Y box-binding protein-1 binds preferentially to single-stranded nucleic acids and exhibits 3'->5' exonuclease activity

Abstract

We have previously shown that Y box-binding protein-1 (YB-1) binds preferentially to cisplatin-modified Y box sequences. Based on structural and biochemical data, we predicted that this protein binds single-stranded nucleic acids. In the present study we confirmed the prediction and also discovered some unexpected functional features of YB-1. We found that the cold shock domain of the protein is necessary but not sufficient for double-stranded DNA binding while the C-tail domain interacts with both single-stranded DNA and RNA independently of the cold shock domain. In an in vitro translation system the C-tail domain of the protein inhibited translation but the cold shock domain did not. Both in vitro pull-down and in vivo co-immunoprecipitation assays revealed that YB-1 can form a homodimer. Deletion analysis mapped the C-tail domain of the protein as the region of homodimerization. We also characterized an intrinsic 3'-->5' DNA exonuclease activity of the protein. The region between residues 51 and 205 of its 324-amino acid extent is required for full exonuclease activity. Our findings suggest that YB-1 functions in regulating DNA/RNA transactions and that these actions involve different domains.

YB-1 and CTCF Differentially Regulate the 5-HTT Polymorphic Intron 2 Enhancer Which Predisposes to a Variety of Neurological Disorders

“…3A, lanes 1-3, 6 -8, and 11-13). Complexes 1, 2, and 3 may reflect the occupancy of different sites within the VNTR domain by YB-1 as increasing amounts of this protein are added to the reaction or result from multimerization of YB-1 (Izumi et al, 2001). All three bands were competed when unlabeled mdr-1 sequences containing the well characterized YB-1 binding site were included in the reaction (Fig.…”

Intact RNA-binding Domains Are Necessary for Structure-specific DNA Binding and Transcription Control by CBTF122 during Xenopus Development

“…1a). The predicted sizes of the expressed protein fragments were 41 and 28.5 kDa, respectively (CBTF 122 (41) and CBTF 122 (28.5)); both contained the nuclear localization signal. We purified these proteins to 95% homogeneity and then tested their ability to bind a dsRNA homologue of the Ϫ69 to Ϫ33 CCAAT box-containing region of the GATA-2 promoter by EMSA (Fig.…”

“…One possible explana- (41)GST forms of the CBTF 122 RNA-binding domains were expressed in E. coli as fusions with glutathione S-transferase. The purified proteins were used in EMSA assays with 0.3 nM of either the dsRNA, dsDNA, or ssRNA version of the CBTF 122 -binding sequence as a probe.…”

The Y-box Binding Protein YB-1 Suppresses Collagen α1(I) Gene Transcription via an Evolutionarily Conserved Regulatory Element in the Proximal Promoter

“…5C). The anti-YB-1 antibody used in this study is directed against epitopes in the C terminus of the protein (13), which also contributes to DNA binding (26). As a result this antibody mainly disrupts DNA binding, leading to diminished bands (bands designated 4 and 5 in lane 2 with SS1 and 9, 10, and 11 in lane 8 with SS2), whereas supershifts are rather weak and are visualized only with prolonged autoradiography (indicated by asterisks in lanes 5 and 11).…”