ISSN 1607�6729, Doklady Biochemistry and Biophysics, 2014, Vol. 455, pp. 68–71. © Pleiades Publishing, Ltd., 2014.Original Russian Text © N.A. Lebedeva, N.I. Rechkunova, O.I. Lavrik, 2014, published in Doklady Akademii Nauk, 2014, Vol. 455, No. 4, pp. 474–477.

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Tyrosyl�DNA phosphodiesterase 1 (Tdp1) is anenzyme that prevents the accumulation of covalentadducts of topoisomerase I (Top1) with DNA, ithydrolyzes 3'�phosphotirosyl bonds [1]. Human Tdp1can also remove other modifying groups from the 3'�end of DNA (e.g., 3'�phosphoglycolates and deoxyri�bose residues); i.e., it functions as a 3'�phosphodi�esterase [2, 3]. We have recently found that humanTdp1 also possesses another activity: it is able to hydro�lyze the apurinic/apyrimidinic (AP) site (both naturaland its synthetic analogue, THF residue) locatedwithin the DNA strand [4]. Mutation of Tdp1, whichcauses spinocerebellar ataxia with axonal neuropathy(SCAN1) [5], leads to the loss of the AP�cleavingactivity of the enzyme [6].

AP sites appear as a result of spontaneous hydroly�sis of the N�glycosidic bond or elimination of a wrongor damaged base by DNA glycosylases. AP sites areamong the most common DNA lesions. According toestimations, in mammalian cells approximately 10 000AP sites are formed per cell under physiological con�ditions [7]. Due to their high reactivity, AP sites arevery toxic to cells. They form covalent crosslinks withthe primary amino groups in proteins through the for�mation of Schiff bases, which entails the formation ofDNA�protein adducts. AP sites are also a source ofDNA�protein crosslinks, crosslinking of DNAstrands, and double strand breaks in DNA at the chro�matin level [8].

The main enzyme cleaving AP sites during baseexcision repair (BER) in mammalian cells is the apu�rinic/apyrimidinic endonuclease 1 (APE1). The abil�ity of the Tdp1 enzyme to cleave AP sites by a differentmechanism, which was discovered by us, suggests itsinvolvement in BER as an alternative of APE1. Tdp1

hydrolyzes an AP site in DNA to form a mononucle�otide gap with phosphate groups at its 3'� and 5'�ends.This activity does not depend on magnesium ions.Polynucleotide kinase/phosphatase (PNKP), exhibit�ing the 3'�phosphatase activity, DNA polymerase β,and DNA ligase III/XRCC1 complex are involved infurther repair process [4].

The process of AP site repair with the involvementof Tdp1 can be attributed to the so�called back�uppathway, which ensure additional reliability of func�tioning of DNA repair mechanisms. According topublished data, BER process in mitochondria dependson the activity of Tdp1, and this enzyme is required forefficient repair of oxidative damages occurring inmitochondrial DNA [9]. We have previously shownthat Tdp1, unlike APE1, much more efficiently cleavesthe AP sites located in single�stranded DNA regionsor opposite to bulky DNA damages in the DNAduplex as compared to the DNA duplex with a regularstructure [6]. This property of Tdp1 suggests that thisenzyme has the potential to be involved in AP siterepair in single�stranded regions of genomic DNA,occurring during its metabolic processes, includingreplication, DNA repair, and transcription. It shouldbe noted that destabilization of the DNA double helixand, consequently, the occurrence of single�strandedregions in it significantly increases the likelihood ofAP sites, as a result of spontaneous apurination/ apy�rimidination of DNA and under the action of DNAglycosylases, which remove the modified (oxidized oralkylated) bases [10].

Figure 1 shows the results of action of Tdp1 andother DNA repair enzymes in the case of DNA con�taining an AP site in a single�stranded regions of a par�tially open DNA duplex. Such DNA structures simu�late the intermediates of the processes that occur withthe formation of locally unwound regions of DNAdouble helix, for example, during the replication initi�ation or in the promoter region of the transcribedgenes. As can be seen from this figure, Tdp1 efficiently

Repair of Apurinic/Apyrimidinic Sites in Single�Stranded DNA Initiated by Tyrosyl�DNA

Phosphodiesterase 1

N. A. Lebedeva, N. I. Rechkunova, and Corresponding Member of the RAS O. I. Lavrik

Received September 6, 2013

DOI: 10.1134/S1607672914020070

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences, pr. Aademika Lavrent’eva 8, Novosibirsk, 630090 Russiae�mail: lavrik@niboch.nsc.ru

BIOCHEMISTRY, BIOPHYSICSAND MOLECULAR BIOLOGY

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REPAIR OF APURINIC/APYRIMIDINIC SITES 69

cleaves the AP site (lane 2) formed after the excision ofthe uridine residue by uracil�DNA glycosylase in thesingle�stranded region. After the removal of the 3'�ter�minal phosphate by PNKP (lane 3), DNA polymeraseβ incorporates into the 3' end of the break one deoxy�nucleoside monophosphate residue of any of the fournucleotides, which are added separately (lanes 4–7) oras a mixture (lane 8), whereupon the ends of the breakcan be covalently joined by DNA ligase (lanes 9–13).In the above�described experiment, the ends of thebreak were ligated using T4 DNA ligase with highactivity. Potentially in higher eukaryotes this functioncan be performed by the DNA or RNA ligases. It isknown that T4 RNA ligase 1 is capable of linking sin�gle�stranded DNA fragments in vitro [11]. Perhapsthat effective ligation of single�stranded DNA endsrequires the involvement of additional protein factors,including the yet unknown. Proposed repair pathwayof the AP sites in single�stranded segment of DNA ini�tiated by Tdp1, is represented schematically in Fig. 2.

Recently, we found [6] that Tdp1 more effectivelycleaves the AP site in a DNA duplex containing abulky substituent in the opposite strand—a fluores�cein residue linked to the base. This substituent mim�ics a DNA lesion that is excised by the nucleotide exci�sion repair (NER) system [12]. The effect of this sub�

stituent, located in one strand of a partially open DNAduplex, on the Tdp1�catalyzed hydrolysis of THF, asynthetic analogue of the AP site, located in the oppo�site strand, was studied (Fig. 3). As can be seen in Fig. 3,the introduction of a bulky substituent does not affectthe binding of Tdp1 to DNA containing a “bubble,”but reduces the efficiency of cleavage of the AP siterelative to the DNA structure containing no bulky sub�stituent, possibly due to a steric hindrance induced bythe bulky substituent. The observed effect may have abiological significance, because such DNA structurerepresents a NER intermediate formed as a result ofpartial unwinding of the DNA duplex around thebulky lesion by the NER factors. At the next stage ofNER, the fragment of the damaged DNA strand isexcised, and the cleavage of the AP site in the oppositestrand would inevitably lead to the formation of a dou�ble stranded break most toxic for cell DNA damage.Thus, until the completion of repair by the NER path�way, the cleavage of the AP site in the opposite strandis a very undesirable event. Since there exists thedescribed pathway of AP site repair in a single�stranded DNA, this control mechanism may preventthe formation of breaks in an intact DNA strand.

1 2 3 4 5 6 7 8 9 10 11 12 13+dATP

+dСTP

+dТTP

+dGTP

+4 dN

TP

+dATP

+dСTP

+dТTP

+dGTP

+4 dN

TP

S

+dNTP3'�OH

3'�P

UDG

Tdp1

PNKP

Polβ

DNA ligase

− + + + + + + + + + + + +

− + + + + + + + + + + + +

− − + + + + + + + + + + +

− − − + + + + + + + + + +

− − − − − − − − + + + + +

Fig. 1. Tdp1�initiated AP site repair in a single�stranded region of a partially open DNA duplex. Designations: lane 2—cleavageof the AP site by Tdp1, lane 3—Removal of the 3'�terminal phosphate by PNKP, lanes 4–8—elongation of the 3' end by DNApolymerase β in the presence of dNTP, lanes 9–13—end ligation, lane 1—control oligonucleotide.

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LEBEDEVA et al.

MATERIALS AND METHODS

Tdp1 recombinant protein was isolated as describedin [6]. The AP site repair in a single�stranded region ofa partly open DNA duplex was performed in 10 μL ofa reaction mixture containing 10 nM 5'�[32P]�labeledDNA, 50 mM Tris�HCl buffer (pH 7.5), 50 mMNaCl, 5 mM MgCl2, 0.5 mM dNTP, 1 mM ATP, andvarious combinations of proteins UDG (0.5 U/μL),Tdp1 (100 nM), Polβ (50 nM), PNKP (300 nM), andT4 DNA ligase (200 U/μL). Reaction mixtures wereincubated at 37°C for 30 min and analyzed by PAGEfollowed by autoradiography with a Molecular ImagerFX Pro + instrument (BioRad, United States).

The binding of Tdp1 to DNA was analyzed by adecrease in the electrophoretic mobility of radiola�beled DNA in complex with protein (gel retardation

method). The reaction was performed in 10 μL of areaction mixture containing 50 mM Tris�HCl buffer(pH 7.5), 50 mM NaCl, 0.1 mg/mL BSA, 10 nM 5'�[32P]�labeled DNA, and various concentrations ofTdp1. The complex between Tdp1 and DNA wasformed by incubation on ice for 5 min. Then, sampleswere supplemented with the loading buffer (1/5 of vol�ume) containing 20% glycerol and 0.015% bromophe�nol blue and analyzed by electrophoresis in nondena�turing 5% PAGE (acrylamide : bis�acrylamide = 60 : 1).Tris�borate�EDTA (TBE) buffer was used as a elec�trode buffer. Electrophoresis was performed at 4°C ata voltage drop of 17 V/cm. The positions of the radio�labeled oligonucleotide and protein�nucleic acidcomplexes were determined by autoradiography.

ACKNOWLEDGMENTS

This study was supported by the Russian Founda�tion for Basic Research (project nos. 12�04�00337, 12�04�33162, and 13�04�40197�comfy), the program ofthe President of the Russian Federation for state sup�port of leading scientific schools (project no. NSh�420.2014.4), and the program of the Russian Academyof Sciences “Molecular and Cellular Biology.”

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5. El�Khamisy, S.F., Saifi, G.M., Weinfeld, M., Johans�son, F., Helleday, T., Lupski, J.R., and Caldecott, K.W.,Nature, 2005, vol. 434, pp. 108–113.

AP site

5' 3' 5' 3'

PP

5'3'5' 3'

5'

5'

3'

3'

PHO

dNTP

Polβ

OHP

PNKP

P

DNA ligase

Tdp1

Fig. 2. Scheme of Tdp1�initiated AP site repair in a single�stranded region of a partially open DNA duplex.

80

70

60

50

40

30

20

10

0

% D

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in c

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ple

x o

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dp

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(b)

Lev

el o

f A

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ite

60

40

20

0hyd

roly

sis,

%

АР site

АР site

Flu

0 10 30 50 75 100[Tdp1], nM

Fig. 3. Effect of a bulky substituent in the opposite strandof a partially open DNA duplex on (a) Tdp1 binding and(b) AP site hydrolysis.

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REPAIR OF APURINIC/APYRIMIDINIC SITES 71

6. Lebedeva, N.A., Rechkunova, N.I., El�Khamisy, S.F.,and Lavrik, O.I., Biochimie, 2012, vol. 94, pp. 1749–1753.

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Translated by M. Batrukova