Effect of multifunctional protein YB-1 on the AP site cleavage by AP endonuclease 1 and tyrosyl phosphodiesterase 1

Apurinic/apyrimidinic sites (AP sites) which represent one of the most abundantly generated DNA lesions in the cell are generally repaired by base excision repair (BER) pathway. Multifunctional protein YB-1 is known to participate in cellular response to genotoxic stress and was shown to interact with several components of BER – DNA glycosylases NTH1, NEIL2, DNA polymerase and DNA ligase III. Therefore, it is of great interest to investigate the influence of YB-1 on one of the major BER enzymes, responsible for AP site cleavage, AP endonuclease APE1, and on tyrosyl phosphodiesterase Tdp1, participating in APE1 independent pathway of AP site repair. Aim. Effect of multifunctional protein YB-1 on the AP site cleavage by the activities of APE1 and Tdp1 was studied. Methods. Gel-mobility shift assays and enzyme activity tests. Results. YB-1 was shown to inhibit the cleavage of AP site located in single-stranded DNA by both APE1 and Tdp1. Stimulation of APE1 activity on protruding double-stranded DNA in the presence of YB-1 was observed, whereas no effect on Tdp1-mediated cleavage of AP site in double-stranded DNA was found. Conclusions. YB-1 can modulate the repair of AP sites in DNA by both positively stimulating APE1 during the classic BER of AP sites and avoiding a possible generation of doublestrand breaks, arising from the cleavage of single-stranded portion of DNA substrate already used by different DNA-processing pathway.

Introduction.AP sites in DNA appear as a result of release of the nucleobase from the nucleotide residue in DNA.The process is going both spontaneously and as DNA glycosylase-driven release of damaged nucleobases repaired by base excision repair (BER) of DNA.AP sites are also representative DNA damages, since absence of coding base in DNA leads to the blockade of replicative DNA polymerases or error-prone TLS synthesis across the lesion [1], which in turn define cytotoxicity and mutations.The major repair pathway of AP sites is BER which consists in the cleavage of DNA, containing AP site, followed by DNA repair synthesis and ligation.The major AP site cleaving activity is attributed to APE1, however, there is evidence of APE1 independent pathway of repair, which utilize the AP lyase activity of bifunctional DNA glycosylases such as NEIL2 or NTH1 and tyrosyl phosphodiesterase Tdp1 [2,3].In recent studies it was shown that Tdp1 can initiate the repair of AP sites in vitro, cleaving DNA at the position of AP site [4].Such pathways can contribute to the backup for APE1-dependent AP site repair.One of the proteins that can affect both the mainstream and backup ways of the repair of AP sites is multifunctional Y-box binding protein YB-1.YB-1 directly interacts with en- zymes responsible for the initial stages of BER -APE1, NEIL2 and NTH1, stimulating the activities of the latter two enzymes, when the repair of oxidatively damaged DNA is performed (reviewed in [5]).We also have shown recently that YB-1 modulates AP lyase activity of bifunctional DNA glycosylase/AP lyase NEIL1 [6].Therefore, the aim of present study was to investigate the influence of YB-1 on the other enzymes responsible for the AP site cleavage during the repair of AP sites -APE1 and Tdp1.
Enzymatic reactions and GMSA experiments were performed in 50 mM sodium phosphate buffer, pH 7.8, 100 mM NaCl, 2 mM EDTA, 0.1 g/l BSA, 0.2 mM DTT, 2.5 % glycerol and in case of APE1 5 mM MgCl 2 .Enzymes (Tdp1 at final concentration 200 nM, APE1 at 0.3 nM and 0.6 nM for dsDNA and ssDNA experiments, respectively), DNA (50 nM final concentration) and YB-1 (if used) were incubated for indicated periods of time or 20 min at 37 o C. Quenching of the reaction and analysis of reaction products were performed essentially as in [6].
Gel-mobility shift assays (GMSA) experiments were performed essentially as in [6].Results and discussion.We assayed AP site cleavage activity of APE1 and Tdp1 on three different AP site-containing substrates -double-stranded DNA, singlestranded DNA and double-stranded DNA, containing fluorescein residue in + 1 position in the complementary strand opposite the AP site (Fig. 1).The latter substrate imitates DNA, bearing clustered lesions that require different DNA repair pathways.
We have found that despite the mechanistic model depicting physical interaction of APE with nucleotides adjacent to the AP site [8], presence of bulky fluorescein residue in close proximity to AP site does not affect endonucleolytic activity of enzyme.Similarly with earlier observations we reveal that introduction of bulky moiety in close proximity to AP site in double-stranded DNA transforms such DNA to a much better substrate for Tdp1.Results from GMSA experiments suggest that YB-1 affinity towards DNA, containing AP site cluste-red with bulky lesion, is higher than to DNA with single AP site, however under reaction conditions used the highest affinity of YB-1 was shown for the single-stranded DNA substrate (Fig. 2).Addition of YB-1 to the reaction mixture affected both APE1 and Tdp1 activities, depending on the DNA substrate used (Fig. 3, 4).Evident inhibition of the AP site cleavage located in the single-stranded DNA was observed for both enzymes (Fig. 3).The same inhibitory effect was shown, when single-stranded AP site-containing DNA of two other lengths -32 and 60 nt were tested (data not shown).Such findings are consistent with published data showing negative regulation of NEIL1 AP lyase activity by YB-1 when single-stranded DNA was used [6].
On the other hand, almost no effect was found when APE1 or Tdp1 activity was assayed in reaction mixtures, containing double-stranded 48-mer DNA substrates with clustered lesion or single AP site, that were Similarly absence of functional interaction between YB-1 and APE1 was reported earlier in experiments with 43-mer double-stranded substrates, when YB-1 was in shortage in comparison to the amount of DNA [9].Under our experimental conditions, however, large (10-fold) molar excess of YB-1 over DNA lead to almost complete inhibition of AP site cleavage.Taking together with YB-1 ability to form multimers [10], data from GMSA experiments (Fig. 1) and NaBH4-mediated crosslinking of YB-1 directly to AP site [6] we assume that at such high concentrations several YB-1 molecules are bound to the DNA substrate in specific conformation forming multimeric complex.Such complex is stabilized by Schiff base formation of primary amino group of protein with aldehyde group of AP site which presumably results in blocking the access of APE1 or Tdp1 to the AP site.
Stimulation of APE1 activity by YB-1 on doublestranded DNA was observed when longer 60-mer AP site-containing DNA substrate was used (Fig. 4, B).These data are in agreement with our previous observations that YB-1 notably stimulates AP lyase activity of NEIL1 when protruding double-stranded substrate was used and may reflect specific interaction of YB-1 with such DNA substrate.
Taken together the data from present and previous studies imply that YB-1 can play a modulatory role during the repair of AP sites in DNA both by positively stimulating the cleavage of AP site located in specific double-stranded substrates and by avoiding the cleavage of single-stranded portion of unwound DNA substrate, which is already used by different DNA-processing pathway (i.e.DNA replication, transcription or different types of repair).YB-1 can affect both mainstream and backup pathways influencing major AP endonuclease -APE1 and enzymes that can serve as a backup for AP site cleavage reaction -bifunctional glycosylase NEIL1 and tyrosyl phosphodiesterase Tdp1.Involvements of YB-1 itself into abovementioned DNA-processing events (transcription activity of YB-1, putative role of YB-1 in DNA replication and repair (reviewed in [5])) would serve as a basis for coordinating these processes with the repair of AP sites.