Isolated human ribosomal protein uS3 has extraribosomal functions including those related to base excision DNA repair, e.g. AP lyase activity that nicks double-stranded (ds) DNA 3' to the abasic (AP) site. However, the ability of uS3 residing within ribosome to recognize and cleave damaged DNA has never been addressed. Here, we compare interactions of single-stranded (ss) DNA and dsDNA bearing AP site with human ribosome-bound uS3 and with the isolated protein, whose interactions with ssDNA were not yet studied. The AP lyase activity of free uS3 was much higher with ssDNA than with dsDNA, whereas ribosome-bound uS3 was completely deprived of this activity. Nevertheless, an exposed peptide of ribosome-bound uS3 located far away from the putative catalytic center previously suggested for isolated uS3 cross-linked to full-length uncleaved ss- DNA, but not to dsDNA. In contrast, free uS3 crosslinked mainly to the 5--part of the damaged DNA strand after its cleavage at the AP site. ChIP-seq analysis showed preferential uS3 binding to nucleolusassociated chromatin domains. We conclude that free and ribosome-bound uS3 proteins interact with AP sites differently, exhibiting their non-translational functions in DNA repair in and around the nucleolus and in regulation of DNA damage response in looped DNA structures, respectively.