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. 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 Biopolymers 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


Introduction
Y-box-binding protein 1 (YB-1) is a multifunctional 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 followed by synthesis of nucleic acid-like polymer 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 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.000954modulation 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 noncanonical DNA repair proteins (usually RNAbinding [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 ligand for YB-1 in the presence of DNA and RNA as the YB-1 targets.

Molecular and Cell Biotechnologies
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 dialysis stages [6].Using histidine-tagged YB-1 is more advantageous since purification of a tagged protein can be performed during singlestep affinity chromatography.Moreover, obtained 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 activation 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.

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 amplified 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 and plate51-reverse in GC-buffer (Biolabmix, Russian Federation) using pET3-I-YB-1 as a template, then purified by isopropanol preci pitation 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 containing 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 column was equilibrated by Ni-A buffer (see

PAR labeling in vitro
Radioactively labelled (or unlabeled) PAR polymer was obtained as described previ-

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 reading 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 undocumented 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 transformed by pLATE-51-YB-1 were optimized to achieve maximal yield of the target protein in soluble form.Since a single-step metal affinity chromatography did not result in pure YB-1 preparation, two additional purification steps were performed (Fig. 2).Using this approach 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 mobili 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 singleand 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 RNAbinding of YB-1 and recruit this protein to Supplementary, Figure S1.Construction of his-tagged YB-1 expression vector (scheme).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 purification 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 concentration 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.

Fig. 1 .
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 rubricated.