BRIEF REPORT

Identification of two novel BRCA1-partner genes in the DNAdouble-strand break repair pathway

Chiara Guglielmi • Iacopo Cerri • Monica Evangelista •

Anita Collavoli • Mariella Tancredi •

Paolo Aretini • Maria Adelaide Caligo

Received: 16 July 2013 / Accepted: 16 September 2013 / Published online: 9 October 2013

� Springer Science+Business Media New York 2013

Abstract M1775R and A1789T are two missense vari-

ants located within the BRCT domains of BRCA1 gene.

The M1775R is a known deleterious variant, while the

A1789T is an unclassified variant that has been analyzed

and classified as probably deleterious for the first time by

our group. In a previous study, we described the expression

profile of HeLa G1 cells transfected with the two variants

and we found that they altered molecular mechanisms

critical for cell proliferation and genome integrity. Con-

sidering that the mutations in the BRCA1 C terminus

(BRCT) domains are associated to a phenotype with an

altered ability in the DNA double-strand break repair, we

chose three of the genes previously identified, EEF1E1,

MRE11A, and OBFC2B, to be tested for an homologous

recombination (HR) in vitro assay. For our purpose, we

performed a gene expression knockdown by siRNA trans-

fection in HeLa cells, containing an integrated recombi-

nation substrate (hprtDRGFP), for each of the target genes

included BRCA1. The knockdown of BRCA1, OBFC2B,

MRE11A, and EEF1E1 reduces the HR rate, respectively,

of 97.6, 28.6, 41.8, and 42.3 % compared to cells trans-

fected with a scrambled negative control duplex and all

these differences are statistically significant (P \ 0.05;

Kruskal–Wallis test). Finally, we analyzed the effect of

target gene depletion both on HR that on cell survival after

DNA-damage induction by ionizing radiation. The clono-

genic assay showed that the down-regulation of the target

genes reduced the cell survival, but the effect on the HR, is

not evident. Only the BRCA1-siRNA confirmed the inhi-

bition effect on HR. Overall these results confirmed the

involvement of MRE11A in the HR pathway and identified

two new genes, OBFC2B and EEF1E1, which according to

these data and the knowledge obtained from literature,

might be involved in BRCA1-pathway.

Keywords Homologous recombination � BRCA1-

partners genes � OBFC2B � EEF1E1 � Breast cancer �Functional assay

Introduction

BRCA1 is a protein with multiple functions. It has a crucial

role in DNA-damage response signaling network (G1/S, S,

and G2/M checkpoints), it is required for TP53 phosphor-

ylation mediated by ATM/ATR (ataxia telangiectasia

mutated/ataxia telangiectasia and Rad3 related) in response

to DNA damage by ionizing or ultraviolet irradiation [1]. It

takes part in maintaining the cell genomic integrity and

participates, with MRN (MRE11A/RAD50/NBN) com-

plex, in DNA double-strand break repair (DSBR) mediated

by homologous recombination (HR) and by non-homolo-

gous end-joining (NHEJ) [2–4]. It is involved in tran-

scriptional regulation [5], in the chromatin remodeling [6]

and, when heterodimerizes with BARD1 (BRCA1-associ-

ated RING domain 1) [7], in the protein ubiquitination.

The protein consists of different functional domains: a

N-terminal RING finger domain, two nuclear localization

C. Guglielmi � I. Cerri � A. Collavoli � M. Tancredi �M. A. Caligo (&)

Section of Genetic Oncology, University Hospital and University

of Pisa, Pisa, Italy

e-mail: adelaide.caligo@do.unipi.it

M. Evangelista

Laboratory of Molecular and Gene Therapy, Institute of Clinical

Physiology, CNR, Pisa, Italy

P. Aretini

Fondazione Pisana per la Scienza Onlus, Pisa, Italy

123

Breast Cancer Res Treat (2013) 141:515–522

DOI 10.1007/s10549-013-2705-9

signals, a ‘‘SQ’’ cluster, a branched DNA-binding domain

and a C-terminal domain containing two BRCT (BRCA1 C

Terminus) repeats [8]. The BRCT repeats regulate the

DNA-damage response facilitating the assembly of DNA-

damage signaling complexes [9]; they also have place in

the transcriptional activity of BRCA1 and they are con-

sidered the most responsible for the BRCA1 tumor sup-

pression activity. [10].

Nonsense and frameshift BRCA1 mutations encoding

for truncated and not functional proteins, predispose

women to early onset breast and ovarian cancer, but for a

significant number of variants, the effect on protein func-

tion is unknown making more difficult to infer the conse-

quences on cancer risks.

So a variety of predictive approaches, including func-

tional assay, have been reported to understand the func-

tional significance of these missense variants of uncertain

pathological significance and to distinguish cancer-related

variants from neutral polymorphisms [11].

In a previous collaborative work [12], we investigated

the effects on human cell transcriptome of two missense

variants, M1775R and A1789T, both located within the

second BRCA1 BRCT domain and identified in breast

cancer patients. The M1775R variant has widely been

described as deleterious by both in silico analysis and

functional assays, while the A1789T variant has been

identified and classified as probably deleterious by our

group. We found, by a microarray experiment, that the

overexpression of the two variants, in HeLa G1 cells,

altered the expression of genes involved in pathway critical

for cell proliferation and genome integrity, suggesting a

causative role of these two variants in breast cancer onset

and development. [12]. Considering that those genes are

probably implicated in BRCA1 pathway and that mutations

in the BRCT domain are associated to phenotype with an

altered ability in the DNA DSBR, we decided, in this study,

to investigate the involvement of some of these genes in the

HR process. In particular, we chose three genes previously

described in literature as involved in DNA repair: EEF1E1,

MRE11A, and OBFC2B.

MRE11A is a member of the MRN complex that pro-

motes the recognition of DSB [13, 14].

OBFC2B (hSSB1) is a single-strand DNA-binding pro-

tein that participates in the recombination and in ATM-

mediated checkpoint pathway [15–18], while EEF1E1

(p18) is a factor associated with the macromolecular tRNA

synthetase complex important for ATM/ATR-mediated

TP53 activation in response to DNA damage [19, 20].

To evaluate the involvement of these genes in the HR,

we specifically constructed clones of HeLa cells with an

integrated recombination substrate (hprtDRGFP) that allow

to estimate the efficiency of HR as number of GFP-positive

cells by fluorescent microscopy or FACS analysis [21]. In

particular, we performed a gene expression knockdown for

each of the tested genes by siRNA transfection and we

evaluated the frequency of recombination in comparison

with the controls.

Moreover, we analyzed the effects of down-expression

of each target gene on HR and on cell survival in a situa-

tion of DNA-damage induced by ionizing radiation.

Materials and methods

Plasmids

To determine if the down-regulation of the studied genes

affects the HR efficiency, we used the hprtDRGFP and the

pCBASce plasmids previously described [21, 22] (Fig. 1).

Cell culture

All the hprtDRGFP HeLa clones were grown in Dulbecco’s

modified Eagle’s medium with 10 % fetal bovine serum, 1 %

glutamate, 1 % penicillin–streptomycin, 1 lg/ml puromycin

(DMEM/FBS).

hprthprt

HR

GFP wt

DSB

ISce1

No GFP

(1 )

(1 )

(2 )

(2 )

SceGFP

puroRiGFP

I-Sce1

hprtDRGFP

Fig. 1 The hprtDRGFP substrate is composed of two differentially

mutated green fluorescent protein (GFP) genes: one contains the

18 bp recognition site for the I-SceI endonuclease (sceGFP) and the

other one is an 812 bp internal GFP fragment (iGFP) that can be used

to correct the mutation in the SceGFP gene to result in a GFP

functional gene. When the I-SceI expression vector is expressed

(pCBASce vector), creates a double-strand break and only the HR

pathway can reconstitute the functional gene (1). So the efficiency of

HR induced by I-SceI is evaluated distinguishing into GFP-positive

and GFP-negative cells. The vector also contains homologous hprt-

targeting arms for integration of the construct into the hprt locus, and

a dominant selectable puroR marker [21]

516 Breast Cancer Res Treat (2013) 141:515–522

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siRNA and pCBASce plasmid transfection

The knockdown of BRCA1, MRE11A, OBFC2B, and

EEF1E1 was performed using the commercial siRNA

(TEMA Ricerca) reported in Table 1. Cells transfected with

BRCA1-siRNA were used as positive control to check the

system functionality and as yardstick for the other genes.

We used, as negative controls, a siRNA directed against an

irrelevant gene, not involved in the DNA repair mechanism

as HPRT and a scrambled negative control duplex (SNCD).

Each experiment was performed in a week:

Day 1 1 9 105 cells of the clone were seeded in a

24-well plate in 0.5 ml of DMEM/FBS.

Day 2 If the cells were *40–50 % confluent, we could

proceed with the transfection by mixing: 10 pmol siR-

NA ? 24 ll Opti-MEM and 0.5 ll Lipofectamine

2000 ? 24,5 ll Opti-MEM. We followed the Lipofect-

amine protocol (Invitrogen).

Day 3 The cells were trypsinized and transferred from

the 24-well plate to a 6-well plate in 2 ml of DMEM/FBS.

Day 4 Cells were transfected by mixing: 50 pmol siR-

NA ? 2 lg pCBASce ? 123 ll Opti-MEM and 5 ll

Lipofectamine 2000 ? 120 ll Opti-MEM.

Day 7 72 h after the transfection, cells were trypsinized

and analysed for GFP expression by fluorescent micros-

copy or by FACS.

This experiment was performed eight times. The num-

ber of GFP-positive cells was evaluated three times by

fluorescent microscopy and five times by FACS analysis.

Analysis by fluorescent microscopy and FACS

For the fluorescent microscopy analysis we spotted on slide

250,000 cells of each well (by Citospin 3—Shandon). The slides

were fixed for 5 min in paraformaldehyde 2 % in phosphate-

buffered saline (PBS), permeabilized for 5 min in PBS-Tween

1 % and washed in PBS 19. We added 3 ll of DAPI II (Vysis)

for each spot of cells and we analyzed 1,000 cells per slide.

For the FACS analysis we analyzed 10,000 cells of each

well.

Evaluation of protein level

72 h after the transfection, aliquots of 4 9 105cells were

washed twice in PBS 19 and lysed in the RIPA buffer 19

(NaPO4 10 mM pH 7.2, NaCl 0.3 M, SDS 0.1 %, NP40

1 %, deoxycholate 1 %, EDTA 2 mM) together with the

Protease Inhibitor Cocktail 19, Sodium orthovanadate 19,

PMSF 19 (Sigma).

The level of protein was analyzed by western blot (WB)

technique as previously reported [23] using the following

primary antibodies: BRCA1 sc-6954 (dilution 1:100, Santa

Cruz Biotechnology.), OBFC2B sc-168826 (dilution 1:400,

Santa Cruz Biotechnology); EEF1E1 sc-68325 (dilution

1:200, Santa Cruz Biotechnology); MRE11A sc-135992

(dilution 1:500, Santa Cruz Biotechnology).

HR and cell survival assay after ionizing radiation

exposure

To investigate the involvement of the studied genes in the

cell survival and in the HR after induction of DNA damage

by ionizing radiation exposure, we first of all made a sur-

vival curve for the hprtRGFP HeLa cells at the doses of 2,

3, 4, and 8 Gy and we chose 3 Gy as dose to use for our

experiments being our LD50. Then we performed a gene

expression knockdown, for each target gene, following the

same protocol described above, but this time we irradiated

the cells 48 h after the second transfection to give them

sufficient time to express the pCBASce vector.

24 h after the irradiation, cells were trypsinized, ana-

lysed by FACS and 100 cells were seeded in 60-mm dishes.

The number of surviving colonies was evaluated by

staining cells with crystal violet 10 days after seeding. We

repeated this experiment three times.

Statistical analysis

All the analyses were performed using MedCal software.

The results were analyzed by the Kruskal–Wallis test, a

Table 1 Commercial siRNA used

Genea DsiRNA duplexb Region/exonc Sequenced

BRCA1 HSC.RNAI.N000059.12.3 30-UTR 50-CCCACAAACUGUAAAUGAAGAUATT-30

MRE11A HSC.RNAI.N005590.12.2 30-UTR 50-GGAGUAAACAUGGAAGUACUAACTT-30

OBFC2B HSC.RNAI.N024068.12.3 50-UTR-CDS/2 50-CCCUGUUAGUAACGGCAAAGAAACC-30

EEF1E1 HSC.RNAI.N001135650.12.1 CDS/2 50-AGAAUACAGGGUCACUCAAGUAGAT-30

a Gene symbolb Commercial catalog ID of the siRNA usedc siRNA locationd siRNA sequence

Breast Cancer Res Treat (2013) 141:515–522 517

123

nonparametric method to test the equality of the medians of

different groups considering as p value cut-off P \ 0.05.

Results

Isolation of HeLa hprtDRGFP clone

We transfected 2 lg of the hprtDRGFP substrate in HeLa

cells and isolated the clones puromycin resistant. (1 lg/ml).

We confirmed the presence of the integrated recombi-

nation substrate transfecting 2 lg of the I-Sce1 expression

vector pCBASce and verifying the presence of GFP-posi-

tive cells by fluorescent microscopy. 4 % of cells were

GFP-positive in all the isolated clones. Moreover, we

amplified the sequence of the construct by PCR with the

following primers forward, 50-AGGGCGGGGTTCGG

CTTCTGG-30 and reverse, 50-CCTTCGGGCATGGCG G

ACTTGA-30 (Fig. 2).

Knockdown of BRCA1-partner genes (BPG) by siRNA

transfection in HeLa hprtDRGFP

To test the involvement of MRE11A, OBFC2B, and

EEF1E1 in the HR, we performed, in the HeLa hprtDRGFP

clone, a knockdown of each of these genes by siRNA

transfection. We compared the frequency of recombination

of cells treated with BPG-siRNA to the one of cells inac-

tivated for BRCA1 and to control cells (cells inactivated

for HPRT and cells transfected with a SNCD).

WB experiments showed that the expression levels of

BRCA1, OBFC2B, EEF1E1, and MRE11A seem to be

lower in cells transfected with each specific siRNA in

comparison with control cells (Fig. 3).

The depletion of OBFC2B, EEF1E1, and MRE11A

reduces the HR frequency: analysis by fluorescent

microscopy

In cells depleted for HPRT and in cells transfected with a

SNCD, the number of GFP-positive cells was, respectively,

38 9 10-3 and 36 9 10-3: these recombination frequen-

cies are comparable to the background frequency in

parental HeLa cell line. In Fig. 4, the mean values of three

experiments are shown.

In cells depleted for BRCA1 the number of GFP-posi-

tive cells is less than 1 9 10-3 (±0.57). The HR frequency

Fig. 2 Amplification by PCR of a sequence of 800 pb of the

hprtDRGFP substrate. Lane 1 marker 1 kb, lane 2 clone A, lane 3

clone B, lane 4 clone C, lane 5 negative control

Fig. 3 Analysis of protein levels in cells transfected with siRNA

directed against (a) BRCA1, (b) OBFC2B, (c) EEF1E1, and

(d) MRE11A by WB. In each experiment, cells transfected with

SNCD and HPRT-siRNA were the controls. In cells transfected,

respectively, with specific siRNA for BRCA1, OBFC2B, EEF1E1,

and MRE11A, the protein levels are lower in comparison with control

cells (1). The b-tubulin expression is use as control for equal protein

loading (2)

- ISce1 HPRT SNCD BRCA1 OBFC2B EEF1E1 MRE11A + ISce1

Fig. 4 Graph representing the mean values and the standard devia-

tions of spontaneous HR frequency obtained from three experiments.

In each experiment we analyzed, by fluorescent microscopy, the

number of GFP-positive cells out of 1,000 cells counted per slide

(n 9 10-3 GFP-positive cells). In clones not transfected with the

I-Sce1 expression vector (pCBASce), but with an empty vector, we

did not detect the presence of GFP-positive cells. Cells depleted for

BRCA1, OBFC2B, EEF1E1, and MRE11A showed a statistically

significant reduction in the HR levels in comparison with control cells

(HPRT and SNCD)-Kruskal–Wallis test, *P \ 0.05

518 Breast Cancer Res Treat (2013) 141:515–522

123

in OBFC2B, MRE11A, and EEF1E1-depleted cells were

15.3 9 10-3 (±2.08), 20.6 9 10-3 (±6.02), and 11.5 9

10-3 (±2.12), respectively.

The statistical analysis showed that all the tested genes

(BRCA1, OBFC2B, EEF1E1, and MRE11A), when less

expressed, lead to a significant reduction of HR events

compared to the controls (P \ 0.05; Kruskal–Wallis test).

Confirmation of the effect of OBFC2B, EEF1E1,

and MRE11A in the HR by FACS analysis

The results obtained were confirmed by FACS analysis, but

this time 10,000 cells per well were analyzed. We repli-

cated the experiments of HR five times. The recombination

frequency for each experiment is reported in Fig. 5, while

the Fig. 6 shows the mean values of HR levels in cells

inactivated for each gene.

The frequency of HR in cells depleted for HPRT and in

cells transfected with the SNCD is, respectively, 2.01 %

(±0.42) and 1.82 % (±0.33). In cells depleted for BRCA1,

the HR frequency is only 0.06 % (±0.02). The depletion of

OBFC2B, MRE11A, and EEF1E1 showed, respectively

1.30 % (±0.31), 1.06 % (±0.23), and 1.05 % (±0.46) of

GFP-positive cells. In all cases the difference is statistically

significant compared to the control HPRT-depleted cells and

to SNCD-transfected cells (P \ 0.05; Kruskal–Wallis test).

HR assay and cell survival assay after ionizing radiation

exposure

The previous experiments showed an involvement of

MRE11A, OBFC2B, and EEF1E1 in the HR. To investi-

gate if the induction of DNA damage by ionizing radiation

exposure could emphasize the effects previously detected,

we repeated the experiments as before and we irradiated

the cells at the doses of 3 Gy 48 h after the second

transfection.

We examined the HR frequency by FACS analysis and

the number of surviving cells by clonogenic assay after

24 h from the irradiation.

There is a reduction in the survival of 65 % for the

BRCA1-inactivated cells, of 26 % for those inactivated for

EEF1E1, 39 % for OBFC2B, and 37 % for MRE11A in

comparison with cells transfected with the SNCD (Fig. 7).

Fig. 5 Spontaneous HR frequency (%) obtained in cells transfected

with a SNCD and in cells depleted, respectively, for the HPRT,

BRCA1, OBFC2B, EEF1E1, and MRE11A proteins. The histograms

represent each of the five experiments performed analyzing 10.000

cells by FACS

HPRT SNCD BRCA1 OBFC2B EEF1E1 MRE11A

Fig. 6 Graph representing the mean values and the standard devia-

tions of spontaneous HR levels (%) of five experiments obtained by

FACS analysis. In cells in which we performed the knockdown of

BRCA1, OBFC2B, EEF1E1 and MRE11A, the reductions of HR

frequencies are all statistically significant compared to the values

obtained in the control HPRT-depleted cells and in cells transfected

with the SNCD (Kruskal–Wallis test, *P \ 0,05)

Fig. 7 Reduction of clonogenic efficiency respect to control (SNCD)

of cells treated with siRNA and ionizing radiation. The data derive

from two experiments normalized for the values of cell survival

obtained transfecting the SNCD

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123

On the other hand, the effect on the HR of gene inhi-

bition following the irradiation is not amplified as we

expected.

In this experiment the frequency of HR in cells treated

with HPRT and SNCD siRNA was 0.59 % (±0.14) and

0.53 % (±0.14), respectively. In cells depleted for BRCA1,

OBFC2B, MRE11A, and EEF1E1, the frequencies of GFP-

positive cells were 0.01 % (±0.01), 0.69 % (±0.25),

0.55 % (±0.33), and 0.58 % (±0.30), respectively. There

are not statistically significant differences between the HR

frequencies of the tested genes and the controls. Only the

BRCA1-siRNA confirmed the inhibition effect on HR

(Fig. 8).

Discussion

BRCA1 regulates a variety of DNA-damage–repair pathway

[24] and, in particular, the BRCT domains are involved in

the assembly of DNA-damage signaling complexes, so

BRCA1 variants localized at BRCT that alter these molec-

ular mechanisms crucial for genome integrity, could have a

putative role in breast cancer pathogenesis [8–10].

M1775R and the A1789T are two BRCA1 missense

variants both located within the same BRCT domain and

both isolated from familial breast cancers patients: the first

is a known deleterious variant, while the A1789T variant

has been identified for the first time by our group [23].

An in vitro functional assay in HeLa G1 cells over-

expressing these two BRCA1 variants showed that the

A1789T alters the DNA DSBR [23].

To understand the molecular basis of such phenotype, we

analyzed, in a microarray experiment, the expression profiles

of HeLa G1 cells overexpressing the two BRCA1 variants

and HeLa G1-overexpressing BRCA1 wild-type. We found

that both variants altered molecular mechanisms critical in

the control of genome integrity, cell proliferation, apoptosis,

metastatic process, angiogenesis, DNA recombination, sug-

gesting a causative role in breast cancer onset and devel-

opment of these two variants with similar mechanisms [12].

In this study, we hypothesized that some genes, identi-

fied in the above reported experiment, in particular the ones

involved in DNA repair mechanism, could be implicated in

BRCA1 pathway. Considering that mutations in the BRCT

domains are associated to phenotype with an altered ability

in the DNA DSBR, we decided to investigate the

involvement of three of these genes in the HR process:

MRE11A, EEF1E1, and OBFC2B.

MRE11A is a component of the MRN complex (together

with RAD50 and NBS1) which plays a central role in

double-strand break (DSB) repair, DNA recombination,

maintenance of telomere integrity and meiosis [13, 14].

The role of this complex in the HR and NHEJ mechanisms

is known, so, we used MRE11A as a sort of positive control

for our experiments and as validation of the system used.

EEF1E1 (p18) encodes a multifunctional protein that

localizes to both the cytoplasm and nucleus. In the cyto-

plasm, the protein is an auxiliary component of the mac-

romolecular aminoacyl-tRNA synthase complex [19].

Elevated expression of EEF1E1 results in elevated p53

levels and also in p53-target levels as p21. The protein

coimmunoprecipitates with ATM/ATR after induction of

DNA damage. The activity of ATM is dependent on the

level of EEF1E: the ectopic expression of EEF1E1

increases the phosphorylation of ATM suggesting the

requirement of EEF1E1 for the activation of ATM [20].

OBFC2B (hSSB1) is a single-stranded DNA (ssDNA)-

binding proteins essential for a variety of DNA metabolic

processes, including replication, recombination, and

detection and repair of DNA damage [15].

It is a component of the SOSS (sensor of single-stranded

DNA) complex that functions downstream of the MRN

complex to promote DNA repair and G2/M checkpoint

[16]. Recently, the group of Richard et al. [25] demon-

strated that OBFC2B binds directly to the N terminus of

NBS1 in a DNA-damage independent manner. ATM

kinase phosphorylates OBFC2B in response to DSBs sta-

bilizating the protein that accumulates in the nucleus and

forms distinct foci with other known repair proteins inde-

pendent of cell-cycle phase.

In the SOSS complex, OBFC2B acts as a sensor of

single-stranded DNA. It plays a key role in ATM activa-

tion, but also in RAD51 recruitment to DNA-damage foci

during the response to genotoxic stresses.

BRCA1 SNCD EEF1E1 HPRT MRE11A OBFC2B

Fig. 8 HR frequency (%) evaluated 24 h after the ionizing radiation

exposure. The histogram shows the mean values and the standard

deviations of three experiments (Kruskal–Wallis test, *P \ 0.05).

Between the control cells (cells transfected with the SNCD and the

HPRT-siRNA) and cells depleted, respectively, for EEF1E1,

MRE11A, and OBFC2B, there are not statistically significant

differences. Only BRCA1-siRNA transfection induces a significant

reduction of the recombination levels

520 Breast Cancer Res Treat (2013) 141:515–522

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Depletion of OFC2B abrogates the cellular response to

DSBs, including activation of ATM and phosphorylation of

ATM targets after ionizing radiation. Cells deficient in

OBFC2B exhibit increased radiosensitivity, defective

checkpoint activation, and enhanced genomic instability

coupled with a diminished capacity for DNA repair [17, 18].

To evaluate the involvement of these genes in the HR

mechanisms, we constructed a clone from HeLa cells

containing an integrated recombination substrate

(hprtDRGFP) that allows to evaluate the events of HR,

after induction of damage by the endonuclease I-SceI

expression, analysing the number of GFP-positive cells

(Fig. 1).

In hprtDRGFP HeLa clone, we performed a knockdown

of each of the target genes, by siRNA transfection, and we

evaluated the effects on the recombination frequency.

Both fluorescent microscopy and FACS analysis revealed

a reduction of the level of HR in cells inactivated for each of

the target genes in comparison with the controls.

In particular the knockdown of BRCA1, OBFC2B,

MRE11A, and EEF1E1 reduces the HR rate, respectively,

of 97.6, 28.6, 41.8, and 42.3 % compared to the controls

and these differences are statistically significant (Fig. 6).

Then, we evaluated the effects of down-regulation of

those genes on HR and on cell survival after induction of

DNA damage by ionizing radiation to emphasize the

effects previously observed.

The clonogenic assay showed that the cell survival was

reduced of 65 % for BRCA1, 26 % for EEF1E1, 39 % for

OBFC2B, and 37 % for MRE11A in comparison with the

cells transfected with a SNCD (Fig. 7).

Differently from what we expected, there is not statis-

tically significant variation between the HR frequencies of

the controls and the tested genes. The only gene that

confirmed the inhibition effect on HR, as in previous

experiments, is BRCA1 (Fig. 8).

Considering the high values of standard deviation and

the reduced basal frequencies of HR present in the controls

(HPRT and SNCD) in comparison with the previous

experiments, we thought that, probably, the irradiation

could introduce additional variables to the system making

more difficult, in this experimental condition, the identifi-

cation of small reduction in the HR levels.

Nevertheless BRCA1 down-regulation in irradiated cells

drastically reduces the levels of recombination as before,

and this, leads us to think that the lack of a significant

difference in the HR frequency in irradiated cells down-

regulated for the target genes, compared to controls, may

have a biological explanation. It is possible that these genes

may not have a so decisive role in the HR after induction in

the cell of a great stress such as exposure to ionizing

radiation and that, in these cases, a leading role could be

played by other genes.

We must consider that the cell after induction of damage

by ionizing radiation exposure, can repair DSBs by HR or

NHEJ. In mammals, the NHEJ pathway predominates in

many phases of the cell cycle, particularly in G0 and G1,

while HR is important particularly during S and G2 phases

[26].

The predominant involvement of BRCA1 in the phases

G2-M is concordant with its drastic effect on HR.

MRE11A, OBFC2B, and EEF1E1, instead, act in dif-

ferent phases of the cell cycle and it would be interesting to

see if, after damage induced by ionizing radiation, they

might have an involvement in the NHEJ pathway.

Despite several works confirm the important role played

by OBFC2B in genomic stability, nothing is known about

its expression levels in cancer tissues.

Concerning EEF1E1, instead, the group of Park BJ et al.

[20] reports that the EEF1E1 heterozygous mice with a

lower level of EEF1E1 as compared to the wild-type, fre-

quently develop various tumors as breast and seminal

vesicle adenocarcinomas, sarcoma, hepatocarcinoma,

lymphoma. Moreover lower EEF1E1 expressions, were

found in different human cancers as in leukemia, liver

cancer [20], gastric and colorectal cancer [27],compared to

control tissues, but not in breast cancer.

Our data regarding a possible involvement of these

genes in the pathway of BRCA1, it makes very interesting

to investigate their role in the breast carcinogenesis by

evaluating their protein expression in breast cancer tissues

and their somatic and germ line mutational profile in breast

cancer patients.

Acknowledgments The authors wish to thank Dr. Michele Me-

nicagli for the use of fluorescent microscopy and Dr. Simone Pacini

for the use of FACS. Moreover, the authors are grateful to Dr. Luigi

Tana for his technical help in the cell exposure to ionizing radiation.

Conflict of interest The authors declare that they have no conflict

of interest.

Ethical standards All the the experiments comply with the current

laws of the country in which they were performed (Italy).

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