Recognition but no repair of abasic site in single-stranded DNA by human ribosomal uS3 protein residing within intact 40S subunit

Abstract

Isolated human ribosomal protein uS3 has extra-ribosomal 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 ssDNA, but not to dsDNA. In contrast, free uS3 cross-linked 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 nucleolus-associated 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.

Protection of abasic sites during DNA replication by a stable thiazolidine protein-DNA cross-link

“…This contrasts with unstable, transient protein-DNA Schiff base crosslinks that rapidly proceed to β-elimination as part of enzymatic strand cleavage reactions catalysed by bifunctional glycosylases and DNA polβ as part of the BER pathway 13–15 . Other proteins, including PARP-1, Histone H4, and Ribosomal protein uS3 can crosslink to AP sites, but in each case the DPC leads to strand scission 18–21 .…”

Ribosomal protein uS3 in cell biology and human disease: Latest insights and prospects

“…The positions of uS3 (pink) and 40S assembly factors Ltv1 (dark blue) and Enp1 (brown) are shown on the pre-40S particle surface together with the uS3 NTD position in the mature 40S subunit (cyan) according to [75] (B) A simplified scheme of the implication of uS3 in the 40S subunit assembly according to [72] uS3: A player in DNA repair New aspects concerning the interactions of uS3 with AP sites in DNA include the discovery that the protein strongly prefers to interact with single-stranded DNA regions and the findings from the identification of human chromatin sites that bind uS3 in vivo. [12] These sites are located predominantly in the pericentromeric regions of chromosomes, where chromatin is less condensed and more accessible for DNA-binding proteins and the formation of loops with single-stranded fragments is very likely. However, it remains to be studied what specific function of uS3 is performed when it binds to these regions, and, besides, the dependence of uS3 affinity for deoxyribooligomers on their sequences [78] raises the issue of identifying DNA sequences preferable for the protein binding.…”

“…Recently, various novel data have been obtained that significantly expand our knowledge in this area (e.g., see refs. [10][11][12][13][14]).…”

In vitro eradication of abasic site-mediated DNA–peptide/protein cross-links by Escherichia coli long-patch base excision repair

“…If left unrepaired, AP sites can react with the N terminal or lysine side chain amines in peptides ( 12 ) and numerous proteins ( 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 ) to form covalent Schiff base DNA–peptide/protein cross-links (DPCs, Fig. 1 B ).…”

“…1 B ). Schiff base AP-protein DPC formation has been demonstrated in vitro using AP site-containing DNA and recombinant proteins ( 13 , 14 , 15 , 16 , 17 , 18 , 19 , 20 , 21 , 22 ) or Escherichia coli ( E. coli ) and yeast cell extracts ( 23 ) and also in human cells with an abundance of ∼1500/cell under normal conditions ( 24 , 25 ). Schiff base DPCs are unstable due to the spontaneous hydrolysis; however, their half-lives can be as long as several hours under physiological pH and temperature ( 14 , 26 ).…”