Ku80 interaction with apurinic/apyrimidinic sites depends on the structure of DNA ends
Aim. The identification of a protein from human cell extract which specifically interacts with the apurinic/apyrimidinic (AP) site in the partial DNA duplex containing 5'and 3'-dangling ends (DDE-AP DNA) and mimicking clustered DNA damage. Methods. The Schiff base-dependent cross-linking o...
Saved in:
| Date: | 2014 |
|---|---|
| Main Authors: | , , |
| Format: | Article |
| Language: | English |
| Published: |
Інститут молекулярної біології і генетики НАН України
2014
|
| Series: | Вiopolymers and Cell |
| Subjects: | |
| Online Access: | http://dspace.nbuv.gov.ua/handle/123456789/153721 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| Journal Title: | Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| Cite this: | Ku80 interaction with apurinic/apyrimidinic sites depends on the structure of DNA ends / A.A. Kosova, S.N. Khodyreva, O.I. Lavrik // Вiopolymers and Cell. — 2014. — Т. 30, № 1. — С. 42-46. — Бібліогр.: 10 назв. — англ. |
Institution
Digital Library of Periodicals of National Academy of Sciences of Ukraine| id |
irk-123456789-153721 |
|---|---|
| record_format |
dspace |
| spelling |
irk-123456789-1537212019-06-15T01:31:31Z Ku80 interaction with apurinic/apyrimidinic sites depends on the structure of DNA ends Kosova, A.A. Khodyreva, S.N. Lavrik, O.I. Structure and Function of Biopolymers Aim. The identification of a protein from human cell extract which specifically interacts with the apurinic/apyrimidinic (AP) site in the partial DNA duplex containing 5'and 3'-dangling ends (DDE-AP DNA) and mimicking clustered DNA damage. Methods. The Schiff base-dependent cross-linking of a protein to AP DNA (borohydride trapping), MALDI-TOF-MS, chromatography, and gel electrophoresis. Results. A human cell extract protein which forms a major covalent adduct with the AP DNA duplex with dangling ends was identified as the Ku80 subunit of Ku antigen by peptide mass mapping based on MALDI-TOF-MS data. The Ku antigen purified from the HeLa cell extract was shown to form the covalent adducts with the same mobility as observed in cell extracts. Conclusions. The Ku80 subunit of Ku antigen can specifically interact with AP DNA forming the Schiff base-mediated adducts which electrophoretic mobility depends on the structure of DNA ends. The difference in electrophoretic mobility can be caused by the cross-linking of AP DNA to distinct target amino acids that appears to reflect unequal positioning of AP DNAs in the complex with Ku antigen. Мета. Ідентифікація білка з екстракту клітин людини, який специфічно взаємодіє з апуриновим/апіримідиновим (AP) сайтом у складі часткового дуплексу ДНК, що містить виступаючі 5'і 3'-кінці та імітує кластерне пошкодження ДНК. Методы. Зшивання білка з AP-ДНК, опосередковане утворенням основи Шиффа, MALDI-TOF мас-спектрометрія, хроматографія і гель-електрофорез. Результаты. Білок клітинних екстрактів людиниа, який формує мажорний ковалентний адукт з AP-ДНК, що містить виступаючі кінці, ідентифіковано як субодиниця Ku80 Ku-антигену методом пептидного картування, заснованого на даних MALDITOF мас-спектрометрії. Показано, що Ku-антиген, виділений з клітин HeLa, формує ковалентні адукти, електрофоретична рухливість яких відповідає рухливості адуктів, утворених білками з клітинних екстрактів. Висновки. Субодиниця Ku80 Ku-антигену може специфічно взаємодіяти з AP-ДНК, формуючи адукти, опосередковані утворенням основи Шиффа, електрофоретична рухливість яких залежить від структури кінців ДНК. Розбіжності в електрофоретичній рухливості можуть обумовлюватися зшиванням AP-ДНК з різними амінокислотними залишками білка, що в свою чергу може відображати різне розташування АP-ДНК у комплексі з Ku-антигеном. Цель. Идентификация белка клеточных экстрактов человека, специфично взаимодействующего с апуриновым/апиримидиновым (AP) сайтом в составе частичного ДНК-дуплекса, содержащего выступающие 5'и 3'-концы и имитирующего кластерное повреждение ДНК. Методы. Сшивка белка с AP-ДНК, опосредованная образованием основания Шиффа, MALDI-TOF масс-спектрометрия, хроматография и гель-электрофорез. Результаты. Белок клеточных экстрактов человека, формирующий мажорный ковалентный аддукт с AP-ДНК, содержащей выступающие концы, идентифицирован как субъединица Ku80 Ku-антигена методом пептидного картирования, основанного на данных MALDI-TOF массспектрометрии. Показано, что Ku-антиген, выделенный из клеток HeLa, формирует ковалентные аддукты, электрофоретическая подвижность которых соответствует подвижности аддуктов, формируемых белками клеточных экстрактов. Выводы. Субъединица Ku80 Ku-антигена может специфично взаимодействовать с AP-ДНК, формируя аддукты, опосредованные образованием основания Шиффа, электрофоретическая подвижность которых зависит от структуры концов ДНК. Различие в электрофоретической подвижности может быть обусловлено сшивкой AP-ДНК с разными аминокислотными остатками белка, что может отражать различное расположение AP-ДНК в комплексе с Ku-антигеном. 2014 Article Ku80 interaction with apurinic/apyrimidinic sites depends on the structure of DNA ends / A.A. Kosova, S.N. Khodyreva, O.I. Lavrik // Вiopolymers and Cell. — 2014. — Т. 30, № 1. — С. 42-46. — Бібліогр.: 10 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.00087B http://dspace.nbuv.gov.ua/handle/123456789/153721 577.112.4 en Вiopolymers and Cell Інститут молекулярної біології і генетики НАН України |
| institution |
Digital Library of Periodicals of National Academy of Sciences of Ukraine |
| collection |
DSpace DC |
| language |
English |
| topic |
Structure and Function of Biopolymers Structure and Function of Biopolymers |
| spellingShingle |
Structure and Function of Biopolymers Structure and Function of Biopolymers Kosova, A.A. Khodyreva, S.N. Lavrik, O.I. Ku80 interaction with apurinic/apyrimidinic sites depends on the structure of DNA ends Вiopolymers and Cell |
| description |
Aim. The identification of a protein from human cell extract which specifically interacts with the apurinic/apyrimidinic (AP) site in the partial DNA duplex containing 5'and 3'-dangling ends (DDE-AP DNA) and mimicking clustered DNA damage. Methods. The Schiff base-dependent cross-linking of a protein to AP DNA (borohydride trapping), MALDI-TOF-MS, chromatography, and gel electrophoresis. Results. A human cell extract protein which forms a major covalent adduct with the AP DNA duplex with dangling ends was identified as the Ku80 subunit of Ku antigen by peptide mass mapping based on MALDI-TOF-MS data. The Ku antigen purified from the HeLa cell extract was shown to form the covalent adducts with the same mobility as observed in cell extracts. Conclusions. The Ku80 subunit of Ku antigen can specifically interact with AP DNA forming the Schiff base-mediated adducts which electrophoretic mobility depends on the structure of DNA ends. The difference in electrophoretic mobility can be caused by the cross-linking of AP DNA to distinct target amino acids that appears to reflect unequal positioning of AP DNAs in the complex with Ku antigen. |
| format |
Article |
| author |
Kosova, A.A. Khodyreva, S.N. Lavrik, O.I. |
| author_facet |
Kosova, A.A. Khodyreva, S.N. Lavrik, O.I. |
| author_sort |
Kosova, A.A. |
| title |
Ku80 interaction with apurinic/apyrimidinic sites depends on the structure of DNA ends |
| title_short |
Ku80 interaction with apurinic/apyrimidinic sites depends on the structure of DNA ends |
| title_full |
Ku80 interaction with apurinic/apyrimidinic sites depends on the structure of DNA ends |
| title_fullStr |
Ku80 interaction with apurinic/apyrimidinic sites depends on the structure of DNA ends |
| title_full_unstemmed |
Ku80 interaction with apurinic/apyrimidinic sites depends on the structure of DNA ends |
| title_sort |
ku80 interaction with apurinic/apyrimidinic sites depends on the structure of dna ends |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| publishDate |
2014 |
| topic_facet |
Structure and Function of Biopolymers |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/153721 |
| citation_txt |
Ku80 interaction with apurinic/apyrimidinic sites depends on the structure of DNA ends / A.A. Kosova, S.N. Khodyreva, O.I. Lavrik // Вiopolymers and Cell. — 2014. — Т. 30, № 1. — С. 42-46. — Бібліогр.: 10 назв. — англ. |
| series |
Вiopolymers and Cell |
| work_keys_str_mv |
AT kosovaaa ku80interactionwithapurinicapyrimidinicsitesdependsonthestructureofdnaends AT khodyrevasn ku80interactionwithapurinicapyrimidinicsitesdependsonthestructureofdnaends AT lavrikoi ku80interactionwithapurinicapyrimidinicsitesdependsonthestructureofdnaends |
| first_indexed |
2025-07-14T05:12:38Z |
| last_indexed |
2025-07-14T05:12:38Z |
| _version_ |
1837597939409616896 |
| fulltext |
UDC 577.112.4
Ku80 interaction with apurinic/apyrimidinic
sites depends on the structure of DNA ends
A. A. Kosova1, 2, S. N. Khodyreva1, 2, O. I. Lavrik1, 2
1Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences
8, Akademika Lavrentieva Ave., Novosibirsk, Russian Federation, 630090
2Novosibirsk State University
2, Pirogova Str., Novosibirsk, Russian Federation, 630090
kosova.anastasiya@gmail.com
Aim. The identification of a protein from human cell extract which specifically interacts with the apurinic/apyri-
midinic (AP) site in the partial DNA duplex containing 5'- and 3'-dangling ends (DDE-AP DNA) and mimicking
clustered DNA damage. Methods. The Schiff base-dependent cross-linking of a protein to AP DNA (borohydride
trapping), MALDI-TOF-MS, chromatography, and gel electrophoresis. Results. A human cell extract protein
which forms a major covalent adduct with the AP DNA duplex with dangling ends was identified as the Ku80 su-
bunit of Ku antigen by peptide mass mapping based on MALDI-TOF-MS data. The Ku antigen purified from the
HeLa cell extract was shown to form the covalent adducts with the same mobility as observed in cell extracts.
Conclusions. The Ku80 subunit of Ku antigen can specifically interact with AP DNA forming the Schiff base-me-
diated adducts which electrophoretic mobility depends on the structure of DNA ends. The difference in electro-
phoretic mobility can be caused by the cross-linking of AP DNA to distinct target amino acids that appears to ref-
lect unequal positioning of AP DNAs in the complex with Ku antigen.
Keywords: Ku antigen, apurinic/apyrimidinic site, protein-DNA cross-linking, clustered DNA damages.
Introduction. The abasic (AP) sites are among the most
frequent DNA damages. Up to 50 000 AP sites arise in a
mammalian cell per day [1] as a result of spontaneous
hydrolysis of N-glycosidic bond or under the action of
DNA glycosylases during the base excision repair [2].
If unrepaired, the AP sites are mutagenic and cytotoxic.
The repair of AP sites in clustered DNA damages pre-
sented by combinations of the AP sites, oxidized bases
and single strand breaks within 1–2 turns of DNA helix
is of particular interest [3]. Such lesions arise in DNA
under the action of ionizing radiation or radiomimetic
drugs.
The cross-linking of proteins to a baseless deoxyri-
bose in DNA via reduction of a Schiff-base interme-
diate is often called «borohydride trapping» (BHT).
BHT in combination with mass spectrometry has been
used in a proteomic approach to identify the human pro-
teins reactive to AP sites [4–6]. The set of DNA dup-
lexes containing the AP site in the middle of 32-mer
oligonucleotide and different structural features has be-
en used. With all AP DNA, except DNA with both 5'- and
3'-dangling ends of 8 nt (DDE-AP DNA), the predomi-
nant products of cross-linking were of the same electro-
phoretic mobility (an apparent molecular mass of 90
kDa). The protein cross-linked to the AP DNA duplex
with blunt ends (BE-AP DNA) was identified as the
Ku80 subunit of Ku antigen [4]. Ku antigen consisting
of two subunits with molecular masses about 70 kDa
(Ku70) and 83 kDa (Ku80) is a eukaryotic DNA-bin-
ding component of DNA-dependent protein kinase.
The main function of Ku antigen is participation in the
double-strand break repair by non-homologous end
joining [7]. In the present work we identified the
protein which specifically interacts with DDE-AP DNA
forming the product with an apparent molecular mass of
100 kDa.
42
ISSN 0233–7657. Biopolymers and Cell. 2014. Vol. 30. N 1. P. 42–46 doi: http://dx.doi.org/10.7124/bc.00087B
Ó Institute of Molecular Biology and Genetics, NAS of Ukraine, 2014
Materials and methods. Chromatographic fractio-
nation of cell extract proteins. HEK 293 cell extract pre-
pared as described elsewhere [8] was diluted 4 times
with the buffer containing 50 mM Tris-HCl (pH 8.0),
1 mM EDTA, 1 mM DTT, 0.025 % NP-40 and was ap-
plied to a heparin-sepharose column. After washing with
the same buffer, the proteins were eluted with linear
gradient of NaCl (0.05–1 M) in the same buffer. The
presence of a target protein in the fractions was deter-
mined by BHT. The fractions were concentrated and
supplemented with glycerol (final concentration of 10 %).
Borohydride trapping and MALDI-TOF-MS ana-
lysis. All procedures were carried out as described pre-
viously [4] with a few exceptions. The concentration of
AP DNA in the reaction mixtures was 0.2 mM. The pre-
parative cross-linking for MALDI-TOF-MS analysis
was performed using a fractionated cell extract enriched
in the target protein by chromatography on heparin-se-
pharose. The peptides derived from the cross-linked
protein were additionally purified on Zip-tip C-18 prior
to the MS analysis.
Purification of Ku antigen. Ku antigen was purified
from the HeLa cell extract prepared as described in [8]
by ammonium sulfate fractionation (45–65 % of satura-
tion) followed by the successive chromatographies on
DEAE-support («BioRad», USA), Q-sepharose («GE
Healthcare») and DS-DNA-cellulose («ICN», USA).
Results and discussion. The BHT experiments using
the DDE- and BE-AP DNAs and several human cell ex-
tracts, including the HeLa and HEK 293 cells, reveal
nearly identical patterns of the cross-linked proteins.
Data for HeLa cell extract are shown in Fig. 1.
For identification of the target protein we used the
fraction of HEK 293 cell extract proteins eluted from
heparin-sepharose at 450–500 mM NaCl and enriched in
the target protein. Use of the enriched extract ensures the
yield of target protein cross-linking with AP DNA and
reduces the likelihood of sample contamination with the
proteins nonspecifically bound to DNA. After the elect-
rophoretic separation of the protein covalent adducts with
biotinylated DNA bound to streptavidin-coated beads,
the gel was stained by Coomassie Brilliant Blue R-250
(Fig. 2, A) and autoradiography was carried out (Fig. 2,
B). Five well-defined protein bands with apparent mole-
cular masses from 100 to 70 kDa (Fig. 2, C) were excised
from the gel and subjected to in-gel trypsin digestion.
The bands N 1–4 have the matching radioactive bands,
thus corresponding to the protein-DNA covalent adducts.
43
KOSOVA A. A., KHODYREVA S. N., LAVRIK O. I.
1 2
100 ®
75 ®
50 ®
37 ®
kDa
Fig. 1. Borohydride trapping of HeLa cell extract proteins with 32P-
labeled AP DNA: 1 – BE-AP DNA; 2 – DDE-AP DNA
1 2 3 4 2 3 4
250 ®
A B
C
150 ®
100 ®
75 ®
50 ®
37 ®
100 ®
75 ®
¬ 1
¬ 2
¬ 3
¬ 4
¬ 5
kDa
kDa
Fig. 2. Purification of cell extract protein(s) cross-linked with AP DNA:
A – Coomassie Brilliant Blue R-250 stained gel (1 – molecular weight
markers; 2 – an aliquot (25 ml) of reaction mixture before incubation
with streptavidin-coated beads; 3 – an aliquot (25 ml) after incubation
(unbound proteins); 4 – covalent adducts and proteins bound to beads);
B – autoradiograph of the gel from Fig. 2, A; C – zoomed fragment of
Fig. 2, A, lane 4
Furthermore, the 100 kDa-band has the highest intensi-
ty with the both methods of visualization. Peptides we-
re analyzed by MALDI-TOF-MS and data were searched
against a database.
The first rank candidates for all the samples with their
scores, the number of the matched peptide mass values
and the protein sequence coverage are shown in Table 1.
Obviously, Ku80 forms several covalent adducts with
DDE-AP DNA. Ku70 is retained on the beads due to
the strong interaction with Ku80 [4, 9].
The data for the matching peptides in the sample N
1 are shown in Table 2. Interestingly, the set of Ku80
peptides identified in this work is almost overlapped
with those obtained in the other works [4, 10]. This is
44
KOSOVA A. A., KHODYREVA S. N., LAVRIK O. I.
Sample number First rank candidate Molecular mass, Da MOWSE score Mass values matched Protein sequence coverage, %
1 Ku80 82652 176 19 27
2 Ku80 82652 149 33 38
3 Ku80 82652 67 13 17
4 N-terminal proteolytic fragment of Ku80 64064 85 19 22
5 Ku70 69799 71 16 35
Table 1
Parameters of the first rank candidates for the samples N 1–5
Position (M + H)obs
+ (M+H)clc
+ Ku80 peptides
37–44 993.5544 992.5477 K.VITMFVQR.Q
37–44 1009.5442 1008.5426 K.VITMFVQR.Q + Oxidation (M)
82–97 1914.9439 1913.9444 R.HLMLPDFDLLEDIESK.I
82–97 1930.9707 1929.9394 R.HLMLPDFDLLEDIESK.I + Oxidation (M)
131–141 1317.6786 1316.6725 R.HIEIFTDLSSR.F
145–155 1266.7332 1265.7343 K.SQLDIIIHSLK.K
185–195 1109.6030 1108.6029 R.LGGHGPSFPLK.G
243–250 1035.4896 1034.4868 R.HSIHWPCR.L
251–260 1073.6282 1072.6240 R.LTIGSNLSIR.I
266–271 745.4070 744.4130 K.SILQER.V
308–315 977.5023 976.4978 K.EDIIQGFR.Y
355–363 1083.5626 1082.5583 R.FFMGNQVLK.V
355–363 1099.5442 1098.5532 R.FFMGNQVLK.V + Oxidation (M)
401–413 1377.7583 1376.7565 R.ANPQVGVAFPHIK.H
444–465 2321.1723 2320.1620 K.YAPTEAQLNAVDALIDSMSLAK.K
470–481 1380.7484 1379.6820 K.TDTLEDLFPTTK.I
546–565 2243.0201 2242.0138 K.DQVTAQEIFQDNHEDGPTAK.K
569–599 3121.5137 3120.4749 K.TEQGGAHFSVSSLAEGSVTSVGSVNPAENFR.V
649–654 795.3550 794.3559 K.FSEEQR.F
Table 2
Observed masses in MALDI-TOF mass spectrum of the sample N 1 which correspond to theoretical peptides of Ku80
probably due to an easy ionization of these peptides un-
der the analysis conditions.
The appearance of the Ku80 adduct with a lowered
electrophoretic mobility characteristic for DDE-AP DNA
can reflect the existence of two different modes of Ku80
binding with DNA (and, consequently, different points
of interaction with the AP site) or the highly efficient
cross-linking of DNA with another Ku80 isoform. In-
deed, the lists of candidates for the samples N 1 and 2
include a Ku80 isoform with higher molecular mass
(93464 Da). The biochemical properties of this isoform
resemble those of the conventional Ku80, but it cannot
fully replace Ku80 [9]. To discriminate these alterna-
tives, we purified the Ku antigen from HeLa cells (see
«Materials and methods») to near homogeneity (Fig. 3)
and then tested the ability of the purified protein to in-
teract with two aforementioned types of AP DNA (Fig.
4). The patterns of cross-linking of two AP DNAs to
the purified Ku antigen and cell extract proteins have
the analogous set of products: more abundant 100 kDa
and less intensive 90 kDa adducts for DDE-AP DNA
(lanes 1, 3) and 90 kDa adduct for BE-AP DNA (lanes
2, 4).
Conclusions. Thus, the Ku80 subunit of Ku antigen
specifically interacts with the AP DNA structure with
5'- and 3'-dangling ends and forms a covalent adduct
with an apparent molecular mass of 100 kDa after the
treatment with NaBH4. Further investigations are requi-
red to determine the role of these modes of the Ku anti-
gen interaction with the AP sites. This type of damaged
DNA structure can be used to monitor the binding acti-
vity of Ku antigen in the extracts of various human cells.
Funding. This work was supported by RFBR, pro-
jects 13-04-01426, 13-04-93107, GDRI program «From
Molecular to Cellular Events in Human Pathologies»,
the RAS program «Molecular and Cellular Biology»,
and the Ministry of Education and Science of Russia,
project 14.B37.21.0188.
Âçàºìîä³ÿ Ku80 ç àïó ðè íî âè ìè/àï³ðèì³äè íî âè ìè ñàé òà ìè
çà ëå æèòü â³ä ñòðóê òó ðè ÄÍÊ
À. À. Êî ñî âà, Ñ. Í. Õî äè ðå âà, Î. ². Ëàâ ðèê
Ðå çþ ìå
Ìåòà. ²äåí òèô³êàö³ÿ á³ëêà ç åêñòðàê òó êë³òèí ëþ äè íè, ÿêèé ñïå -
öèô³÷íî âçàºìî䳺 ç àïó ðè íî âèì/àï³ðèì³äè íî âèì (AP) ñàé òîì ó
ñêëàä³ ÷àñ òêî âî ãî äóï ëåê ñó ÄÍÊ, ùî ì³ñòèòü âèñ òó ïà þ÷³ 5'- ³
3'-ê³íö³ òà ³ì³òóº êëàñ òåð íå ïî øêîä æåí íÿ ÄÍÊ. Ìå òî äû. Çøè -
âàí íÿ á³ëêà ç AP-ÄÍÊ, îïî ñå ðåä êî âà íå óòâî ðåí íÿì îñíî âè Øèô ôà,
MALDI-TOF ìàñ-ñïåê òðîìåòð³ÿ, õðî ìà òîã ðàô³ÿ ³ ãåëü-åëåê òðî -
ôî ðåç. Ðå çóëü òà òû. Á³ëîê êë³òèí íèõ åêñòðàêò³â ëþ äè íèà, ÿêèé
ôîð ìóº ìà æîð íèé êî âà ëåí òíèé àäóêò ç AP-ÄÍÊ, ùî ì³ñòèòü âè-
ñòó ïà þ÷³ ê³íö³, ³äåí òèô³êî âà íî ÿê ñóá îäè íè öÿ Ku80 Ku-àí òè ãå íó
ìå òî äîì ïåïòèä íî ãî êàð òó âàí íÿ, çà ñíî âà íî ãî íà äà íèõ MALDI-
TOF ìàñ-ñïåê òðî ìåòð³¿. Ïî êà çà íî, ùî Ku-àí òè ãåí, âèä³ëå íèé ç êë³-
òèí HeLa, ôîð ìóº êî âà ëåíòí³ àäóê òè, åëåê òðî ôî ðå òè÷ íà ðóõ ëè-
â³ñòü ÿêèõ â³äïîâ³äຠðóõ ëè âîñò³ àäóêò³â, óòâî ðå íèõ á³ëêà ìè ç êë³-
òèí íèõ åêñòðàêò³â. Âèñ íîâ êè. Ñó áî äè íè öÿ Ku80 Ku-àí òè ãå íó ìî-
æå ñïå öèô³÷íî âçàºìîä³ÿòè ç AP-ÄÍÊ, ôîð ìó þ ÷è àäóê òè, îïî ñå -
ðåä êî âàí³ óòâî ðåí íÿì îñíî âè Øèô ôà, åëåê òðî ôî ðå òè÷ íà ðóõ -
ëèâ³ñòü ÿêèõ çà ëå æèòü â³ä ñòðóê òó ðè ê³íö³â ÄÍÊ. Ðîçá³æíîñò³ â
åëåê òðî ôî ðå òè÷í³é ðóõ ëè âîñò³ ìî æóòü îá óìîâ ëþ âà òè ñÿ çøè -
âàí íÿì AP-ÄÍÊ ç ð³çíè ìè àì³íî êèñ ëîò íè ìè çà ëèø êà ìè á³ëêà, ùî
â ñâîþ ÷åð ãó ìîæå â³äîá ðà æà òè ð³çíå ðîç òà øó âàí íÿ ÀP-ÄÍÊ ó
êîì ïëåêñ³ ç Ku-àí òè ãå íîì.
45
Ku80 INTERACTION WITH APURINIC/APYRIMIDINIC SITES
1 2
250 ®
150 ®
100 ®
75 ®
50 ®
37 ®
25 ®
kDa
Fig. 3. Purified Ku antigen (Coomassie Brilliant Blue R-250 stained
gel): 1 – molecular weight markers; 2 – purified Ku antigen (0.6 mg)
1 2 3 4
100 ®
75 ®
50 ®
37 ®
kDa
Fig. 4. Borohydride trapping of purified Ku antigen (1, 2 – 0.6 mg and
3, 4 – 1.2 mg of Ku antigen): 1, 3 – DDE-AP DNA; 2, 4 – BE-AP DNA
46
KOSOVA A. A., KHODYREVA S. N., LAVRIK O. I.
Êëþ ÷îâ³ ñëî âà: Ku-àí òè ãåí, àïó ðè íî âèé/àï³ðèì³äè íî âèé ñàéò,
çøè âàí íÿ á³ëîê–ÄÍÊ, êëàñ òåðí³ ïî øêîä æåí íÿ ÄÍÊ.
Âçà è ìî äå éñòâèå Ku80 ñ àïó ðè íî âû ìè/àïè ðè ìè äè íî âû ìè
ñàé òà ìè çà âè ñèò îò ñòðóê òó ðû êîí öîâ ÄÍÊ
À. À. Êî ñî âà, Ñ. Í. Õî äû ðå âà, Î. È. Ëàâ ðèê
Ðå çþ ìå
Öåëü. Èäåí òè ôè êà öèÿ áåë êà êëå òî÷ íûõ ýêñ òðàê òîâ ÷å ëî âå êà,
ñïå öè ôè÷ íî âçà è ìî äå éñòâó þ ùå ãî ñ àïó ðè íî âûì/àïè ðè ìè äè íî âûì
(AP) ñàé òîì â ñî ñòà âå ÷àñ òè÷ íî ãî ÄÍÊ-äóï ëåê ñà, ñî äåð æà ùå ãî
âû ñòó ïà þ ùèå 5'- è 3'-êîí öû è èìè òè ðó þ ùå ãî êëàñ òåð íîå ïî âðåæ-
äåíèå ÄÍÊ. Ìå òî äû. Ñøèâ êà áåë êà ñ AP-ÄÍÊ, îïîñ ðå äî âàí íàÿ îá-
ðàç îâà íè åì îñíî âà íèÿ Øèô ôà, MALDI-TOF ìàññ-ñïåê òðîìåò ðèÿ,
õðî ìà òîã ðà ôèÿ è ãåëü-ýëåê òðî ôî ðåç. Ðå çóëü òà òû. Áå ëîê êëå òî÷-
íûõ ýêñ òðàê òîâ ÷å ëî âå êà, ôîð ìè ðó þ ùèé ìà æîð íûé êî âà ëåíò-
íûé àä äóêò ñ AP-ÄÍÊ, ñî äåð æà ùåé âû ñòó ïà þ ùèå êîí öû, èäåí -
òè ôè öè ðî âàí êàê ñóá ú å äè íè öà Ku80 Ku-àí òè ãå íà ìå òî äîì ïåï-
òèä íî ãî êàð òè ðî âà íèÿ, îñíî âàí íî ãî íà äàí íûõ MALDI-TOF ìàññ-
ñïåê òðî ìåò ðèè. Ïî êà çà íî, ÷òî Ku-àí òè ãåí, âû äå ëåí íûé èç êëå -
òîê HeLa, ôîð ìè ðó åò êî âà ëåí òíûå àä äóê òû, ýëåê òðî ôî ðå òè -
÷åñ êàÿ ïîä âèæ íîñòü êî òî ðûõ ñî îò âå òñòâó åò ïîä âèæ íîñ òè àä-
äóê òîâ, ôîð ìè ðó å ìûõ áåë êà ìè êëå òî÷ íûõ ýêñ òðàê òîâ. Âû âî äû.
Ñóáú å äè íè öà Ku80 Ku-àí òè ãå íà ìî æåò ñïå öè ôè÷ íî âçà è ìî äåé-
ñòâî âàòü ñ AP-ÄÍÊ, ôîð ìè ðóÿ àä äóê òû, îïîñ ðå äî âàí íûå îá ðà -
çî âà íè åì îñíî âà íèÿ Øèô ôà, ýëåê òðî ôî ðå òè ÷åñ êàÿ ïîä âèæ -
íîñòü êî òî ðûõ çà âè ñèò îò ñòðóê òó ðû êîí öîâ ÄÍÊ. Ðàç ëè ÷èå â
ýëåê òðî ôî ðå òè ÷åñ êîé ïîä âèæ íîñ òè ìî æåò áûòü îá óñëîâ ëå íî
ñøèâ êîé AP-ÄÍÊ ñ ðàç íû ìè àìè íî êèñ ëîò íû ìè îñòàò êà ìè áåë -
êà, ÷òî ìî æåò îò ðà æàòü ðàç ëè÷ íîå ðàñ ïî ëî æå íèå AP-ÄÍÊ â
êîì ïëåê ñå ñ Ku-àí òè ãå íîì.
Êëþ ÷å âûå ñëî âà: Ku-àí òè ãåí, àïó ðè íî âûé/àïè ðè ìè äè íî âûé
ñàéò, ñøèâ êà áå ëîê–ÄÍÊ, êëàñ òåð íûå ïî âðåæ äå íèÿ ÄÍÊ.
REFERENCES
1. Atamna H, Cheung I, Ames BN. A method for detecting abasic
sites in living cells: age-dependent changes in base excision re-
pair. Proc Natl Acad Sci USA. 2000; 97(2):686–91.
2. McCullough AK, Dodson ML, Lloyd RS. Initiation of base
excision repair: glycosylase mechanisms and structures. Annu
Rev Biochem. 1999; 68:255–85.
3. Georgakilas AG, O’Neill P, Stewart RD. Induction and repair of
clustered DNA lesions: what do we know so far? Radiat Res.
2013; 180(1):100–9.
4. Ilina ES, Lavrik OI, Khodyreva SN. Ku antigen interacts with
abasic sites. Biochim Biophys Acta. 2008; 1784(11):1777–85.
5. Khodyreva SN, Prasad R, Ilina ES, Sukhanova MV, Kutuzov MM,
Liu Y, Hou EW, Wilson SH, Lavrik OI. Apurinic/apyrimidinic
(AP) site recognition by the 5'-dRP/AP lyase in poly(ADP-
ribose) polymerase-1 (PARP-1). Proc Natl Acad Sci USA. 2010;
107(51):22090–5.
6. Prasad R, Liu Y, Deterding LJ, Poltoratsky VP, Kedar PS, Hor-
ton JK, Kanno S, Asagoshi K, Hou EW, Khodyreva SN, Lavrik
OI, Tomer KB, Yasui A, Wilson SH. HMGB1 is a cofactor in
mammalian base excision repair. Mol Cell. 2007; 27(5):829–41.
7. Gullo C, Au M, Feng G, Teoh G. The biology of Ku and its poten-
tial oncogenic role in cancer. Biochim Biophys Acta. 2006; 1765
(2):223–34.
8. Biade S, Sobol RW, Wilson SH, Matsumoto Y. Impairment of pro-
liferating cell nuclear antigen-dependent apurinic/apyrimidinic
site repair on linear DNA. J Biol Chem. 1998; 273(2):898–902.
9. Koike M, Yutoku Y, Koike A. KARP-1 works as a heterodimer
with Ku70, but the function of KARP-1 cannot perfectly replace
that of Ku80 in DSB repair. Exp Cell Res. 2011; 317(16):
2267–5.
10. Fransson J, Borrebaeck CA. The nuclear DNA repair protein
Ku70/80 is a tumor-associated antigen displaying rapid receptor
mediated endocytosis. Int J Cancer. 2006; 119(10):2492–6.
Received 15.09.13
|