu1035del) located in the coiled-coil
159
domain of RAD50 (Figures 1B, 1C and Figure S2A). This deletion corresponded to a de novo mutation
160
since it was absent in siblings and parents (Figure S2A) while microsatellite analysis ascertained for the
161
genetic paternity of the P1's father (Figure S2B). Both RAD50 mutations were absent from gnomAD
162
database (>120,000 individuals tested). Sequencing of RAD50 cDNA from P1's blood cells did not detect
163
the c.2165dup; p.Glu723Glyfs*5 mutation likely owed to nonsense mediated decay (NMD) (not shown).
164
Hence, the only RAD50 transcripts present in P1's cells encoded a RAD50 protein lacking a unique
165
glutamic acid residue in the coiled-coil domain of RAD50 (p.Glu1035del, hereafter dubbed RAD50E1035Δ)
166
(Figure 1C). RAD50 immunoblots with B lymphoblastoid cell line (B-LCL) lysates from P1 and his
167
mother revealed approximately half of RAD50 amount as compared to B-LCL lysate from a healthy
168
donor (Figure 1D). This result was consistent with the presence of a null allele in both P1 and his mother
169
(i.e. c.2165dup; p.Glu723Glyfs*5). Moreover, this finding indicated that RAD50E1035Δ was correctly
170
expressed and as stable as its WT counterpart. Congruent with an interdependent stability of the human
171
MRN components(Stewart et al., 1999; Waltes et al., 2009), we also noticed a reduced amount of NBS1
172
and MRE11 in cell lysates from P1 and his mother (Figure 1D). The RAD50E1035Δ mutation was located
173
in the coiled-coil structure composed of heptad amino acid repeats commonly labeled abcdefg, where a
174
and d represent hydrophobic positions(Truebestein and Leonard, 2016) (Figure 1E). The program
175
Marcoil that calculates coiled-coil probability(Delorenzi and Speed, 2002) indicated that the loss of the
176
residue E1035 led to a break in the heptad repeats (Blue arrow in Figure 1F) that was predicted to disrupt
177
the RAD50 coiled-coil structure nearby the mutation, as inferred by the drop in coiled-coil probability in
178
this region (Blue arrow in Figure 1G). Thus, we decided to investigate the functional consequence of this
179
peculiar mutation in P1's cells.
180 181
RAD50E1035Δ mutation impairs DNA repair
182
First, we assessed whether P1's cells were able to cope with various DNA lesions produced by
183
distinct genotoxic agents. P1's SV40-transformed fibroblasts, similar to cells from an ATM-deficient
184
patient, exhibited a strong sensitivity to ionizing radiation (IR) suggesting a DNA repair defect (Figure
185
2A). P1's cells also had a pronounced sensitivity to the DSB-inducing agent phleomycin (Figure 2B) and
186
to etoposide (Figure 2C), a drug that generates topoisomerase2-DNA adducts. P1's cells were also
187
sensitive to the DNA interstrand crosslink (ICL)-inducing agent mitomycin C (MMC), although at a
188
lesser extent than cells from a Fanconi anemia patient (Figure 2D), and not associated with an impaired
189
MMC-induced FANCD2 ubiquitination (not shown). Of note, a human HT1080 cell line carrying a
190
nonsense mutation on one allele of the RAD50 gene (RAD50+/-) generated by CRISPR/Cas9 only
191
exhibited a modest sensitivity to genotoxics at higher doses, ruling out haploinsufficiency as the cause of
192
the severe DNA repair defect in P1's cells (Figure S3). Taken together, these results demonstrated that
193
P1's cells have a general DNA repair defect that is likely the cause of the bone marrow failure and
194
developmental anomalies found in this patient.
195
To verify that the RAD50 deficiency in P1’s cells was responsible for the DNA repair defect, we
196
transduced the cells with a vector expressing wtRAD50 (Figure S4). While empty vector had no effect,
197
wtRAD50-expressing vector complemented the phleomycin sensitivity of P1's cells (Figure 2E). We
198
further confirmed this result by a multicolor competition assay after transduction of P1 and control cells
199
with a lentiviral Ires-mCherry vector containing or not the wtRAD50 coding sequence (Smogorzewska et
200
al., 2007). A mix of transduced (mCherry+) and nontransduced (mCherry-) cells was analyzed for
201
selective advantage in culture, as evaluated by the mCherry index of transduced (mCherry+) over
202
nontransduced (mCherry-) cells upon treatment with phleomycin (Figure 2F). Ectopic expression of
203
mCherry/wtRAD50 but not mCherry alone gave in P1's cells a strong selective advantage over
204
nontransduced cells upon treatment with phleomycin, as determined by the 40-fold increase in
205
mCherry/wtRAD50-expressing cells after 18 days in this culture condition (Figure 2F). Taken together,
206
these results established a causal link between the DNA repair defect and the expression of RAD50E1035Δ
in P1's cells.
208
209
RAD50E1035Δ mutation does not affect DNA damage sensing and signaling
210
To determine whether the DNA repair defect in P1's cells could be associated with an impaired
211
MRN recruitment at DNA damage sites, we monitored the dynamics of NBS1 at DSBs. We transfected
212
cells with a vector expressing GFP-NBS1 fusion protein and analyzed by live-microscopy the GFP-NBS1
213
behavior following the generation of localized DNA damages induced by a laser microbeam (Zhang et al.,
214
2016). Kinetics and intensity of GFP-NBS1 recruitment at DNA lesions was similar in control and P1's
215
cells (Figures 2G and 2H) indicating that RAD50E1035Δ
216
217
We next assessed whether the DNA repair defect in P1's cells was associated with impaired
ATM-218
dependent DDR functions (Stracker and Petrini, 2011). As expected, KAP1 phosphorylation (P-KAP1),
219
analyzed by immunofluorescence, was undetectable in primary fibroblasts from ATM- and
NBS1-220
mutated patients demonstrating the need for functional ATM and MRN in this process (Figure 2I). To
221
our surprise, IR-induced P-KAP1 was readily detected in primary fibroblasts from P1 suggesting that
222
RAD50E1035Δ did not impair ATM-dependent KAP1 phosphorylation. Furthermore, Western blot analysis
223
demonstrated that P1's SV40-fibroblasts normally induced phosphorylation of NBS1 and CHK2
224
following IR, while ATM-deficient control cells did not (Figure 2J). Consistent with a normal
IR-225
induced CHK2 phosphorylation, P1's SV40-transformed fibroblasts exhibited functional G2/M
226
checkpoint, as determined by the reduction of phospho-histone 3 in G2 phase, a marker of chromosome
227
condensation, following IR (Figure 2K). As expected, ATM-deficient cells used as negative control had a
228
defective G2/M checkpoint (Figure 2K).
229
Collectively, these results demonstrated that P1's cells behaved differently from ATM- and
NBS1-230
mutated cells and provided evidence that the human RAD50E1035Δ mutation, although leading to a DNA
repair defect, did not impair recruitment of the MRN complex at DNA damage sites or the subsequent
232
activation of ATM.
233 234
RAD50E1035Δ mutation impairs DNA end resection and homology directed repair
235
MRE11 initiates double-stranded DNA end resection via its nuclease activity, a process that
236
culminates in the formation of single stranded DNA (ssDNA) that is used to scan sequence homology and
237
promote homology directed repair (HDR) (Stracker and Petrini, 2011; Syed and Tainer, 2018). During
238
this process the ssDNA molecules are coated by RPA and then by RAD51 (Zhao et al., 2019). To get
239
further insight into the DNA repair pathway that might be affected in this patient, we assessed DNA end
240
resection following IR-treatment in P1's cells. In contrast to P-KAP1, which was readily detected in P1's
241
primary fibroblasts 6h post IR (Figure 3A), RPA (Figure 3B) and RAD51 (Figures 3C and Figure S5;
242
P<0.0001) foci were virtually absent in these cells, suggesting impaired DNA end resection at IR-induced
243
DSB. The impaired RAD51 foci formation upon IR treatment was further confirmed in SV40-hTERT
244
P1's fibroblasts (P<0.0001; Figure 3D). Transduction with wtRAD50 but not with an empty vector
245
complemented the impaired IR-induced RAD51 foci formation in P1's cells, a result demonstrating the
246
causal link between compromised DNA end resection and RAD50 deficiency in these cells (Figure 3D).
247
We next tested HDR pathway by measuring the efficiency of insertion in a chromosomal context of a
248
DNA sequence encoding Clover, a green fluorescent protein variant, mediated by
CRISPR/Cas9-249
mediated HDR within the first exon of Lamin-A gene (Figure S6A) (Pinder et al., 2015). Cells were
250
transfected with a mCherry-expressing vector to gate on transfected cells (mCherry+) in combination with
251
the Clover-donor vector with or without the CRISPR/Cas9 vector targeting the Lamin-A locus (Pinder et
252
al., 2015). This approach induced a significant increase in Clover positive cells in control's cells
253
transfected with both the donor and the CRISPR/Cas9 vectors (P <0.0001), asserting for efficient HDR in
254
these cells (Figure 3E). In contrast, in the same experimental conditions, we did not detect a significant
255
increase in Clover expression in P1's cells, suggesting that HDR was impaired in these cells (Figure 3E).
256
Since the HDR pathway is mainly active during the S and G2 phases, when a DNA template generated by
DNA replication is available (Jasin and Rothstein, 2013), we analyzed the cell cycle profile of P1's cells.
258
As determined by the combined detection of BrdU incorporation and propidium iodide staining, the cell
259
cycle profiles in control and P1's cells were similar, ruling out that the defective HDR in P1's cells was
260
caused by abnormal cell cycle (Figure S6B).
261
We concluded from these experiments that the DNA repair defect observed in P1's cells was
262
associated with a reduced resection of IR-induced DSBs and impaired HDR efficiency.
263 264
RAD50E1035Δ mutation prevents nascent DNA degradation after replication stress.
265
In the absence of genotoxic stress, spontaneous 53BP1 focus formation is an indicator of
266
replicative stress (Pasero and Vindigni, 2017). Strikingly, we noticed an increase in 53BP1 foci in P1's
267
primary fibroblasts in the absence of any exogenous stress (Figures 4A). Automated detection and
268
quantification of 53BP1 foci reported a mean of 7 events/nucleus in P1's cells (n=9,894) and 2.7 in
269
control (n= 19,632; P <0.0001) (Figure 4B), with 63.9% of P1's cells exhibiting 5 or more 53BP1 foci
270
versus 16.3% in control (P <0.0001; Figure 4C). We then performed replication-combing assay (RCA), a
271
technique that enables the measurement of fork speed, fork asymmetry, and inter-origin distance (Bianco
272
et al., 2012; Schurra and Bensimon, 2009) (Figure 4D). SV40-transformed fibroblasts from P1
273
consistently exhibited defective DNA replication inferred by a significant reduction of fork speed (P
274
<0.0001) (Figure 4E). We also noticed a shortening of inter-origin distance in P1's cells (P <0.01)
275
(Figure 4F), suggesting an increased dormant origin firing to compensate the reduced fork speed, as
276
previously described (Anglana et al., 2003; Courbet et al., 2008; Mokrani-Benhelli et al., 2013). P1's cells
277
also showed an increase in asymmetric replicons (P <0.001) likely attributable to stochastic replication
278
fork stalling and/or collapse (Figure 4G). Transduction of P1's cells with a lentiviral vector allowing the
279
expression of wtRAD50 restored a fork speed comparable to the control's cells, demonstrating a causal
280
link between the replication defects and RAD50 deficiency in P1's cells (Figure 4H).
281
Fork restart depends on fork resection initiated by the nuclease activity of MRE11 that degrades
282
newly synthesized DNA strands (Bryant et al., 2009; Pasero and Vindigni, 2017; Trenz et al., 2006). To
test whether replicative stress observed in P1's cells could be accompanied by a defect in DNA resection
284
at arrested forks, after successive pulses of CldU and IdU, we treated cells with a high dose of HU for 3
285
hours to provoke replicative stress, and examined DNA resection by molecular combing (Coquel et al.,
286
2019) (Figure 4I). As expected, HU treatment led to nascent DNA degradation in control cells, as
287
revealed by ratio IdU/CldU <1 (Figures 4I-J). Contrastingly, IdU track length did not change after HU
288
treatment in P1's cells (Figure 4I-J) suggesting that RAD50E1035Δ mutation led to an impaired DNA
289
resection at arrested forks. Transduction of wtRAD50 in P1's cells complemented this defect (Figure
290
4K), further supporting the notion that RAD50E1035Δ impaired DNA resection at arrested forks.
291
Collectively, the sharp reduction of DNA end resection at IR-induced DSBs (Figures 3B, 3C,3D,
292
and S5) and at arrested forks (Figures 4J) observed in P1's cells suggested that the RAD50E1035Δ mutation
293
impairs MRE11 nuclease activity.
294 295
Impairment of nuclease activity of recombinant MR(N) complex containing RAD50E1035Δ
296
As complete loss of MRE11 nuclease activity is incompatible with cellular viability (Buis et al.,
297
2008; Hoa et al., 2015; Hoa et al., 2016), we surmised that RAD50E1035Δ could not fully abolish this
298
process. To directly evaluate the impact of RAD50E1035Δ on MRE11 nuclease activity we expressed and
299
purified human MRE11 and RAD50 (MR) as a complex from baculovirus-infected Spodoptera
300
frugiperda 9 (Sf9) cells as previously described (Anand et al., 2016) (Figure S7A). For simplicity we
301
denominated MR WT the wild-type MRE11-RAD50 complex and MR E1035 the MRE11-RAD50
302
complex containing RAD50E1035Δ. As depicted in Figure 5A, MR WT and MR E1035 were similarly
303
purified, reinforcing our previous results showing that the RAD50E1035Δ mutation did not affect protein
304
stability (Figures 1D, 2J, and S4A). Electrophoretic mobility shift assay (EMSA) using a 70bp probe
305
revealed a slight decrease in DNA binding capacity of the MR E1035 complex as compared to its WT
306
counterpart (Figures 5B, 5C). In vitro assay with 50bp unprotected DNA substrates revealed a consistent
307
decreased exonuclease activity of MR complexes with MR E1035 as compared to MR WT (Figures 5D,
308
5E). In particular, the lower products corresponding to the most resected DNA substrates were sharply
underrepresented with MR E1035 asserting for a reduced exonuclease activity (Figure 5D, 5F). CtIP is
310
a cofactor that potentiates the DNA binding activity of the MR complex especially in its
non-311
phosphorylated form (Anand et al., 2016). We produced recombinant CtIP treated or not with lambda
312
phosphatase to generate either non-phosphorylated CtIP (CtIP) or phosphorylated CtIP (pCtIP),
313
respectively (Figure S7B). As previously described (Anand et al., 2016), the adding of the
non-314
phosphorylated recombinant CtIP led to a strong increase in the DNA binding capacity of the MR
315
complex that, in this condition, was similar with MR E1035 and MR WT (Figures S7C and S7D). This
316
result indicated that in the presence of CtIP, the RAD50 E1035 mutation did not impair DNA binding
317
capacity of the MR complex. However, in the presence of pCtIP, we observed a slight reduction in DNA
318
binding capacity of the mutated MR E1035 complex (Figures S7C and S7D). Next, we added
MBP-319
NBS1 and pCtIP to the complex in order to assess the endonuclease activity of MRE11 as described
320
previously (Anand et al., 2019). This analysis revealed a subtle reduction of endonuclease activity of the
321
MR E1035 complex, evident only at higher concentration (Figures 5G, 5H). Salt titration experiments
322
performed to examine whether the effect of the mutant may be more apparent after more restrictive
323
conditions confirmed the slight decreased endonuclease activity of the MR E1035 complex and further
324
showed that high salt concentration did not worsen this defect (Figures S7E and S7F). The detection of
325
MRE11 endonuclease activity, which depends on ATP hydrolysis (Anand et al., 2016; Cannavo et al.,
326
2019; Deshpande et al., 2017; Hopfner et al., 2000; Liu et al., 2016; Paull and Gellert, 1999), suggested
327
that recombinant RAD50E1035Δ did not abolish ATP hydrolysis, at least in vitro. Accordingly, in vitro
328
ATPase activity was similar with MR E1035 and MR WT (Figure 5I, J).
329
In summary, our experiments using recombinant proteins indicated that the RAD50E1035Δ
-330
containing MRN complex exhibits defects in MRE11 nuclease activity in vitro, a result congruent with
331
the conclusions drawn from the observations obtained in RAD50E1035Δ-expressing P1's cells.
332
333
Modeling the human RAD50E1035Δ mutation in Saccharomyces cerevisiae
334
We next modeled the human RAD50E1035Δmutation in S. cerevisiae by producing a yeast strain deleted
of the corresponding residue, i.e. the glutamic acid at position 1042 (rad50-E1042) (Figure 6A). In
336
addition, we generated a strain lacking a glutamic acid located nearby, at position 1044 (rad50-E1044;
337
Figure 6A) and also created two other yeast strains carrying missense mutations instead of deletion at
338
position 1042 where the glutamic acid residue was substituted by either an alanine (rad50-E1042A) or a
339
lysine (rad50-E1042K). Western blot and co-immunoprecipitation experiments showed a normal
340
expression and interaction with Mre11 of the deletion and missense mutants at both 30°C and 37°C,
341
indicating that these mutations did not impair Mre11 complex formation and stability (Figure 6B).
342
However, unlike wt strain, rad50-E1042 and rad50-E1044 deletion mutants exhibited high sensitivity
343
to camptothecin (CPT) (Figure 6C), methyl methane sulfonate (MMS) (Figure 6D), and hydroxyurea
344
(HU) (Figure 6E) at 37°C (but not at 30°C), indicating a strong temperature-sensitive effect of these
345
mutations on DNA repair. In striking contrast, both rad50-E1042A and rad50-E1042K missense mutants
346
showed WT levels of survival in the presence of CPT, MMS, and HU at both 30°C and 37°C (Figures
347
6C, 6D, 6E). These observations provided evidence that the glutamic acid residue at position 1042 (or
348
1044) in the coiled-coil domain of Rad50 is not critical for DNA repair. Rather, the presence of any
349
residue at this position is required, presumably to preserve the phasing of the local heptad repetitive
350
disposition of the coiled coil region. Consistent with this result, Marcoil analysis predicted that Rad50
351
deletion mutations caused a heptad break in the coiled-coil domain while this structure was not affected in
352
missense mutants (Figure S8A). Thus, this observation supported the hypothesis that the temperature
353
dependent DNA repair defect observed in rad50-E1042 and rad50-E1044 strains resulted from an
354
abnormal coiled-coil conformation.
355
In S. cerevisiae telomere maintenance relies on a functional Tel1ATM pathway. Both rad50-E1042
356
and rad50-E1044 deletion mutants, but not the rad50-E1042A and rad50-E1042K missense mutants,
357
exhibited shorter telomeres at 37°C (and to a lesser extent at 30°C) than in WT strain, suggesting
358
impaired Tel1 activation in deletion mutants (Figure 6F and Figure S8B). We previously showed that
359
MMS sensitivity of mec1ATRΔ cells is strongly rescued by sae2CtIPΔ, as mec1Δ sae2Δ cells hyperactivate
Tel1 kinase signaling and compensate for a lack of Mec1 kinase (Park et al., 2017; Usui et al., 2001).
361
Thus, to further examine Tel1 function, we evaluated the phosphorylation of Rad53CHK2 and thesurvival
362
upon MMS treatment of Rad50 mutants deficient for both Mec1 and Sae2. Rad50-E1042Δ mec1Δ sae2Δ
363
and rad50-E1044Δ mec1Δ sae2Δ mutant strains exhibited defective survival (Figure 6G) and Rad53
364
phosphorylation (Figure S8C) upon MMS exposure at 37°C, confirming impaired Tel1 signaling at this
365
temperature. In contrast, rad50-E1042A mec1Δ sae2Δ and rad50-E1042K mec1Δ sae2Δ mutants behaved
366
as WT at both 30°C and 37°C (Figure 6G and Figure S8C).
367
Taken together, the experiments conducted in yeast demonstrated that the loss of a unique residue
368
breaking the heptad repeats in the coiled-coil domain of Rad50 did not impact Rad50 expression or
369
Mre11 complex formation but severely affected DNA repair and Tel1 checkpoint signaling in a
370 temperature-dependent manner. 371 372
Discussion
373 374In this study, we identified biallelic mutations in the RAD50-encoding gene in an individual (P1)
375
exhibiting bone marrow failure, immunodeficiency, microcephaly, and developmental defects.
376
Consistently with the function of RAD50 as a part of the MRE11-RAD50-NBS1 complex, most of the
377
P1's clinical characteristics were reminiscent, although apparently less severe, than those found in the
378
autosomal-recessive diseases Nijmegen breakage syndrome (NBS) and ataxia-telangiectasia-like disorder
379
(ATLD), respectively caused by biallelic mutations in the genes encoding NBS1 and MRE11 (O'Driscoll,
380
2012; Stracker and Petrini, 2011). The symptoms observed in these disorders as well as in P1 likely
381
originate from the genome instability and DNA damage accumulation that particularly affect proliferating
382
tissues and lead to apoptotic attrition in those contexts (Taylor et al., 2019). Of note, P1 also developed a
383
cataract at 10 years of age that necessitated surgical intervention. To our knowledge, cataracts have not
384
been reported in patients with NBS1 and MRE11 deficiencies. However, a mouse model has implicated
385
Nbs1 in terminal differentiation of the lens fiber cells and cataractogenesis (Yang et al., 2006). Thus, the
386
early onset cataract in P1 suggests that RAD50 might also participate to the development and
maintenance of the lens in humans.
388
To the best of our knowledge, RAD50 hypomorphism has only been reported twice in two
389
individuals presenting with microcephaly, mental retardation, and short stature but no immunodeficiency
390
(Taylor et al., 2019; Waltes et al., 2009). These patients carried biallelic RAD50 mutations that strongly
391
reduced the expression of RAD50 (Ragamin et al., 2020; Waltes et al., 2009). Waltes et al. and Ragamin
392
et al. demonstrated that the highly pronounced reduction of functional MRN in the cells from these
393
patients caused DNA repair defects, genome instability, and impaired ATM-dependent DDR similar to
394
that observed in cells from patients with ATM, MRE11, and NBS1 mutations (Carney et al., 1998; Gatei
395
et al., 2011; Shiloh and Lederman, 2017; Stewart et al., 1999; Taylor et al., 2019; Varon et al., 1998;
396
Waltes et al., 2009). In sharp contrast, the present study showed that the cells from patient P1 exhibited
397
severe defects in DNA replication, DNA repair, and DNA end resection while the ATM-dependent DDR
398
remained intact (Figure S9A). Since one of the P1's RAD50 mutations generated a null allele, we
399
attributed this unprecedented phenotype to the RAD50E1035Δ mutation producing a normally expressed and
400
stable RAD50 protein that lacks a unique glutamic acid causing a break in the heptad repeats within the
401
coiled-coil domain. Knowing that the complete loss of Rad50 is lethal in mice (Adelman et al., 2009) and
402
having observed that P1's cells were proficient in ATM-dependent DDR, we deduced that
403
RAD50E1035Δmutation is hypomorphic.
404
Remarkably, P1's cells expressing RAD50E1035Δ mirrored the phenotype observed in mouse
405
embryonic fibroblasts (MEFs) expressing the Mre11 nuclease dead mutant (Mre11H129N) (Buis et al.,
406
2008). Indeed, in both Mre11H129N MEFs and RAD50E1035Δ P1's cells, MRN stability and recruitment to
407
DSB as well as ATM-dependent DDR were functional (Buis et al., 2008). Contrastingly, both Mre11H129N
408
MEFs and RAD50E1035Δ P1's cells exhibited increased sensitivity to genotoxics, defective IR-induced
409
RPA and RAD51 focus formation, and reduced HDR efficiency (Buis et al., 2008; Myler et al., 2017).
410
This resemblance is consistent with the interpretation that the RAD50E1035Δ mutation impacted the
411
MRE11 nuclease activity in vivo. This hypothesis was further supported by in vitro assays with purified
412
recombinant proteins that showed impaired MRE11 exonuclease activity when in complex with
RAD50E1035Δ while endonuclease activity was only slightly affected. However, since animal model
414
expressing the Mre11 nuclease dead mutant at a homozygous status (Mre11H129N/H129N) and human
415
MRE11-/H129N lymphoblast cell lines are unviable (Buis et al., 2008; Hoa et al., 2015; Hoa et al., 2016), we
416
surmise that RAD50E1035Δdoes not totally abolish MRE11 nuclease activity in vivo and/or that an
417
alternative mechanism allows to cope with defective MRE11-dependent DNA end resection in P1.
418
Hence, RAD50E1035Δ mutation represents the first human RAD50 separation-of-function mutation
419
impairing DNA repair, DNA replication, and DNA end resection without affecting MRN recruitment to
420
DSBs, and ATM-dependent DDR (Figure S9A). It is unlikely that RAD50E1035Δ affected RAD50-ATP
421
binding and hydrolysis since (i) this step is required for ATM-dependent DDR (Cannavo and Cejka,
422
2014; Deshpande et al., 2014; Lee et al., 2013), (ii) we observed an in vitro ATP hydrolysis with
423
recombinant RAD50E1035Δ (Figure 5), (iii) in vitro MRE11 endonuclease activity, which depends on ATP
424
hydrolysis (Anand et al., 2016; Cannavo et al., 2019; Deshpande et al., 2017; Hopfner et al., 2000; Liu et
425
al., 2016; Paull and Gellert, 1999), was detected with recombinant RAD50E1035Δ (Figure 5). Our
426
observation that recombinant MRE11-RAD50E1035Δ complex exhibited a reduced DNA binding and an
427
impaired exonuclease activity in vitro (Figure 5) combined with the reduction of IR-induced RPA and
428
RAD51 foci and HDR in P1's cells (Figure 3) rather supports the notion that RAD50E1035Δ impairs the
429
MRN complex nuclease activity required to promote DNA end resection.
430
These results led us to propose a unified speculative model in which the human RAD50E1035Δ
431
mutation creates (or loses) a structural constraint in the coiled-coil domain that propagates to the globular
432
domain to hinder the proper conformational transition of the MRN intracomplex from the ring to the rod
433
shape (Figure S9B). This abnormally structured MRN intracomplex would retain its ATP binding and
434
hydrolysis, as well as most of its capacity to stimulate MRE11 endonuclease activity. However, the
435
mutant would corrupt subsequent conformational change required for MRE11-dependent exonuclease
436
activity and DNA end resection (Shibata et al., 2014). Since it has been suggested that the MRE11
437
intracomplex in its rod shaped conformation clamps DNA (Kashammer et al., 2019), the observation of a
438
reduced capacity of recombinant MRE11-RAD50E1035Δ proteins to interact with DNA in vitro further
supports this model. One cannot however rule out that other functionally important coiled-coil
440
interactions both within the anti-parallel coiled-coil and between the dimeric coiled-coil assembly could
441
be affected by RAD50E1035Δ. Alternatively, we cannot exclude that RAD50E1035Δ mutation could
442
compromise MRN to interact and/or activate other factors such as EXO1, DNA2, and EXD2 required for
443
efficient DNA end resection in vivo (Broderick et al., 2016; Cejka et al., 2010; Delamarre et al., 2020;
444
Myler et al., 2017; Pasero and Vindigni, 2017; Paull, 2018; Stracker and Petrini, 2011; Syed and Tainer,
445
2018; Zhu et al., 2008).
446
Notably, our analysis of rad50-E1042∆ yeast strain modeling the human RAD50E1035Δ mutation
447
pinpointed a phenotype strikingly different from human cells. Indeed, E1042∆ strain (and
rad50-448
E1044∆) behaved as wt at 30°C while it was almost as severe as rad50∆ strain for both DNA repair and
449
Tel1ATM activation at 37°C. To our knowledge this extremely severe thermosensitive phenotype in
rad50-450
E1042∆ (and rad50E1044∆) is unprecedented in Rad50 mutants. Furthermore, the observation that yeast
451
strains carrying missense mutations at position E1042 (rad50-E1042A and rad50-E1042K) behaved as wt
452
at both 30°C and 37°C proved that this is the deletion of a single amino-acid at this position that is
453
deleterious and not the nature of the residue. From these yeast experiments we propose a model in which
454
the rad50E1042∆ (and rad50E1044∆) mutations, by breaking the heptad repeats, increase the flexibility
455
of the coiled-coil at 37°C but not at 30°C. This temperature dependent change would ultimately
456
destabilize either the coiled-coil interface-dependent dimerization, the conformation of the globular
457
domain, the Zn-hook-mediated dimerization, or a combination of these modifications that would result in
458
the extremely severe phenotype at 37°C. Interestingly, the phenotype of rad50-E1042∆ strain at 37°C was
459
similar to the one of yeast mutant lacking the Zn-hook in which MR intra- and intercomplex formations
460
are abolished (Hopfner et al., 2002; Stracker and Petrini, 2011). Moreover, it has been recently reported
461
that the Zn-hook-proximal coiled-coil participated in the stabilization of intracomplex Mre11 complex
462
assembly (Park et al., 2017). One can therefore hypothesize that the temperature sensitivity of the
rad50-463
E1042∆ mutant results from defective MRN intracomplex assembly at 37°C (Hohl et al., 2011; Wiltzius
464
et al., 2005). Interestingly, other yeast strains carrying mutations affecting the Rad50 Zn-hook and/or
coiled-coil domains exhibited thermosensitive phenotypes (Hohl et al., 2015; Hohl et al., 2011). However,
466
the molecular cause of the temperature dependent phenotype in these Rad50 mutants remains unclear and
467
future studies are warranted to get further into the structural and functional consequences of these
468
mutants.
469
In summary, our study provided evidence that a single amino-acid deletion that breaks the heptad
470
repeats in the coiled-coil domain of the yeast and human RAD50 compromised MRE11 functions.
471
Furthermore, our demonstration that human RAD50E1035Δ is a separation-of-function mutation that
472
impairs DNA repair, DNA replication, and DNA end resection without affecting ATM-dependent DDR
473
supports the idea that the integrity of the RAD50 coiled-coil domain is essential to enable switch of the
474
MRE11-RAD50 intracomplex toward functional rod shaped conformation promoting DNA clamping and
475
DNA end resection (Hopfner, 2014; Kashammer et al., 2019; Park et al., 2017; Rojowska et al., 2014).
476
Further studies using cryo-EM, atomic force, and high-throughput single-molecule microscopy are
477
warranted to better characterized the structural hindrance caused by RAD50E1035Δ and decipher the rules
478
delineating the communication between the hook, the coiled-coil, and the globular domains of the
479
MRE11-RAD50 complex in mammals. Lastly, the development of a Rad50E1035Δ mouse model should
480
provide crucial information on the functional consequence of compromised Mre11-dependent DNA end
481 resection in vivo. 482 483 484 485 486 487 488 489 490 491
Acknowledgments
492 493
General: We thank the patient and his family for their generous assistance with samples and information,
494
which made this research possible. We acknowledge M. Garfa Traore and the imaging facility of Imagine
495
Institute for help with microlaser irradiation, the Bioinformatics Department of Imagine Institute for help
496
with Whole exome sequencing, N. Lambert (CEDI, AP-HP, Necker Hospital, Paris, France) for
497
microsatellite analysis, A. Fernandes from the Centre de ressource biologique, Imagine Institute, who
498
generated B-LCL. We thank Dr B. Lopez for RAD50 construct used as matrix and technical advice for
499
RAD51 and RPA immunofluorescence study. We thank Dr G. Dellaire for the HDR assay components
500
(Pinder et al., 2015). Dr P. Pasero is acknowledged for his advice with replication combing assays. P.R.
501
thanks Dr A. Decottignies, Dr P. Kannouche, and Dr E. Brunet for critical reading of the manuscript. P.R.
502
is a scientist from Centre National de la Recherche Scientifique (CNRS).
503
504
Funding: Work performed in the GDIS lab was supported by institutional grants from INSERM, Ligue
505
Nationale contre le Cancer (Equipe Labellisée "LIGUE 2020"), INCa (PLBIO19-027; INCA_13766),
506
GIS-Institut des maladies rares, and state funding from the Agence Nationale de la Recherche under
507
“Investissements d’avenir” program (ANR-10-IAHU-01). This work was supported by GM56888 and
508
MSK Cancer Center Core Grant P30 CA008748 (J.H.J.P.) and Swiss National Science Foundation
509
(31003A_17544) and European Research Council grant (681-630) to P.C. M.M. is supported by the
510
French National League Against Cancer (EL2028.LNCC/MaM), the French National Cancer Institute
511
(PLBIO2017-167). M.C.D-C. beneficiated from scholarships from Association Nationale Recherche
512
Technologie (ANRT) and La Ligue contre le Cancer. P.R. is a scientist from Centre National de la
513 Recherche Scientifique (CNRS). 514 515 516 517 Author contributions 518
M.C.D-C. carried out most of the experimental work on human cells assisted by P.R. and L.K. T.I.
519
identified the affected patient and assisted with related clinical and laboratory studies. I.C. and P.C.
520
produced recombinant MRN complex and performed in vitro experiments. M.H and J.H.J.P conceived
521
and performed experiments on yeast strains. P.R. conceived the project and wrote the manuscript with
522
editing contributions from J-P.V., I.C., M.H., P.C., and J.H.J.P.
Main figure titles and legends
524
Figure 1. Biallelic RAD50 mutations identified in individual P1. (A) (upper panel) Pedigree of the P1's
525
family. (lower panel) Genetic approach used to identify the disease-causing gene. (B) Sequencing of
526
cloned RAD50 PCR products in patient. (C) Schematic of the domain architecture of human RAD50
527
showing the position of disease-associated mutations. NBD: nucleotide binding domain. (D) (left)
528
Immunoblot of indicated proteins in B-LCL lysates from a healthy donor, P1 and his mother. Ku70 was
529
used as loading control. (right) Relative abundance of indicated proteins normalized to Ku70. (E) (left)
530
Schematic view of a RAD50 protein. (right) Schematic view of MRN complex in its intracomplex
531
conformation13. (F) Marcoil analysis revealed a break in the heptad repeats nearby the E1035Δ mutation
532
(blue arrow). (G) (Up) Representative view of the coiled-coil probability along the full WT and
533
RAD50E1035Δ sequences determined by Marcoil tool. The red area corresponds to the probable coiled-coil
534
regions. (Down). Zoom of the region encompassing the E1035Δ mutation. Blue arrow indicates a break
535
in the coiled-coil probability.
536 537
Figure 2. Defective DNA repair but normal DNA damage sensing and signaling in P1's cells. (A)
538
Survival of SV40-fibroblasts after IR (A), Phleomycin (B), Etoposide (C) and MMC (D) treatment.
539
Results are expressed as the fraction of surviving cells in relation to untreated cells. Each point represents
540
the mean value and standard deviation of three separate determinations. ATM and Cernunnos deficient
541
cells were used as sensitive control. Control sensitive fibroblasts were from ATM, Cernunnos and
542
Fanconi deficient patients. (E) Sensitivity of the P1's cells to phleomycin after wtRAD50 or empty vector
543
transduction. This experiment was performed three times. Control sensitive fibroblasts were from a
544
Cernunnos deficient patient. (F) Functional complementation of phleomycin sensitivity provided by
545
wtRAD50 transduced into the patient’s cells as compared with the empty vector. The selective growth
546
advantage is scored as the increase in the index of mCherry-positive cells/mCherry-negative cells at
547
various times compared with the initiation of the culture (index = 1). This experiment was performed
548
twice. (G) Recruitment of GFP-NBS1 to DNA damage upon laser microirradiation in indicated cells.
549
GFP-NLS (nuclear localization signal) is used as negative control. Irradiated zones are located in the red
550
circles. Representative of three independent experiments. (H) Kinetics of recruitment of GFP-NBS1 and
551
GFP-NLS to DNA damage in control and patient's cells. (I) Immunofluorescence detection of
552
phosphorylated KAP1 (P-KAP1) in primary fibroblasts before and 1 hour after 0.5 Gy irradiation. ATM-
553
and NBS1- deficient cells are used as negative controls. Image representative of three independent
554
experiments. (J) Phosphorylated forms of CHK2 and NBS1 detected by Western blot analysis of whole
555
cell lysates from SV40-fibroblasts from a healthy control, an ATM-deficient patient and patient P1
556
untreated or 1 hour after 5 or 10 Gy irradiation. Vinculin immunoblot is used as loading control. Asterisk
557
indicates a non specific band. (K) (Left) The DNA content and phosphorylation of histone H3 in G2 (pink
558
rectangle) were analyzed by FACS. (Middle) FACS images of P-H3 in G2 in untreated and irradiated
559
cells. (Right) G2/M checkpoint was measured by the % inhibition of entry into mitosis in cells after IR.
560
Results represents the mean and SD from at least three independent experiments. Statistical significances
561
are noted.
562 563
Figure 3. P1's cells exhibit reduced DNA end resection and homology directed repair.
564
Immunofluorescence detection of phosphorylated KAP1 (A), RPA32 (B), and RAD51 (C) in primary
565
fibroblasts from healthy control and P1 before and 6 hours after 10 Gy irradiation. Image representative
566
of three independent experiments. (D) Quantitative analysis of RAD51 foci before and after IR in
hTERT fibroblasts untransduced or transduced with empty or wtRAD50- expressing vector. The number
568
of nuclei scored and statistical significances are noted. (E) Efficiency of homology directed repair
569
assessed by the Cas9-directed knock-in of Clover in the LMNA coding sequence. Cells were transfected
570
with the mCherry vector alone (used to control transfection efficiency), or in combination with the repair
571
template sequence containing the Clover coding sequence in conjunction or not with the pX330-LMNA1
572
vector encoding Cas9 and the gRNAs targeting the LMNA sequence (noted CRISPR/Cas9). Relative
573
HDR efficiency was measured by the percentage of Clover+cells triple transfected relative to cells
574
transfected without the CRISPR/Cas9-gRNA expressing vector. Results represents the mean and SD of
575
triplicates from three independent experiments.
576 577
Figure 4. DNA replication and resection at stalled forks in P1's cells. (A) Detection of 53BP1 foci in
578
primary fibroblasts from patient and a healthy control at similar passage. (B) Mean of 53BP1 foci in
579
primary fibroblasts from healthy control and patient. The number of nuclei scored and statistical
580
significances are noted. (C) The percentages of fibroblasts from a control and patient P1 with the
581
indicated number of 53BP1 foci are indicated. Statistical significances are noted. (D) (Up) Representative
582
image of combed DNA (yoyo labeling). (Down) Picture representing the detection of newly synthesized
583
DNA by molecular combing (successive pulse labelings of IdU and CldU). DNA is detected by
584
counterstaining (blue). (E) Fork velocity in SV40-fibroblasts from 3 independent experiments in control
585
and P1 are represented. (F) Inter-origin distances in SV40-fibroblasts from control and P1are represented.
586
The statistical significances are noted. (G) Left. Asymmetric replicon with sister forks with more than a
587
25% length difference in SV40-fibroblasts from control and P1. Plots of the distances covered by
right-588
moving and left-moving sister forks during the IdU and CldU pulses from two independent experiments.
589
(Right) Percentage of replicons with more of 25% asymmetry in SV40-transformed fibroblasts from
590
control and P1. The statistical significance is noted. (H) Fork velocity of SV40-transformed fibroblasts
591
from control and P1 before or after transduction with a wtRAD50 expressing vector or empty vector.
592
Results representative of two independent experiments. The statistical significances are noted
(Mann-593
Whitney statistical test). (I) (Up) Protocol used to measure DNA resection after replicative stress
594
induction by HU. (Down) Representative tracks of CldU (red) and IdU (green) incorporation in control
595
and patient's cells with or without HU treatment. DNA is counterstained in blue. (J) DNA resection at
596
stalled forks is determined by the ratio between CldU and IdU track lengths after 3 hours of HU treatment.
597
Results representative of two independent experiments. (K) DNA resection at stalled forks after 3 hours
598
of HU in control and P1 cells before or after transduction with an empty or wtRAD50 expressing vector.
599
Results representative of two independent experiments. The statistical significances are noted
(Mann-600
Whitney statistical test).
601 602 603
Figure 5. Impairment of nuclease activity of recombinant MRE11 and RAD50E1035Δ proteins. (A)
604
Recombinant MRE11-RAD50 (MR) with WT RAD50 (MR WT) or mutated RAD50E1035Δ (MR
605
E1035Δ) used in this study. (B) Representative DNA-binding analysis by electrophoretic mobility shift
606
assay with recombinant MR WT and MR 1035Δ, using 70-bp-long dsDNA as a substrate. (C)
607
Quantitation of experiments shown in (B). Error bars, SEM; n=3. (D) Exonuclease activity of MR WT or
608
MR E1035Δ using a 50-bp-long dsDNA substrate. (E) Plots of signals shown in (D) reveal a reduction of
609
the most resected DNA substrate (higher migration) with MR E1035Δ as compared to MR WT. (F)
Quantitation of experiments shown in (C). Error bars, SEM; n=3. (G) Representative nuclease assay with
611
MR WT or MR E1035Δ in presence of MBP-NBS1 and pCtIP on 5'-end-labeled 70-bp dsDNA with all
612
ends blocked with streptavidin. (H) Quantitation of experiments shown in (G). Error bars, SEM; n=3. (I)
613
ATP hydrolysis activity of MR WT or MR E1035Δ with or without MBP-NBS1. The MRN complexes
614
containing the RAD50 K40A (noted MR(KA)N) and RAD50 K40R (noted MR(KR)N) mutants, defective
615
in ATP binding and hydrolysis (Cannavo et al., 2019; Chen et al., 2005), were used as negative controls.
616
(J) Quantitation of experiments shown in (I). n=2.
617 618 619
Figure 6. Modeling the human RAD50E1035Δ mutation in Saccharomyces cerevisiae. (A) Schematic
620
representation of the Rad50 structure and multiple sequence alignment of C-terminal coil domain of the
621
Rad50 carrying the human E1035Δ mutation. The human E1035 residue and corresponding amino acid in
622
Saccharomyces cerevisiae and Mus musculus are highlighted in green. (Sc, S. cerevisiae; Mm, M.
623
musculus; Hs, H. sapiens). (B) Coimmunoprecipitation and western blot with Rad50 or Mre11 antisera
624
assessed the Mre11 complex integrity in wild-type (WT) and indicated Rad50 mutants. IP,
625
immunoprecipitation. (C, D, E) Sensitivities of WT and indicated Rad50 mutant to the indicated
626
concentration of CPT (C), MMS (D), and HU (E). Plates were incubated at 30°C and 37°C as indicated.
627
(F) Telomere lengths of wild-type (WT) and rad50 mutants after 4 days of growth at 30°C and 37°C.
628
Heterozygote diploids were included as 0 generation of growth. (G) Cell survival of rad50 mutants in
629
Mec1- and Mec1- Sae2- deficient backgrounds upon MMS treatment at 30°C and 37°C.
630 631 632 633 634 635 636 637 638 639 640 641 642 643 644
STAR METHODS 645 646 RESOURCE AVAILABILITY 647 Lead Contact 648
Further information and requests for resources and reagents should be directed to and will be fulfilled by
649
the Lead Contact, Patrick Revy ([email protected]).
650 651
Material availability
652
All unique/stable reagents generated in this study are available from the Lead Contact with a completed Materials 653
Transfer Agreement. 654
655
Data and Code Availability
656
All used software is listed in the Key Resources Table. This study did not generate any unique datasets or new code. 657
658
EXPERIMENTAL MODEL AND SUBJECT DETAILS
659
Human fibroblasts used in this study were obtained from skin biopsies from pediatric healthy donors (3
660
years of age) and patients. When indicated transformed (with large T antigen from SV40T) and
661
immortalized (after transduction with a hTERT-expressing vector, Addgene #85140) fibroblasts were used.
662
All cell lines were from male individuals and were checked for mycoplasma contamination. Informed and
663
written consent was obtained from donors and patients. The study and protocols comply with the 1975
664
Declaration of Helsinki as well as with the local legislation and ethical guidelines from the Comité de
665
Protection des Personnes de l’Ile de France II and the French advisory committee on data processing in
666
medical research.
667
Saccharomyces cerevisiae strains used in this paper are listed in Table S2. Yeast strains were grown in
668
liquid cultures and on plates in YPD media supplemented with 100 mg/L adenine. Further specifications
669
are mentioned within the Methods Details section.
670 671 672 METHOD DETAILS 673 674 Study approval 675
Informed and written consent was obtained from donors and patients. The study and protocols comply with
676
the 1975 Declaration of Helsinki as well as with the local legislation and ethical guidelines from the
677
Comité de Protection des Personnes de l’Ile de France II and the French advisory committee on data
678
processing in medical research.
679 680
Cells
681
Control fibroblasts were obtained from skin biopsies from pediatric healthy donors (3 years of age). When
682
indicated transformed (with large T antigen from SV40T as previously described (Buck et al, 2006)) and
683
immortalized (after transduction with a hTERT-expressing vector, Addgene #85140) fibroblasts were used.
684
All cell lines were checked for mycoplasma contamination.
685 686
Yeast
687
Yeast strains were generated by integration of the rad50-coiled coil mutants at their native chromosomal
688
locus in a diploid WT strain. rad50 mutant spores were obtained by tetrad dissection and verified by PCR
689
genotyping and sequencing using genomic DNA. Details of yeast strains and plasmid constructions are