Conversion of the Bifunctional 8-Oxoguanine/β-δ Apurinic/Apyrimidinic DNA Repair Activities ofDrosophila Ribosomal Protein S3 into the Human S3 Monofunctional β-Elimination Catalyst through a Single Amino Acid Change

Abstract

The Drosophila S3 ribosomal protein has important roles in both protein translation and DNA repair. In regards to the latter activity, it has been shown that S3 contains vigorous N-glycosylase activity for the removal of 8-oxoguanine residues in DNA that leaves baseless sites in their places. Drosophila S3 also possesses an apurinic/apyrimidinic (AP) lyase activity in which the enzyme catalyzes a ␤-elimination reaction that cleaves phosphodiester bonds 3 and adjacent to an AP lesion in DNA. In certain situations, this is followed by a ␦-elimination reaction that ultimately leads to the formation of a single nucleotide gap in DNA bordered by 5-and 3-phosphate groups. The human S3 protein, although 80% identical to its Drosophila homolog and shorter by only two amino acids, has only marginal N-glycosylase activity. Its lyase activity only cleaves AP DNA by a ␤-elimination reaction, thus further distinguishing itself from the Drosophila S3 protein in lacking a ␦-elimination activity. Using a hidden Markov model analysis based on the crystal structures of several DNA repair proteins, the enzymatic differences between Drosophila and human S3 were suggested by the absence of a conserved glutamine residue in human S3 that usually resides at the cleft of the deduced active site pocket of DNA glycosylases. Here we show that the replacement of the Drosophila glutamine by an alanine residue leads to the complete loss of glycosylase activity. Unexpectedly, the ␦-elimination reaction at AP sites was also abrogated by a change in the Drosophila glutamine residue. Thus, a single amino acid change converted the Drosophila activity into one that is similar to that possessed by the human S3 protein. In support of this were experiments executed in vivo that showed that human S3 and the Drosophila site-directed glutamine-changed S3 performed poorly when compared with Drosophila wildtype S3 and its ability to protect a bacterial mutant from the harmful effects of DNA-damaging agents.Aerobic respiration produces electrophilic oxidants known as reactive oxygen species, which are potentially deleterious to the stability and integrity of DNA. Beyond the internal cellular production of reactive oxygen species, the burden of free radical attack on cellular DNA is increased through exposure to ionizing radiation (1), cigarette tar and smoke (2), and the recently discovered presence of stable free radicals associated with particulate matter generated by combustion (3).The impact that free radicals have on DNA is varied and profound. For example, single-and double-strand breaks, along with the production of baseless sites in DNA, are a common result of free radical damage to DNA (4). Oxidatively damaged DNA bases also result from the presence of free radicals; the most notable of such bases is 7, 8-dihydro-8-oxoguanine (8-oxoG). This lesion frequently mispairs with adenine, resulting in G:C 3 T:A transversion mutations (5) that are exceedingly common in somatic mutations found in human cancers and, importantly, are abundant in the tumor supp...

Translocation of human ribosomal protein S3 to sites of DNA damage is dependant on ERK-mediated phosphorylation following genotoxic stress

“…coli strain RPC501 was used for overexpressing human GST-S3 [18]. Overnight E. coli cultures bearing human GST-S3 plasmid construct were grown at 37 °C up to A 595 of 0.5 OD and induced with 1 mM Isopropyl-1-thio-β-D-galactopyranoside (IPTG) for 3 h at 27 °C.…”

Ribosomal Protein S3 Negatively Regulates Unwinding Activity of RecQ-like Helicase 4 through Their Physical Interaction

“…After revealing this domain as the major RECQL4 interaction domain responsible for helicase activity inhibition, we studied whether the C-RPS3 is responsible for AP endonuclease activity of human RPS3. This domain was previously reported to have homology with HhH motif of the Escherichia coli AP endonuclease, endonuclease III (24). We assessed the AP endonuclease activity of purified FL-RPS3, N-RPS3, and C-RPS3 on circular double-stranded depurinated substrates using a plasmid nicking assay.…”

“…Together, these data confirmed that the N-terminal 1-320 aa of RECQL4 (N-RECQL4) and the C-terminal 94 -244 aa of RPS3 (C-RPS3) are the main interaction domains. Human N-RECQL4 (1-320 aa) is known to have a Sld2 homologous region (9), whereas the C-RPS3 (94 -244 aa) domain has a putative hairpin-helix-hairpin (HhH) motif known for AP endonuclease activity (24).…”

“…We attribute these differences in the activity of RECQL4 to the variations in substrate type, buffer conditions, purification strategies, and overall quality of final protein fractions. Various extraribosomal functions of nuclear RPS3 have been reported such as its role as an NF-B subunit in apoptosis and as an AP endonuclease in base excision repair (19,24). RPS3 localizes to nucleus, both with or without various stimulations (25,27,31).…”

“…These patients also show increased sensitivity to UV light, which is also characteristics of RTS patients (22,23). Drosophila RPS3 exhibits 8-oxoguanine glycosylase activity along with potent AP endonuclease activity (24). Other accessory roles of RPS3 include acting as a NF-B complex subunit (25) and serving as an assistant for microtubules (26).…”