Optimization of nucleosome assembly from histones and model DNAs and estimation of the reconstitution efficiency
Nucleosome core particles (NCPs) are basic units of chromatin organization; they represent the most convenient model system for the study of key DNA-dependent processes. Therefore, a robust method of nucleosome assembly is important for research. To prepare NCPs, purified histones and DNAs with sequ...
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Інститут молекулярної біології і генетики НАН України
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| Цитувати: | Optimization of nucleosome assembly from histones and model DNAs and estimation of the reconstitution efficiency / M.M. Kutuzov, T.A. Kurgina, E.A. Belousova, S.N. Khodyreva, O.I. Lavrik // Вiopolymers and Cell. — 2019. — Т. 35, № 2. — С. 91-98. — Бібліогр.: 12 назв. — англ. |
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irk-123456789-1543982019-07-07T13:03:15Z Optimization of nucleosome assembly from histones and model DNAs and estimation of the reconstitution efficiency Kutuzov, M.M. Kurgina, T.A. Belousova, E.A. Khodyreva, S.N. Lavrik, O.I. Structure and Function of Biopolymers Nucleosome core particles (NCPs) are basic units of chromatin organization; they represent the most convenient model system for the study of key DNA-dependent processes. Therefore, a robust method of nucleosome assembly is important for research. To prepare NCPs, purified histones and DNAs with sequences providing strong positioning of DNA relative to histone octamer are commonly used, and a method to control the efficacy of NCP reconstruction is required. Aim. To optimize the procedure for NCP reconstitution from purified histone octamers and different types of synthetic model DNAs and develop a new approach for express analysis of the efficacy of NCP reconstitution. Methods. Dialysis, PAAG, fluorescence measurement using the Eva-Green dye. Results. We first developed a convenient procedure for NCP assembly in a low-salt buffer with a variable DNA-histone ratio at the first stage. Once the optimal ratio was determined, NCPs could be assembled by a slow gradient dialysis. The efficacy of NCP assembly can be estimated directly in the solution using the Eva-Green dye. Conclusions. The efficacy of dye intercalation into DNA duplex was sharply reduced in the nucleosomal context. The main benefits of the proposed approach are the rapid analysis directly in the solution and possibility to use DNAs without any special tags. Нуклеосомна корова частка, НКЧ, являє собою зручну модельну систему для вивчення ключових ДНК-залежних процесів, оскільки є елементарною одиницею структури хроматину. Саме тому основним завданням представленого дослідження було створення універсального методу збірки нуклеосом. Для реконструкції НКЧ використовуються очищені гістони і ДНК-структури з певними послідовностями, що забезпечують чітке позиціонування ДНК відносно октамера гістонів. Це вимагає наявності способу контролю ефективності реконструкції НКЧ. Мета. Оптимізувати процедуру реконструкції НКЧ з очищених октамер гістонів і різних типів синтезованих модельних ДНК і запропонувати підхід для експрес-аналізу ефективності цього процесу. Методи. Діаліз, поділ в ПААГ, вимір флуоресценції з використанням барвника Eva-Green. Результати. Ми розробили зручну процедуру складання НКЧ в слабо сольовому буфері із змінним співвідношенням ДНК-гістони на першому етапі. Після визначення оптимального співвідношення реконструкція НКЧ може бути проведена за допомогою повільного градиентного діалізу. На цьому етапі ефективність процесу може бути оцінена безпосередньо в розчині з використанням барвника Eva-Green. Висновки. Було показано, що ефективність интеркаляции барвника в дуплекс ДНК в контексті нуклеосоми різко знижується. До основних переваг запропонованого підходу можна віднести швидкий аналіз суміші безпосередньо в розчині і можливість використання ДНК, що не містять будь-яких спеціальних міток. Нуклеосомная коровая частица, НКЧ, представляет собой удобную модельную систему для изучения ключевых ДНК-зависимых процессов, поскольку является элементарной единицей структуры хроматина. Именно поэтому основной задачей представленного исследования было создание универсального метода сборки нуклеосом. Для реконструкции НКЧ используются очищенные гистоны и ДНК-структуры с определенными последовательностями, обеспечивающими четкое позиционирование ДНК относительно октамера гистонов. Это требует наличия способа контроля эффективности реконструкции НКЧ. Цель. Оптимизировать процедуру реконструкции НКЧ из очищенных октамеров гистонов и различных типов синтезированных модельных ДНК и предложить подход для экспресс-анализа эффективности этого процесса. Методы. Диализ, разделение в ПААГ, измерение флуоресценции с использованием красителя Eva-Green. Результаты. Мы разработали удобную процедуру сборки НКЧ в слабо солевом буфере с изменяемым соотношением ДНК-гистоны на первом этапе. После определения оптимального соотношения реконструкция НКЧ может быть проведена с помощью медленного градиентного диализа. На этом этапе эффективность процесса может быть оценена непосредственно в растворе с использованием красителя Eva-Green. Выводы. Было показано, что эффективность интеркаляции красителя в дуплекс ДНК в контексте нуклеосомы резко снижается. К основным преимуществам предложенного подхода можно отнести быстрый анализ смеси непосредственно в растворе и возможность использования ДНК, не содержащих каких-либо специальных меток. 2019 Article Optimization of nucleosome assembly from histones and model DNAs and estimation of the reconstitution efficiency / M.M. Kutuzov, T.A. Kurgina, E.A. Belousova, S.N. Khodyreva, O.I. Lavrik // Вiopolymers and Cell. — 2019. — Т. 35, № 2. — С. 91-98. — Бібліогр.: 12 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.00099A http://dspace.nbuv.gov.ua/handle/123456789/154398 577.29 + 57.015.5 en Вiopolymers and Cell Інститут молекулярної біології і генетики НАН України |
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Digital Library of Periodicals of National Academy of Sciences of Ukraine |
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English |
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Structure and Function of Biopolymers Structure and Function of Biopolymers |
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Structure and Function of Biopolymers Structure and Function of Biopolymers Kutuzov, M.M. Kurgina, T.A. Belousova, E.A. Khodyreva, S.N. Lavrik, O.I. Optimization of nucleosome assembly from histones and model DNAs and estimation of the reconstitution efficiency Вiopolymers and Cell |
| description |
Nucleosome core particles (NCPs) are basic units of chromatin organization; they represent the most convenient model system for the study of key DNA-dependent processes. Therefore, a robust method of nucleosome assembly is important for research. To prepare NCPs, purified histones and DNAs with sequences providing strong positioning of DNA relative to histone octamer are commonly used, and a method to control the efficacy of NCP reconstruction is required. Aim. To optimize the procedure for NCP reconstitution from purified histone octamers and different types of synthetic model DNAs and develop a new approach for express analysis of the efficacy of NCP reconstitution. Methods. Dialysis, PAAG, fluorescence measurement using the Eva-Green dye. Results. We first developed a convenient procedure for NCP assembly in a low-salt buffer with a variable DNA-histone ratio at the first stage. Once the optimal ratio was determined, NCPs could be assembled by a slow gradient dialysis. The efficacy of NCP assembly can be estimated directly in the solution using the Eva-Green dye. Conclusions. The efficacy of dye intercalation into DNA duplex was sharply reduced in the nucleosomal context. The main benefits of the proposed approach are the rapid analysis directly in the solution and possibility to use DNAs without any special tags. |
| format |
Article |
| author |
Kutuzov, M.M. Kurgina, T.A. Belousova, E.A. Khodyreva, S.N. Lavrik, O.I. |
| author_facet |
Kutuzov, M.M. Kurgina, T.A. Belousova, E.A. Khodyreva, S.N. Lavrik, O.I. |
| author_sort |
Kutuzov, M.M. |
| title |
Optimization of nucleosome assembly from histones and model DNAs and estimation of the reconstitution efficiency |
| title_short |
Optimization of nucleosome assembly from histones and model DNAs and estimation of the reconstitution efficiency |
| title_full |
Optimization of nucleosome assembly from histones and model DNAs and estimation of the reconstitution efficiency |
| title_fullStr |
Optimization of nucleosome assembly from histones and model DNAs and estimation of the reconstitution efficiency |
| title_full_unstemmed |
Optimization of nucleosome assembly from histones and model DNAs and estimation of the reconstitution efficiency |
| title_sort |
optimization of nucleosome assembly from histones and model dnas and estimation of the reconstitution efficiency |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| publishDate |
2019 |
| topic_facet |
Structure and Function of Biopolymers |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/154398 |
| citation_txt |
Optimization of nucleosome assembly from histones and model DNAs and estimation of the reconstitution efficiency / M.M. Kutuzov, T.A. Kurgina, E.A. Belousova, S.N. Khodyreva, O.I. Lavrik // Вiopolymers and Cell. — 2019. — Т. 35, № 2. — С. 91-98. — Бібліогр.: 12 назв. — англ. |
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Вiopolymers and Cell |
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91
M. M. Kutuzov, T. A. Kurgina, E. A. Belousova
© 2019 M. M. Kutuzov 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 577.29 + 57.015.5
Optimization of nucleosome assembling from histones and model
DNAs and estimation of the reconstitution efficiency
M. M. Kutuzov1,2, T. A. Kurgina1,2, E. A. Belousova1,2, S. N. Khodyreva1, 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
lavrik@niboch.nsc.ru
Nucleosome core particles (NCPs) are basic units of chromatin organization; they represent
the most convenient model system for the study of key DNA-dependent processes. Therefore,
a robust method of nucleosome assembly is important for research. To prepare NCPs, purified
histones and DNAs with sequences providing strong positioning of DNA relative to histone
octamer are commonly used, and a method to control the efficacy of NCP reconstruction is
required. Aim. To optimize the procedure for NCP reconstitution from purified histone octam-
ers and different types of synthetic model DNAs and develop a new approach for express
analysis of the efficacy of NCP reconstitution. Methods. Dialysis, PAAG, fluorescence meas-
urement using the Eva-Green dye. Results. We first developed a convenient procedure for
NCP assembly in a low-salt buffer with a variable DNA-histone ratio at the first stage. Once
the optimal ratio was determined, NCPs could be assembled by a slow gradient dialysis. The
efficacy of NCP assembly can be estimated directly in the solution using the Eva-Green dye.
Conclusions. The efficacy of dye intercalation into DNA duplex was sharply reduced in the
nucleosomal context. The main benefits of the proposed approach are the rapid analysis di-
rectly in the solution and possibility to use DNAs without any special tags.
K e y w o r d s: nucleosome core particle, assembly, chromatin, core histones
Introduction
The eukaryotic genome exists in a form of
chromatin. This supramolecular nucleoprotein
complex provides a compaction of billions of
nucleotide units of DNA within a nucleus. The
primary unit of DNA compaction is nucleo-
some. Thus, to perform any DNA-dependent
process such as replication, repair or transcrip-
tion it is necessary to affect the chromatin
context in whole and to nucleosomes in par-
ticular (1–3). There are multiple studies testi-
ISSN 1993-6842 (on-line); ISSN 0233-7657 (print)
Biopolymers and Cell. 2019. Vol. 35. N 2. P 91–98
doi: http://dx.doi.org/10.7124/bc.00099A
mailto:lavrik@niboch.nsc.ru
92
M. M. Kutuzov, T. A. Kurgina, E. A. Belousova et al.
fying the influence of nucleosomal organiza-
tion on the efficiency of DNA-dependent pro-
cesses and demonstrating the importance of
nucleosomal DNA context to both the nucleo-
some organization and the process efficacy
(1–3). The performance of such investigations
implicates the usage of reconstituted nucleo-
somes with the defined DNA sequence. This
entails the necessity to control the efficiency
of reconstitution of nucleosome core particles,
NCP. Here, we present a method to test the
efficacy of NCP reconstitution and to produce
NCPs free from naked DNA. Histone octamers
used for the NCP formation were purified from
chicken erythrocytes, one of the most popular
sources of natural histones due to their avail-
ability. There is a number of protocols descri-
bing the purification of the core histone octa-
mers (4–7). We analyzed them and proposed
a combined version with several original mod-
ifications. Here, we presented an overview of
this protocol.
Materials and Methods
Erythrocytes purification. All procedures need
to be performed at 4°C unless otherwise spec-
ified. Fresh chicken blood was collected to the
anticoagulation buffer at an accurate proportion
of 50 ml of blood per 200 ml of the buffer, and
then centrifuged for 10 min at 1500 g. Here
and elsewhere it is necessary to avoid the con-
centration higher than 50 ml of blood/erythro-
cytes/pellet per 200 ml of the buffer. The buffer
contained 30 mM sodium citrate, pH 7, 0.1 M
NaCl, 0.1 mM PMSF. Sodium citrate can be
replaced by 2500 IU of heparin. Collected
blood can be stored for a few hours at 4°C.
After careful removing of supernatant, the
pellet was resuspended in the citrate buffer
followed by centrifugation at 1500 g for
10 min. The washing was repeated twice. The
collected pellet was resuspended in sucrose
buffer (60 mM KCl, 15 mM NaCl, 2 mM
spermidine, 340 mM sucrose, 0.1 mM PMSF,
15 mM Tris-HCl pH 7.5) with 2.5 mM EDTA,
0.5 mM EGTA, and centrifuged at 1500 g for
10 min. The washing was repeated twice.
Nuclei purification. The erythrocyte pellet
was resuspended in the sucrose buffer with
2.5 mM EDTA, 0.5 mM EGTA, and 0.5 %
NP-40 and centrifuged at 1500 g for 10 min.
The washing was repeated until the pellet be-
comes white. After that, the pellet was resus-
pended in the sucrose buffer and cent ri fuged
at 1500 g for 10 min. The washing was re-
peated twice. The purified nuclei can be kept
at -70°C in the storage buffer (50 % glycerol,
0.15 M NaCl, 2 mM spermidine, 0.1 mM
EDTA, 0.1mM PMSF, 1mM DTT, 10 mM
Tris-HCl pH 8.0).
Histones purification. The nuclei were re-
suspended in the sucrose buffer with 0.5 mM
spermidine and 1 mM MgCl2, 1 mM CaCl2 to
the final concentration of DNA 3 mg/ml. The
DNA concentration can be estimated from the
absorption of resuspended nuclei at 260 nm.
Then MNase was added to a final concentration
of 30 units per mg of DNA in the solution. The
mixture was incubated for 4 hours at 37°C
under gentle agitation, transferred on ice, and
supplemented with 3 mM EDTA. The pellet
was collected by centrifugation at 7000 g for
10 min, resuspended in the buffer containing
0.25 mM EDTA, pH 8.0, 0.1mM PMSF, stirred
for one hour at 4°C, centrifuged at 7000 g for
20 min. The supernatant containing mononu-
cleosomes was concentrated via the 10 kDa
centrifugal concentrator and diluted with phos-
93
Optimization of nucleosome assembling from histones and model DNAs and estimation of the reconstitution efficiency
phate buffer (25 mM K2HPO4, 25 mM KH2PO4)
with NaCl to a final concentration of 500 mM.
The solution was loaded onto HAP-column
equilibrated with the buffer consisting of 25
mM K2HPO4, 25 mM KH2PO4, 500 mM NaCl.
The column was washed with the same buffer
and with phosphate buffer containing 0.75 M
NaCl. The core histones were eluted with phos-
phate buffer containing 2.2 M NaCl. Pooled
fractions can be concentrated via the 10 kDa
centrifugal concentrator. The samples supple-
mented with 0.1 mM PMSF and 40 % glyc-
erol can be stored at -70°C. To estimate the
concentration of histones the aliquot of purified
proteins was diluted in bidistilled water. The
absorption at 230 nm was measured using spec-
trophotometer. The concentration in mg/ml is
determined by dividing the absorption value by
the extinction coefficient of 4.3.
DNA preparation. The DNA amplification
was provided by conventional PCR. The DNA
template in concentration 1 ng/µl was supple-
mented with 0.5 µM primers and standard
PCR buffer components. The program for
amplification:
95°C 3 min
94°C 15 sec
65°C 15 sec (the decrement of 1°C per
cycle from 65°C to 61°C)
72°C 10 sec
5 cycles
94°C 15 sec
60°C 15 sec
72°C 10 sec
15 cycles
4°C hold
To remove the excess of primers the sam-
ples were treated with ExoI exonuclease by
supplemented with 10 x buffer containing
67 mM MgCl2, 10 mM DTT, 670 mM glycine-
KOH pH 9.5 to 1x and 0.1 U/µl ExoI. The
DNA was precipitated by addition of NaOAc
up to 0.3 M and supplemented by ethanol up
to 70 % and incubated at -20°C for 1 hour.
After centrifugation at 14000 g for 10 min the
pellet was washed twice with 70 % ethanol
followed by dissolution in bidistilled water.
The concentration of DNA was measured using
spectrophotometer at 260 nm.
Nucleosome assembling. Quick-time recon-
stitution. In this approach either radioactive- or
fluorescent-labelled DNA should be used. The
DNA in final concentration of 0.1 µM was
mixed with histones in different concentrations
in low-salted buffer (10 mM NaCl, 0.2 mM
EDTA, 5 mM beta mercaptoethanol, 0.1 %
NP-40, 10 mM Tris-HCl pH 7.5), incubated
for 15 min at 37°C and analyzed by 4 % PAGE
under non-denaturing conditions. The minimal
DNA:histone ratio corresponding to the mix-
ture with no naked DNA should be used for
preparative reconstitution.
Preparative reconstitution. The DNA and
histones should be taken in the concentrations
determined under “quick-time reconstitution”,
mixed in the high-salted buffer containing 2 M
NaCl, and dialyzed against the buffer with
gradient of NaCl from 2 M to 250 mM during
6 hours at 4°C with gentle stirring. Then the
probes were dialyzed against the buffer with
10 mM NaCl overnight at 4°C with gentle
stirring.
Analysis of nucleosome reconstitution using
Eva-Green dye. The samples of 10 µl contain-
ing 50 mM NaCl, 10 mM DTT, 50 mM Tris-
94
M. M. Kutuzov, T. A. Kurgina, E. A. Belousova et al.
HCl pH 8.0, 50 nМ nucleosome (the concen-
tration was defined by nucleic acid component)
and 0.5х Eva-Green (Biotium) were placed to
the 384-well round bottom microplate to esti-
mate the fluorescence intensity. The measure-
ment was performed in microplate reader
CLARIOstar (BMG Labtech) at 520 nm. The
average value of five independent measure-
ments was normalized to fluorescent level of
naked dsDNA and analyzed using MARS Data
Analysis Software (BMG Labtech).
Results and Discussion
The design of DNA substrates was based on
nucleotide sequences named clones 603 and
605 that were constructed and published by
Lowary and Widom (8). The length of these
DNA duplexes is 147 nucleotides, which ex-
actly corresponds to the “core” part of nucleo-
somes in vivo. The feature of these sequences
consists in the precise positioning of the his-
tone octamer on DNA during the formation of
NCP. Additionally, the histone octamer has a
high affinity to these DNAs. Thus, along with
5S ribosomal cDNA, these DNAs are widely
used in the assembling of NCP in vitro (9,10).
First, the preparative amounts of plasmid
DNA encoding the 603 and 605 sequences
were produced in E. coli strain according to a
standard protocol (11). The DNA was isolated,
and the regions of interest were amplified by
PCR. The PCR primers were chemically syn-
thesized to obtain the final DNA containing or
not the discriminant mark in the 5’-part of the
DNA duplex (Figure 1, A). Such approach
permits to produce the target DNA constructs
Fig. 1. A — Electrophoretic analysis of PCR products. The electrophoretic mobility of the plasmid DNA encoding
the Widom clones 603 and 605 in 1 % agarose gel in non-denaturing conditions with SYBR Green-1 staining.
Lane 1 — marker DNAs, lane 2 — clone 603, 3 — clone 605. B — Nucleosome assembling. The electrophoretic
mobility of 5’-FAM labelled products after the process of NCP reconstruction from model FAM-DNA and histone
octamers in 4 % PAAG under non-denaturing conditions. Lane 1 — free DNA; lanes 2-5 — samples with an increas-
ing ratio of DNA to proteins from 1:0.3 to 1:0.6 with the increment of 0.1 assembled by quick-time method; lanes 6-9 —
NCP assembled by gradient dialysis with the ratio of DNA to proteins from 1:0.2 to 1:0.5 with the increment of 0.1.
Ns — nucleosome particles, DNA — naked DNA.
A B
95
Optimization of nucleosome assembling from histones and model DNAs and estimation of the reconstitution efficiency
with the specific damage at the defined posi-
tion or special label at the 5’-end of the result-
ing DNA. This is important to study various
mechanisms of DNA repair in context of NCP.
Some experimental approaches using recon-
stituted nucleosomes are sensitive to contam-
ination by single-stranded DNA. The nucleo-
somal DNAs prepared by PCR often contain
the unspent primer molecules. We used treat-
ment with Exonuclease I to remove any single-
stranded DNAs including PCR primers. After
the ExoI treatment and DNA precipitation with
ethanol the amplicons were used to obtain the
target model NCPs.
At the next stage, we worked out the proce-
dure for nucleosome assembling using purified
histone octamers and amplified model DNAs.
First of all, to assemble the nucleosomes cor-
rectly, the attention should be paid to the ratio
of concentrations of DNA and histone octamer
samples. Basically, this ratio should be equi-
molar to escape, on the one hand, the excess
of free DNA in the final sample solution, and
on the other hand, the formation of high-mo-
lecular weight DNA-protein complexes upon
histone abundance. However, due to highly
basic nature of the histones it is impossible to
determine precise concentration of protein in
the samples using standard Bradford protocol.
Therefore, the absorbance measurements at
230 nm are commonly used, but mostly small
molecules absorb at this wavelength. Thus, it
is necessary to select the DNA-protein ratio in
mixture for each sample. We proposed the fast
assembling of NCPs in low-salted nucleosome
buffer under titration of the DNA-histone ratio.
The analysis in non-denaturing 4 % PAAG al-
lows selection of the nucleic acid to protein
proportion providing the correct assembling of
NCP (Fig. 1, B). Then the octamers were ini-
tially mixed with DNA in the buffer containing
2 M NaCl and subjected to a gradient dialysis
at +4°C for about 6 hours to decrease the salt
concentration to 250 mM. Finally, the NCP
solution was dialyzed overnight against the
buffer with 10 mM NaCl at 4°C. The effi-
ciency of nucleosome assembling was analyzed
by classical method with an electrophoretic
separation in 4 % PAAG under non-denaturing
conditions. In Fig. 1, B it is presented an ex-
ample of electrophoregram of the products of
the nucleosome assembling after the selection
of the DNA-protein ratio for preparative recon-
struction of NCP using DNA with the 5’-fluo-
rescent label.
This standard approach has an obvious dis-
advantage as far as it does not allowed the
estimation of percent of out-of-nucleosome
DNA directly in the solution under equilibrium
conditions. This problem could be solved by
utilizing an external fluorescent dye in the
solution after the assembling procedure.
Accordingly, we suggested an alternative ap-
proach for analyzing the efficiency of nucleo-
some assembling in solution using the Eva-
Green dye. It is an intercalating dye, the spe-
cific characteristic of which is to effectively
interact only with double-stranded DNA (12).
The fluorescent signal from Eva-Green is non-
essential by itself due to self-extinguishing,
but its level dramatically increased upon ds-
DNA binding. Noteworthy, at the formation of
DNA-protein complex the fluorescent signal
can be obtained from all DNA forms, which
are accessible for Eva-Green intercalation. In
this case, at the formation of compact NCP the
efficiency of dye intercalation should sharply
reduce and the consequent fluorescent level of
96
M. M. Kutuzov, T. A. Kurgina, E. A. Belousova et al.
DNA/Eva-Green complex should decrease.
Indeed, in the experiments we obtained a re-
duction of the fluorescent level of DNA/Eva-
Green consisting of nucleosomes compared to
the initial DNA/Eva-Green fluorescence.
Whereas the change in fluorescent level at the
quick-time reconstitution was significantly
lower (Fig. 2). It should be mentioned that
these results are in full agreement with the data
obtained by electrophoretic analysis under
non-denaturing conditions.
Therefore, we suggested the optimized ap-
proach to track the NCP assembly. According
to it, the quick-time assembling of NCP in
low-salted buffer at the first stage allows the
selection of precise DNA-histone ratio. After
that the quality of slow nucleosome assembling
could be estimated directly in the solution.
Moreover, it does not require the presence of
any labels in DNA to monitor the nucleosome
formation during the preparative reconstitu-
tion. The main advantages of this approach are
the performance of the study in the solution,
which is fast and convenient, and the oppor-
tunity to use different types of DNA required
for further research procedures.
Ascknowlegement
The work was supported by RSF grant 17-74-
20075.
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Оптимізація протоколу збірки нуклеосом
з використанням гістонів і модельних ДНК-
дуплексів і оцінка ефективності процесу
реконструкції
М. М. Кутузов, Т. А. Кургіна, Є. А. Белоусова,
С. Н. Ходирева, О. І. Лаврік
Нуклеосомна корова частка, НКЧ, являє собою зруч-
ну модельну систему для вивчення ключових ДНК-
залежних процесів, оскільки є елементарною оди-
ницею структури хроматину. Саме тому основним
завданням представленого дослідження було ство-
рення універсального методу збірки нуклеосом. Для
реконструкції НКЧ використовуються очищені гіс-
тони і ДНК-структури з певними послідовностями,
що забезпечують чітке позиціонування ДНК від-
носно октамера гістонів. Це вимагає наявності спо-
собу контролю ефективності реконструкції НКЧ.
Мета. Оптимізувати процедуру реконструкції НКЧ
з очищених октамер гістонів і різних типів синтезо-
ваних модельних ДНК і запропонувати підхід для
експрес-аналізу ефективності цього процесу.
Методи. Діаліз, поділ в ПААГ, вимір флуоресценції
з використанням барвника Eva-Green. Результати.
Ми розробили зручну процедуру складання НКЧ в
слабо сольовому буфері із змінним співвідношенням
ДНК-гістони на першому етапі. Після визначення
оптимального співвідношення реконструкція НКЧ
може бути проведена за допомогою повільного гра-
диентного діалізу. На цьому етапі ефективність про-
цесу може бути оцінена безпосередньо в розчині з
використанням барвника Eva-Green. Висновки. Було
показано, що ефективність интеркаляции барвника
в дуплекс ДНК в контексті нуклеосоми різко знижу-
ється. До основних переваг запропонованого під-
ходу можна віднести швидкий аналіз суміші безпо-
середньо в розчині і можливість використання ДНК,
що не містять будь-яких спеціальних міток.
К л юч ов і с л ов а: збірка нуклеосомних корових
частинок, хроматин, корові гістони
Оптимизация протокола сборки нуклеосом
с использованием гистонов и модельных
ДНК-дуплексов и оценка эффективности
процесса реконструкции
М. М. Кутузов, Т. А. Кургина, Е. А, Белоусова,
С. Н. Ходырева, О. И. Лаврик
Нуклеосомная коровая частица, НКЧ, представляет
собой удобную модельную систему для изучения клю-
чевых ДНК-зависимых процессов, поскольку является
элементарной единицей структуры хроматина. Именно
поэтому основной задачей представленного исследо-
вания было создание универсального метода сборки
нуклеосом. Для реконструкции НКЧ используются
очищенные гистоны и ДНК-структуры с определен-
ными последовательностями, обеспечивающими чет-
кое позиционирование ДНК относительно октамера
гистонов. Это требует наличия способа контроля эф-
фективности реконструкции НКЧ. Цель. Опти ми зи-
ровать процедуру реконструкции НКЧ из очищенных
октамеров гистонов и различных типов синтезирован-
98
M. M. Kutuzov, T. A. Kurgina, E. A. Belousova et al.
ных модельных ДНК и предложить подход для экс-
пресс-анализа эффективности этого процесса. Методы.
Диализ, разделение в ПААГ, измерение флуоресцен-
ции с использованием красителя Eva-Green.
Результаты. Мы разработали удобную процедуру
сборки НКЧ в слабо солевом буфере с изменяемым
соотношением ДНК-гистоны на первом этапе. После
определения оптимального соотношения реконструк-
ция НКЧ может быть проведена с помощью медлен-
ного градиентного диализа. На этом этапе эффектив-
ность процесса может быть оценена непосредственно
в растворе с использованием красителя Eva-Green.
Выводы. Было показано, что эффективность интерка-
ляции красителя в дуплекс ДНК в контексте нуклео-
сомы резко снижается. К основным преимуществам
предложенного подхода можно отнести быстрый ана-
лиз смеси непосредственно в растворе и возможность
использования ДНК, не содержащих каких-либо
специальных меток.
К л юч е в ы е с л ов а: сборка нуклеосомных коровых
частиц, хроматин, коровые гистоны.
Received 02.01.2019
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