Production, purification of the recombinant analog of Y-box-binding protein and its interaction with poly(ADP-ribose), RNA, single- and double-stranded DNAs
Aim. Production and purification of the recombinant histidine-tagged Y-box- binding protein and study of its interaction with DNA and poly(ADP-ribose). Methods. Ligation-independent cloning, PCR, Sanger sequencing, protein chromatography, polyacrylamide gel electrophoresis, and electrophoresis mobil...
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| Cite this: | Production, purification of the recombinant analog of Y-box-binding protein and its interaction with poly(ADP-ribose), RNA, single- and double-stranded DNAs / E.E. Alemasova, K.N. Naumenko, P.E. Pestryakov, O.I. Lavrik // Вiopolymers and Cell. — 2017. — Т. 33, № 3. — С. 214-220. — Бібліогр.: 8 назв. — англ. |
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Alemasova, E.E. Naumenko, K.N. Pestryakov, P.E. Lavrik, O.I. 2019-06-13T11:55:43Z 2019-06-13T11:55:43Z 2017 Production, purification of the recombinant analog of Y-box-binding protein and its interaction with poly(ADP-ribose), RNA, single- and double-stranded DNAs / E.E. Alemasova, K.N. Naumenko, P.E. Pestryakov, O.I. Lavrik // Вiopolymers and Cell. — 2017. — Т. 33, № 3. — С. 214-220. — Бібліогр.: 8 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.000954 https://nasplib.isofts.kiev.ua/handle/123456789/152976 577.112.083 Aim. Production and purification of the recombinant histidine-tagged Y-box- binding protein and study of its interaction with DNA and poly(ADP-ribose). Methods. Ligation-independent cloning, PCR, Sanger sequencing, protein chromatography, polyacrylamide gel electrophoresis, and electrophoresis mobility shift assay. Results. cDNA coding for the YB-1 protein has a previously undocumented two single nucleotide polymorphisms. The expression construct for production of the his-tagged YB-1 protein was designed to simplify the purification procedure and an appropriate protocol for protein purification was developed. Using electrophoresis mobility shift assay, we have shown that poly(ADP-ribose) competes with a double- and single-stranded DNA and RNA for binding to purified recombinant his-tagged YB-1. Conclusions. In the present work we developed and optimized the procedure of the recombinant YB-1 protein production and purification from bacterial cells. We found that poly(ADP-ribose) at high concentration is able to recruit YB-1 protein from the YB-1-DNA and YB-1-RNA complexes, suggesting a possible YB-1 involvement in DNA repair. Мета. Отримання рекомбінантного гістидин-міченого Y-бокс-зв’язуючий білрк 1 і дослідження його взаємодії з ДНК, РНК та полі (АДФ-рибозою). Методи. Безлігазне клонування, ПЛР, секвенування по Сенгеру, хроматогра-фія, електрофорез в поліакриламідному гелі та метод затримки в гелі. Результати. кДНК YB-1 містить дві раніше недокументовані поодинокі нуклеотидні заміни. Сконструйовано вектор для експресії гистидин-міченого білка YB-1 і розроблена відповідна методика очищення білка. Методом затримки в гелі показано, що полі (АДФ-рибоза) конкурує з одно- і дволанцюговою ДНК, а також РНК, за зв›язування ре-комбінантного гістидин-міченого білка YB-1. Висновки. У цій роботі ми розробили та оптимізували процедуру отримання рекомбінантного білка YB-1 з бактеріальних клітин. Ми встановили, що полі (АДФ-рибоза) у високій концентрації здатна витісняти білок YB-1 з його комплексів з ДНК і РНК, що вказує на можливість участі YB-1 в репарації ДНК. Цель. Получение рекомбинантного гистидин-меченого Y-бокс-связывающего белка 1 и исследование его взаимо-действий с ДНК, РНК и поли(АДФ-рибозой). Методы. Безлигазное клонирование, ПЦР, секвенирование по Сэнге-ру, хроматография, электрофорез в полиакриламидном геле и метод задержки в геле. Результаты. кДНК YB-1 со-держит две ранее недокументированные одиночные нуклеотидные замены. Сконструирован вектор для экспрес-сии гистидин-меченого белка YB-1 и разработана соответствующая методика очистки белка. Методом задержки в геле показано, что поли(АДФ-рибоза) конкурирует с одно- и двухцепочечными ДНК, а также РНК, за связывание рекомбинантного гистидин-меченого белка YB-1. Выводы. В настоящей работе мы разработали и оптимизировали процедуру получения рекомбинантного белка YB-1 из бактериальных клеток. Мы установили, что поли(АДФ-рибоза) в высокой концентрации способна вытеснять белок YB-1 из его комплексов с ДНК и РНК, что указывает на возможность участия YB-1 в репарации ДНК. This work was supported by RFBR grant 16-54-76010; GDRI program; Russian Ministry of Science and Education under 5-100 Excellence Program; Russian State funded budget project (VI.57.1.2, 0309-2016-0001); and educational fellowship from President of Russian Federation to young scientists and PhD students to AEE. en Інститут молекулярної біології і генетики НАН України Вiopolymers and Cell Molecular and Cell Biotechnologies Production, purification of the recombinant analog of Y-box-binding protein and its interaction with poly(ADP-ribose), RNA, single- and double-stranded DNAs Отримання рекомбінантного аналога Y-бокс-зв’язуючого білка 1 і його взаємодію з полі (АДФ-рибозою), РНК, одно- і дволанцюговою ДНК Получение рекомбинантного аналога Y-бокс-связывающего белка 1 и его взаимодействие с поли(АДФ-рибозой), РНК, одно- и двухцепочечной ДНК Article published earlier |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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| title |
Production, purification of the recombinant analog of Y-box-binding protein and its interaction with poly(ADP-ribose), RNA, single- and double-stranded DNAs |
| spellingShingle |
Production, purification of the recombinant analog of Y-box-binding protein and its interaction with poly(ADP-ribose), RNA, single- and double-stranded DNAs Alemasova, E.E. Naumenko, K.N. Pestryakov, P.E. Lavrik, O.I. Molecular and Cell Biotechnologies |
| title_short |
Production, purification of the recombinant analog of Y-box-binding protein and its interaction with poly(ADP-ribose), RNA, single- and double-stranded DNAs |
| title_full |
Production, purification of the recombinant analog of Y-box-binding protein and its interaction with poly(ADP-ribose), RNA, single- and double-stranded DNAs |
| title_fullStr |
Production, purification of the recombinant analog of Y-box-binding protein and its interaction with poly(ADP-ribose), RNA, single- and double-stranded DNAs |
| title_full_unstemmed |
Production, purification of the recombinant analog of Y-box-binding protein and its interaction with poly(ADP-ribose), RNA, single- and double-stranded DNAs |
| title_sort |
production, purification of the recombinant analog of y-box-binding protein and its interaction with poly(adp-ribose), rna, single- and double-stranded dnas |
| author |
Alemasova, E.E. Naumenko, K.N. Pestryakov, P.E. Lavrik, O.I. |
| author_facet |
Alemasova, E.E. Naumenko, K.N. Pestryakov, P.E. Lavrik, O.I. |
| topic |
Molecular and Cell Biotechnologies |
| topic_facet |
Molecular and Cell Biotechnologies |
| publishDate |
2017 |
| language |
English |
| container_title |
Вiopolymers and Cell |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| format |
Article |
| title_alt |
Отримання рекомбінантного аналога Y-бокс-зв’язуючого білка 1 і його взаємодію з полі (АДФ-рибозою), РНК, одно- і дволанцюговою ДНК Получение рекомбинантного аналога Y-бокс-связывающего белка 1 и его взаимодействие с поли(АДФ-рибозой), РНК, одно- и двухцепочечной ДНК |
| description |
Aim. Production and purification of the recombinant histidine-tagged Y-box- binding protein and study of its interaction with DNA and poly(ADP-ribose). Methods. Ligation-independent cloning, PCR, Sanger sequencing, protein chromatography, polyacrylamide gel electrophoresis, and electrophoresis mobility shift assay. Results. cDNA coding for the YB-1 protein has a previously undocumented two single nucleotide polymorphisms. The expression construct for production of the his-tagged YB-1 protein was designed to simplify the purification procedure and an appropriate protocol for protein purification was developed. Using electrophoresis mobility shift assay, we have shown that poly(ADP-ribose) competes with a double- and single-stranded DNA and RNA for binding to purified recombinant his-tagged YB-1. Conclusions. In the present work we developed and optimized the procedure of the recombinant YB-1 protein production and purification from bacterial cells. We found that poly(ADP-ribose) at high concentration is able to recruit YB-1 protein from the YB-1-DNA and YB-1-RNA complexes, suggesting a possible YB-1 involvement in DNA repair.
Мета. Отримання рекомбінантного гістидин-міченого Y-бокс-зв’язуючий білрк 1 і дослідження його взаємодії з ДНК, РНК та полі (АДФ-рибозою). Методи. Безлігазне клонування, ПЛР, секвенування по Сенгеру, хроматогра-фія, електрофорез в поліакриламідному гелі та метод затримки в гелі. Результати. кДНК YB-1 містить дві раніше недокументовані поодинокі нуклеотидні заміни. Сконструйовано вектор для експресії гистидин-міченого білка YB-1 і розроблена відповідна методика очищення білка. Методом затримки в гелі показано, що полі (АДФ-рибоза) конкурує з одно- і дволанцюговою ДНК, а також РНК, за зв›язування ре-комбінантного гістидин-міченого білка YB-1. Висновки. У цій роботі ми розробили та оптимізували процедуру отримання рекомбінантного білка YB-1 з бактеріальних клітин. Ми встановили, що полі (АДФ-рибоза) у високій концентрації здатна витісняти білок YB-1 з його комплексів з ДНК і РНК, що вказує на можливість участі YB-1 в репарації ДНК.
Цель. Получение рекомбинантного гистидин-меченого Y-бокс-связывающего белка 1 и исследование его взаимо-действий с ДНК, РНК и поли(АДФ-рибозой). Методы. Безлигазное клонирование, ПЦР, секвенирование по Сэнге-ру, хроматография, электрофорез в полиакриламидном геле и метод задержки в геле. Результаты. кДНК YB-1 со-держит две ранее недокументированные одиночные нуклеотидные замены. Сконструирован вектор для экспрес-сии гистидин-меченого белка YB-1 и разработана соответствующая методика очистки белка. Методом задержки в геле показано, что поли(АДФ-рибоза) конкурирует с одно- и двухцепочечными ДНК, а также РНК, за связывание рекомбинантного гистидин-меченого белка YB-1. Выводы. В настоящей работе мы разработали и оптимизировали процедуру получения рекомбинантного белка YB-1 из бактериальных клеток. Мы установили, что поли(АДФ-рибоза) в высокой концентрации способна вытеснять белок YB-1 из его комплексов с ДНК и РНК, что указывает на возможность участия YB-1 в репарации ДНК.
|
| issn |
0233-7657 |
| url |
https://nasplib.isofts.kiev.ua/handle/123456789/152976 |
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Production, purification of the recombinant analog of Y-box-binding protein and its interaction with poly(ADP-ribose), RNA, single- and double-stranded DNAs / E.E. Alemasova, K.N. Naumenko, P.E. Pestryakov, O.I. Lavrik // Вiopolymers and Cell. — 2017. — Т. 33, № 3. — С. 214-220. — Бібліогр.: 8 назв. — англ. |
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214
E. E. Alemasova, K. N. Naumenko, P. E. Pestryakov
© 2017 E. E. Alemasova et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Bio-
polymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License
(http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium,
provided the original work is properly cited
UDC 577.112.083
Production, purification of the recombinant analog of Y-box-binding protein 1
and its interaction with poly(ADP-ribose), RNA, single- and double-stranded
DNAs
E. E. Alemasova1, K. N. Naumenko1,2, P. E. Pestryakov1, O. I. Lavrik1,2
1 Novosibirsk Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences
8, Akademika Lavrentieva Ave., Novosibirsk, Russian Federation, 630090
2 Novosibirsk State University
2, Pirogova Str., Novosibirsk, Russian Federation, 630090
lisenok.istreb@gmail.com
Aim. Production and purification of the recombinant histidine-tagged Y-box- binding protein 1
and study of its interaction with DNA, RNA and poly(ADP-ribose). Methods. Ligation-
independent cloning, PCR, Sanger sequencing, protein chromatography, polyacrylamide gel
electrophoresis, and electrophoresis mobility shift assay. Results. cDNA coding for the YB-1
protein has a previously undocumented two single nucleotide polymorphisms. The expression
construct for production of the his-tagged YB-1 protein was designed to simplify the purifica-
tion procedure and an appropriate protocol for protein purification was developed. Using
electrophoresis mobility shift assay, we have shown that poly(ADP-ribose) competes with a
double- and single-stranded DNA and RNA for binding to purified recombinant his-tagged
YB-1. Conclusions. In the present work we developed and optimized the procedure of the
recombinant YB-1 protein production and purification from bacterial cells. We found that
poly(ADP-ribose) at high concentration is able to recruit YB-1 protein from the YB-1-DNA
and YB-1-RNA complexes, suggesting a possible YB-1 involvement in DNA repair.
K e y w o r d s: YB-1, protein purification, poly(ADP-ribose) (PAR), DNA repair.
Introduction
Y-box-binding protein 1 (YB-1) is a multifunc-
tional cellular factor increasingly considered
as a potential universal regulator of different
DNA repair systems [1]. Recent findings of
our laboratory demonstrated YB-1 interplay
with PARP1, the key regulatory protein of base
excision repair pathway [2]. PARP1 binding
to damaged DNA results in its activation fol-
lowed by synthesis of nucleic acid-like poly-
mer called poly(ADP-ribose) (PAR) using
NAD+ as a precursor. The functions of PAR
in the regulation of DNA repair are amazingly
numerous and include chromatin remodeling,
recruitment of downstream repair enzymes and
Molecular and Cell
Biotechnologies
ISSN 1993-6842 (on-line); ISSN 0233-7657 (print)
Biopolymers and Cell. 2017. Vol. 33. N 3. P 214–220
doi: http://dx.doi.org/10.7124/bc.000954
215
Y-box-binding protein and its interaction with poly(ADP-ribose), RNA, single- and double-stranded DNA
modulation of interactions within the DNA
repair complex [2]. PAR was also shown to
effectively outcompete binding of histones to
DNA [3] and proposed to assemble the non-
canonical DNA repair proteins (usually RNA-
binding [4]) at the damage site in a similar
way [5]. However, it is not entirely clear if
poly(ADP-ribose) may act as a preferable li-
gand for YB-1 in the presence of DNA and
RNA as the YB-1 targets.
For further examination of YB-1 role in
DNA repair it is necessary to provide sufficient
amounts of the recombinant protein required
for in vitro studies. The existing method of
YB-1 purification is time-consuming since it
includes three chromatographic and two di-
alysis stages [6]. Using histidine-tagged YB-1
is more advantageous since purification of a
tagged protein can be performed during single-
step affinity chromatography. Moreover, ob-
tained his-tagged YB-1 protein can be also
used in pull-down assay for searching YB-1
protein partners in cell. Here we describe a
producing strain for his-tagYB-1 expression
in bacteria. Optimized purification procedure
allowed us to obtain preparative quantities of
the target protein. We also optimized the pro-
tocol for preparation of poly(ADP-ribose) free
from DNA cofactor used for the PARP1 activa-
tion and perform electrophoresis mobility shift
assay (EMSA) to show that YB-1 binding to
DNA, RNA or PAR may be regulated by PAR/
DNA or PAR/RNA ratio.
Methods
Construction of pLATE-51-his-tagYB-1
expression vector
The plasmid pET-3-1-YB-1 containing cDNA
fragment of human YB-1 was a generous gift
from Lev P. Ovchinnikov and Dmitry Kretov
(Institute of Protein Research RAS, Moscow,
Russian Federation). The plasmid was ampli-
fied in E.coli XL1Blue and isolated according
to standard protocol. The presence of YB-1
cDNA insert was confirmed by PCR (with
pET-U/pET-R primers, Table 1). The presence
of mutations in YB-1 cDNA was analyzed by
Sanger sequencing.
cDNA encoding YB-1 protein was cloned in
pLATE-51 expression vector using aLICator
LIC Cloning & Expression System (Thermo
Scientific, USA). YB-1 cDNA was amplified
by PCR with specific primers plate51-forward
Table 1. Oligonucleotide sequences and designations
pET-U 5’-AGCCAACTCAGCTTCCTTTC-3’
pET-R 5’-ATAGGGAGACCACAACGGTT-3’
Plate-51
Forward 5’-GGAGATGGGAAGTCATTACTCAGCCCCGCCCTG-3’
Plate-51
Reverse 5’-GGTGATGATGATGACAAGAGCAGCGAGGCCGA-3’
ssDNA 5’-CGGTATCCACCAGGTCUGAGACAACGATGAAGCCCAAGCCAGATGAAATGTAGTC-3’
dsDNA 5’-CGGTATCCACCAGGTCUGAGACAACGATGAAGCCCAAGCCAGATGAAATGTAGTC-3’
3’-GCCATAGGTGGTCCAGACTCTGTTGCTACTTCGGGTTCGGTCTACTTTACATCAG-5’
RNA 5’-gggaga aaaaag aaagaa auguuc uucuuc uaagaa gaaaga aaagaa aaagaa aaaaga caaaga cacgaa ggaaga-3’
216
E. E. Alemasova, K. N. Naumenko, P. E. Pestryakov et al.
and plate51-reverse in GC-buffer (Biolabmix,
Russian Federation) using pET3-I-YB-1 as a
template, then purified by isopropanol preci pi-
tation and annealed with linearized pLATE-51
vector. E. coli DH5alpha competent cells were
transformed by electroporation with resulting
plasmid. The presence of pLATE51-YB-1 in
transformant colonies, grown on medium con-
taining ampicillin as selective antibiotic, was
analyzed by colony PCR with specific primers
plate51-forward and plate51-reverse.
Purification of the recombinant his-
tagYB-1 protein
Expression of open reading frame encoding
the his-tagYB-1 protein in E.coli BL21(DE3)
was performed in the auto-induction system
described by Studier [7]. The biomass (~16 g)
was lysed by lysozyme treatment, sonicated
and centrifuged to pellet cell debris. As the
initial purification step we used metal affinity
chromatography on a Ni-NTA resin. The co-
lumn was equilibrated by Ni-A buffer (see
Table 2 for details), then the supernatant was
adjusted and washed with 10 column volumes
(CV) of Ni-A buffer. Protein elution from the
column was performed by step gradient of
imidazole from 20 mM (0 % Ni-B buffer) to
250 mM (50 % Ni-B). For additional purifica-
tion we used ion exchange chromatography.
After Mono S column (GE Healthcare, UK)
equilibration by 25 % Ion-B buffer, the sample
was diluted in Ion-A (to 0.5 M NaCl), ad-
justed, washed by 25 % Ion-B and eluted by
linear gradient of KCl from 0.5 M (25 %
Ion-B) to 2 M (100 % Ion-B). Gel filtration
was employed as a final ‘polishing’ step. In
this regard, the sample was concentrated by
repeated chromatography on Mono S column
using step gradient of KCl from 0.5 M (25 %
Ion-B) to 1.5 M (75 % Ion-B), adjusted on
16/600 Superdex 75 per grade column (GE
Healthcare) equilibrated by GF-buffer and
isocratically eluted. The resulting sample was
concentrated by Vivaspin Turbo 15 (Sartorius,
Germany) in Storage buffer (Table 2).
PAR labeling in vitro
Radioactively labelled (or unlabeled) PAR
polymer was obtained as described previ-
Table 2. Purification details
Purification step Column Buffers
1. Metal affinity
chromatography
Econo-Column (BioRad),
L × I.D. 5 cm × 10 mm + GE
HealthCare Ni-NTA resin
Ni-A: 20 mM Tris-HCl pH 8.0, 1.5 M NaCl, 20 mM imidazole,
10 % glycerol, 0,1 % NP-40
Ni-B: 20 mM Tris-HCl pH 8.0, 1.5 M NaCl, 500 mM imidazole,
10 % glycerol, 0,1 % NP-40
2. Ion exchange
chromatography
MonoS 5/50 GL (GE
HealthCare), L × I.D. 5 cm ×
5 mm
Ion-A: 20 mM Hepes-KOH pH 8.0, 10 % glycerol, 0,1 % NP-40
Ion-B: 20 mM Hepes-KOH pH 8.0, 2 M KCl, 10 % glycerol,
0,1 % NP-40
3. Gel filtration
16/600 Superdex 75 pg (GE
HealthCare), L × I.D. 60 cm ×
16 mm
GF-buffer: 20 mM Hepes-KOH pH 8.0, 1 M KCl
4. His-tagYB-1
concentration
Vivaspin Turbo 15 (Sartorius)
10 kDa MWCO Storage buffer: 20 mM KH2PO4, 0.5 M KCl, 20% glycerol
217
Y-box-binding protein and its interaction with poly(ADP-ribose), RNA, single- and double-stranded DNA
ously [9] with minor modifications. Briefly,
DNA cofactor was removed by benzonase
treatment and PAR was isolated from result-
ing sample by phenol:chloroform:isoamyl
alcohol (25:24:1) extraction. PAR was addi-
tionally purified by ethanol precipitation and
dissolved in 1x reaction buffer (RB) (50 mM
Tris-HCl pH 8.0, 40 mM NaCl, 8 mM MgCl2,
1 mM DTT) to the final concentration 1 A260
units/ml.
His-tagYB-1 interaction with PAR, DNA
and RNA
To study PAR ability to compete with DNA
for YB-1 binding, we used unlabeled PAR and
radioactively labeled DNA. Reaction mixtures
contained 1x RB, 400 nM his-tagYB-1, 50 nM
ssDNA or dsDNA, and 0-8 μl of PAR. To study
DNA and RNA ability to compete with PAR
for YB-1 binding, we used radioactively la-
beled PAR and unlabeled DNA (or RNA).
ssRNA used in this study was kindly provided
by Dmitriy Sharifulin (ICBFM SB RAS,
Novosibirsk, Russia). Reaction mixtures con-
tained 1x RB, 400 nM his-tag YB-1, 1 μl of
PAR and 0-10 μM DNA or RNA. His-tagYB-1
binding to DNA and PAR was performed for
5 min at 37 ○C. Samples were supplemented
by loading buffer (20 mM Tris-HCl pH 8.0,
10 % glycerol, 0.025 % bromphenol blue) and
analyzed by electrophoresis mobility shift as-
say at 10 V/cm (PAAG contained 7 % acry la-
mide, 0.09 % bis-acrylamide, 0.5x TBE buf-
fer). Positions of YB-1-DNA or YB-1-PAR
complexes were visualized by phosphori ma-
ging with Typhoon FLA 7000 (GE Healthcare).
Results and Discussion
The YB-1 cDNA primary structure derived
from Sanger sequencing was compared with
the reference sequence of YBX1 gene coding
for the YB-1 protein (NM_001082785.1). The
data obtained testify that the YB-1 open rea-
ding frame contains two mutations: Pro-312
→ Glu-312 (codon change CCG → CAG) and
Ser-313 → Arg-313 (codon change AGT →
CGT) (Fig. 1). We suppose that these amino
acid changes may represent previously un-
documented YB-1 cDNA polymorphisms.
To obtain a target expression construct that
contains YB-1 cDNA, N-terminally flanked by
hexahistidine coding sequence, we used ligase
independent cloning system (Supplementary,
Fig. S1). The conditions of cell culture trans-
formed by pLATE-51-YB-1 were optimized
to achieve maximal yield of the target protein
Fig. 1. Comparison of YB-1 cDNA
sequence with reference sequence of
YBX1 gene (R) and a fragment of
chromatogram obtained after Sanger
sequencing. Mutated codons are
boxed, nucleotide changes are rubri-
cated.
218
E. E. Alemasova, K. N. Naumenko, P. E. Pestryakov et al.
in soluble form. Since a single-step metal af-
finity chromatography did not result in pure
YB-1 preparation, two additional purification
steps were performed (Fig. 2). Using this ap-
proach we obtained about 3 mg of ~90 %
purity his-tagYB-1 protein per 1 l of bacterial
culture. Thus, the yield of the target protein
obtained by our purification protocol is three
times higher compared to previously reported
approach [6].
Previously, YB-1 was shown to bind
poly(ADP-ribose) even in the presence of da-
ma ged DNA serving as a cofactor for the
PARP1 activation [8]. However, the ability of
PAR to compete with DNA for binding to
YB-1 was not demonstrated yet. In the present
study we employed an electrophoresis mobi-
li ty shift assay to estimate the relative affinity
of poly(ADP-ribose), DNA and RNA for his-
tagYB-1 protein. First of all, we modified the
protocol for PAR preparation [8] in order to
eliminate DNA cofactor from the sample.
Purified PAR was able to compete with single-
and double-stranded DNA for binding to his-
tagYB-1 (Fig. 3A). Reciprocally, ssDNA,
dsDNA and RNA were shown to disrupt YB-1
complexes with PAR (Fig. 3B). Since PAR
and nucleic acids can compete for YB-1, we
propose that in the cellular context the YB-1
functions may be regulated by PAR/DNA and
PAR/RNA ratio. In this regard, an increase of
PAR level induced by genotoxic stress may
dynamically outcompete DNA- and RNA-
binding of YB-1 and recruit this protein to
Supplementary, Figure S1. Con-
struction of his-tagged YB-1 expres-
sion vector (scheme).
Fig. 2. Purification of his-
tagged YB-1(Coomassie
Brilliant Blue R-250 stai-
ned gel). Lane 1 – his-tag-
ged YB-1protein after pu-
rification on Ni-NTA col-
umn; lane 2 – after Mono
S column; lane 3 – after
gel-filtration (final protein
sample).
219
Y-box-binding protein and its interaction with poly(ADP-ribose), RNA, single- and double-stranded DNA
DNA damage sites. Such mechanism might
be similar to that proposed for RNA-binding
proteins [5].
Conclusions
In the present work we developed a simple and
efficient technique for the production and pu-
rification of histidine-tagged recombinant
analog of YB-1 protein from bacterial cells.
We found that poly(ADP-ribose) is able to
compete with DNA and RNA for binding to
YB-1 protein. We propose that high PAR con-
centration at the sites of genomic lesions may
induce transient YB-1 relocalization from its
complexes with nucleic acids to DNA damage
sites. PAR-mediated recruitment of YB-1 to
DNA repair foci provides a basis for YB-1
involvement in the DNA repair process.
Acknowledgements
This work was supported by RFBR grant 16-
54-76010; GDRI program; Russian Ministry
of Science and Education under 5-100
Excellence Program; Russian State funded
budget project (VI.57.1.2, 0309-2016-0001);
and educational fellowship from President of
Russian Federation to young scientists and
PhD students to AEE.
REFERENCES
1. Lyabin DN, Eliseeva IA, Ovchinnikov LP. YB-1
protein: functions and regulation. Wiley Interdiscip
Rev RNA. 2014; 5(1): 95–110.
2. D’Amours D, Desnoyers S, D’Silva I, Poirier GG.
Poly(ADP-ribosyl)ation reactions in the regulation
of nuclear functions. Biochem J. 1999; 342(Pt 2):
249–68.
3. Muthurajan UM, Hepler MR, Hieb AR, Clark NJ,
Kramer M, Yao T, Luger K. Automodification
switches PARP-1 function from chromatin architec-
tural protein to histone chaperone. Proc Natl Acad
Sci U S A. 2014; 111(35): 12752–57.
4. Rulten SL, Rotheray A, Green RL, Grundy GJ,
Moore DA, Gómez-Herreros F, Hafezparast M,
Caldecott KW. PARP-1 dependent recruitment of
the amyotrophic lateral sclerosis-associated protein
FUS/TLS to sites of oxidative DNA damage. Nu-
cleic Acids Res. 2014; 42(1): 307–14.
5. Teloni F, Altmeyer M. Readers of poly(ADP-ribose):
designed to be fit for purpose. Nucleic Acids Res.
2016; 44(3): 993–1006.
6. Kretov DA, Curmi PA, Hamon L, Abrakhi S, Des-
forges B, Ovchinnikov LP, Pastré D. mRNA and DNA
selection via protein multimerization: YB-1 as a case
study. Nucleic Acids Res. 2015; 43(19): 9457–73.
7. Studier FW. Stable expression clones and auto-in-
duction for protein production in E. coli. Methods
Mol Biol. 2014; 1091: 17–32.
A
Fig. 3A. EMSA of YB-1 binding to radioactively labelled
50 nM ssDNA (lanes 2–7) and 50 nM dsDNA (lanes 9–14)
in the presence of varying amounts of poly(ADP-ribose)
(0–0.008 A260 units/ml). C1 – control for ssDNA, C2 –
control for dsDNA.
B
Fig. 3B. EMSA of YB-1 binding to radioactively labelled
PAR (0.001 A260 units/ml) in the presence of ssDNA, ds-
DNA and RNA (0-10 μM).
220
E. E. Alemasova, K. N. Naumenko, P. E. Pestryakov et al.
8. Alemasova EE, Moor NA, Naumenko KN, Kutu-
zov MM, Sukhanova MV, Pestryakov PE, Lavrik OI.
Y-box-binding protein 1 as a non-canonical factor
of base excision repair. Biochim Biophys Acta. 2016;
1864(12): 1631–40.
Отримання рекомбінантного аналога Y-бокс-
зв’язуючого білка 1 і його взаємодію з полі (АДФ-
рибозою), РНК, одно- і дволанцюговою ДНК
Е. Е. Алемасова, К. Н. Науменко,
П. Е. Пестряков, О. І. Лаврик
Мета. Отримання рекомбінантного гістидин-міченого
Y-бокс-зв’язуючий білрк 1 і дослідження його взаємо-
дії з ДНК, РНК та полі (АДФ-рибозою). Методи.
Безлігазне клонування, ПЛР, секвенування по Сенгеру,
хроматографія, електрофорез в поліакриламідному гелі
та метод затримки в гелі. Результати. кДНК YB-1
містить дві раніше недокументовані поодинокі нукле-
отидні заміни. Сконструйовано вектор для експресії
гистидин-міченого білка YB-1 і розроблена відповідна
методика очищення білка. Методом затримки в гелі
показано, що полі (АДФ-рибоза) конкурує з одно- і
дволанцюговою ДНК, а також РНК, за зв›язування
ре комбінантного гістидин-міченого білка YB-1.
Висновки. У цій роботі ми розробили та оптимізува-
ли процедуру отримання рекомбінантного білка YB-1
з бактеріальних клітин. Ми встановили, що полі (АДФ-
рибоза) у високій концентрації здатна витісняти білок
YB-1 з його комплексів з ДНК і РНК, що вказує на
можливість участі YB-1 в репарації ДНК.
К л юч ов і с л ов а: YB-1, отримання рекомбінант-
ного білка, поли (АДФ-рибоза) (PAR), репарація ДНК
Получение рекомбинантного аналога Y-бокс-
связывающего белка 1 и его взаимодействие
с поли(АДФ-рибозой), РНК, одно-
и двухцепочечной ДНК
Е. Э. Алемасова, К. Н. Науменко,
П. Е. Пестряков, О. И. Лаврик
Цель. Получение рекомбинантного гистидин-мечено-
го Y-бокс-связывающего белка 1 и исследование его
взаимодействий с ДНК, РНК и поли(АДФ-рибозой).
Методы. Безлигазное клонирование, ПЦР, секвениро-
вание по Сэнгеру, хроматография, электрофорез в
полиакриламидном геле и метод задержки в геле.
Результаты. кДНК YB-1 содержит две ранее недоку-
ментированные одиночные нуклеотидные замены.
Сконструирован вектор для экспрессии гистидин-ме-
ченого белка YB-1 и разработана соответствующая
методика очистки белка. Методом задержки в геле
показано, что поли(АДФ-рибоза) конкурирует с одно-
и двухцепочечными ДНК, а также РНК, за связывание
рекомбинантного гистидин-меченого белка YB-1.
Выводы. В настоящей работе мы разработали и оп-
тимизировали процедуру получения рекомбинантного
белка YB-1 из бактериальных клеток. Мы установили,
что поли(АДФ-рибоза) в высокой концентрации спо-
собна вытеснять белок YB-1 из его комплексов с ДНК
и РНК, что указывает на возможность участия YB-1 в
репарации ДНК.
К л юч е в ы е с л ов а: YB-1, получение рекомбинант-
ного белка, поли(АДФ-рибоза) (PAR), репарация ДНК
Received 14.03.2017
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