Interactions of human ribosomal protein S3 with intact and damaged DNA

Human S3 (hS3) is a structural component of the ribosome and, in addition to its role in translation, possesses apurinic/apyrimidinic (AP) lyase activity, characteristic of DNA repair enzymes. Recombinant hS3 was isolated from inclusion bodies, refolded under different conditions, and tested for the ability to bind and cleave oligodeoxyribonucleotide substrates with various lesions abundant in genomic DNA: AP sites, uracil, 8-oxoguanine, 8-oxoadenine, 5,6-dihydrouracil, and hypoxanthine. It was found that hS3 is capable of cleaving AP sites via the β-elimination mechanism, producing a Schiff base covalent intermediate, but cannot cleave substrates with the other lesions. Refolding in the presence of Fe²⁺ and S^2? did not increase hS3 activity, suggesting the absence of an iron-sulfur cluster. The binding of hS3 with DNA ligands containing oxidized or deaminated bases was less efficient than with intact DNA. It was assumed that the catalytic activity of hS3 towards AP sites is most likely unimportant for global DNA repair in vivo, but is possibly involved in repairing DNA sites in certain genome regions.     

The high binding affinity of human ribosomal protein S3 to 7,8-dihydro-8-oxoguanine is abrogated by a single amino acid change

Previous studies have shown that human ribosomal protein S3 (hS3) has a high apparent binding affinity for 7,8-dihydro-8-oxoguanine (8-oxoG) residues in DNA and interacts with the human base excision repair (BER) proteins OGG1 and APE/Ref-1. We used a combination of computational and experimental approaches to understand the role of hS3 in BER and its potential to hinder repair of 8-oxoG lesions by OGG1 and APE/Ref-1. Sequence analysis was employed to identify hS3 residues likely to be involved in binding to 8-oxoG. One putative site, lysine 132 (K132), located in a helix-hairpin-helix DNA binding motif, was mutated to alanine (K132A). The hS3-K132A mutant retained the ability to cleave abasic DNA, but its capacity to bind 8-oxoG was abrogated completely. The ability of OGG1 to cleave an 8-oxoG-oligonucleotide substrate pre-incubated with hS3 or hS3-K132A was also tested. Pre-incubations with wild-type hS3 and 8-oxoG-containing oligonucleotides completely prevented the subsequent removal of 8-oxoG by OGG1. On the other hand, OGG1 incubations combined with hS3-K132A stimulated cleavage of 8-oxoG in excess of two-fold, confirming previous observations that hS3 positively interacts with OGG1, but only under conditions in which the binding of hS3 to 8-oxoG is limited. Overall, the ability of OGG1 to repair 8-oxoG is compromised when hS3 is bound to 8-oxoG sites. Conversely, in the absence of DNA binding, hS3 interacts positively with OGG1 to produce a more robust removal of 8-oxoG residues in DNA.     

Human ribosomal protein S3 interacts with DNA base excision repair proteins hAPE/Ref-1 and hOGG1

The human ribosomal protein S3 (hS3) possesses associated activities that suggest alternative roles beyond its participation in protein translation. For example, it is capable of cleaving apurinic/apyrimidinic (AP) DNA via a beta-elimination reaction, an activity that is missing in partially purified extracts of xeroderma pigmentosum group-D fibroblasts. In a recent study, we showed by surface plasmon resonance (SPR) that hS3 also has a very high apparent binding affinity for 7,8-dihydro-8-oxoguanine (8-oxoG) and AP sites in DNA. Using the same SPR technology, it is shown here that hS3 positively interacts with the human base excision repair (BER) enzymes N-glycosylase/AP lyase OGG1 and APE/Ref-1. Using a DNA substrate that allows for the detection of 8-oxoG repair, we also show that hOGG1 N-glycosylase activity becomes increasingly more robust in the presence of hS3. Human S3 was found to co-immunoprecipitate with both hOGG1 and APE/Ref-1, indicating that these proteins physically interact with one another. These results raise the possibility that hS3 not only functions as a ribosomal protein but, in addition, may influence repair activities at sites of DNA damage.     

Characterization of human ribosomal protein S3 binding to 7,8-dihydro-8-oxoguanine and abasic sites by surface plasmon resonance

The human ribosomal protein S3 (hS3) possesses multifunctional activities that are involved in both protein translation, as well as the ability of cleaving apurinic/apyrimidinic (AP) DNA via a beta-elimination reaction. We recently showed that hS3 also has a surprising binding affinity for an 7,8-dihydro-8-oxoguanine (8-oxoG) residue embedded in a 5' end labeled 37mer DNA oligonucleotide. To understand the interaction of hS3 and DNA templates containing 8-oxoG, we carried out real-time analysis using surface plasmon resonance (SPR). Notably, hS3 was found to have an apparent three orders of magnitude higher binding affinity (KD) for 8-oxoG than the human N-glycosylase/AP lyase base excision repair (BER) enzyme OGG1. An even more dramatic five orders of magnitude higher binding affinity for AP DNA was found for hS3 as opposed to hOGG1. These results suggest that ribosomal protein hS3 may have a multifunctional role that may also affect functions associated with DNA base excision repair transactions.     

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

Besides its role in translation and ribosome maturation, human ribosomal protein S3 (hS3) is implicated in DNA damage recognition as reflected by its affinity for abasic sites and 7,8-dihydro-8-oxoguanine (8-oxoG) residues in DNA in vitro. Here, we demonstrate that hS3 is capable of carrying out both roles by its ex vivo translocation from the cytoplasm to the nucleus as a consequence of genotoxic stress. The translocation of hS3 is dependent on ERK1/2-mediated phosphorylation of a threonine residue (T42) of hS3. Two different ectopically expressed site-directed mutants of T42 failed to respond to conditions of genotoxic stress, thus providing a link between DNA damage and ERK1/2 dependent phosphorylation of hS3. Lastly, hS3 was traced in exposed cells to its co-localization with 8-oxoG foci, raising the possibility that hS3 is a member of a cellular DNA damage response pathway that results in its interaction with sites of DNA damage.     

Affinity labeling of flap-endonuclease FEN-1 by photoreactive DNAs

Eukaryotic flap-endonuclease (FEN-1) is 42-kD single-subunit structure-specific nuclease that cleaves 5"-flap strands of the branched DNA structure and possesses 5"-exonuclease activity. FEN-1 participates in DNA replication, repair, and recombination. The interaction of FEN-1 with DNA structures generated during replication and repair was studied using two types of photoreactive oligonucleotides. Oligonucleotides bearing a photoreactive arylazido group at the 3"-end of the primer were synthesized in situ by the action of DNA polymerase using base-substituted photoreactive dUTP analogs as the substrates. The photoreactive group was also bound to the 5"-end phosphate group of the oligonucleotide by chemical synthesis. Interaction of FEN-1 with both 5"- and 3"-ends of the nick or with primer–template systems containing 5"- or 3"-protruding DNA strands was shown. Formation of a structure with the 5"-flap containing the photoreactive group results in decrease of the level of protein labeling caused by cleavage of the photoreactive group due to FEN-1 endonuclease activity. Photoaffinity labeling of proteins of mouse fibroblast cell extract was performed using the radioactively labeled DNA duplex with the photoreactive group at the 3"-end and the apurine/apyrimidine site at the 5"-end of the nick. This structure is a photoreactive analog of an intermediate formed during DNA repair and was generated by the action of cell enzymes from the initial DNA duplex containing the 3-hydroxy-2-hydroxymethyltetrahydrofurane residue. FEN-1 is shown to be one of the photolabeled proteins; this indicates possible participation of this enzyme in base excision repair.     

Knockdown of ribosomal protein S3 protects human cells from genotoxic stress

Human ribosomal protein S3 (hS3) has a high apparent binding affinity for the oxidative lesion 7,8-dihydro-8-oxoguanine (8-oxoG). The hS3 ribosomal protein has also been found to inhibit the base excision repair (BER) enzyme hOGG1 from liberating 8-oxoG residing in a 5'-end-labeled oligonucleotide. To understand the in vivo involvement of hS3 in BER, we have turned to RNA interference to generate knockdown of hS3 in cells exposed to DNA damaging agents. Here we show that a 40% knockdown of hS3 resulted in as much as a seven-fold increase in the 24h survival-rate of HEK293 cells exposed to hydrogen peroxide. Significant protection to the alkylating agent methyl methanesulfonate (MMS) was also observed. Protection to the chemotherapeutic alkylating agent Thio-TEPA was only revealed at longer exposure times where the agent became more toxic to untransfected human cells. Overall, these results raise the possibility that hS3 interferes with the repair of the DNA lesions produced by genotoxic agents that potentially could play a role in the onset of cancer and other pathological states such as aging.     

A Drosophila ribosomal protein contains 8-oxoguanine and abasic site DNA repair activities.

Ionizing radiation and normal cellular respiration form reactive oxygen species that damage DNA and contribute to a variety of human disorders including tumor promotion and carcinogenesis. A major product of free radical DNA damage is the formation of 8-oxoguanine, which is a highly mutagenic base modification produced by oxidative stress. Here, Drosophila ribosomal protein S3 is shown to cleave DNA containing 8-oxoguanine residues efficiently, The ribosomal protein also contains an associated apurinic/apyrimidinic (AP) lyase activity, cleaving phosphodiester bonds via a beta,delta elimination reaction. The significance of this DNA repair activity acting on 8-oxoguanine is shown by the ability of S3 to rescue the H2O2 sensitivity of an Escherichia coli mutM strain (defective for the repair of 8-oxoguanine) and to abolish completely the mutator phenotype of mutM caused by 8-oxoguanine-mediated G-->T transversions. The ribosomal protein is also able to rescue the alkylation sensitivity of an E.coli mutant deficient for the AP endonuclease activities associated with exonuclease III (xth) and endonuclease IV (nfo), indicating for the first time that an AP lyase can represent a significant source of DNA repair activity for the repair of AP sites. These results raise the possibility that DNA repair may be associated with protein translation.     

Characterization of a wide range base-damage-endonuclease activity of mammalian rpS3

Mammalian rpS3, a ribosomal protein S3 with a DNA repair endonuclease activity, nicks heavily UV-irradiated DNA and DNA containing AP sites. RpS3 calls for a novel endonucleolytic activity on AP sites generated from pyrimidine dimers by T4 pyrimidine dimer glycosylase activity. This study revealed that rpS3 cleaves the lesions including AP sites, thymine glycols, and other UV damaged lesions such as pyrimidine dimers. This enzyme does not have a glycosylase activity as predicted from its amino acid sequence. However, it has an endonuclease activity on DNA containing thymine glycol, which is exactly overlapped with UV-irradiated or AP DNAs, indicating that rpS3 cleaves phosphodiester bonds of DNAs containing altered bases with broad specificity acting as a base-damage-endonuclease. RpS3 cleaves supercoiled UV damaged DNA more efficiently than the relaxed counterpart, and the endonuclease activity of rpS3 was inhibited by MgCl2 on AP DNA but not on UV-irradiated DNA.     

Mechanisms of radioresistance in terminally differentiated cells of mature retina

Retinopathy of animals is induced by many DNA-damaging agents. This fact shows that DNA lesions may initiate retinal degeneration. The aim of our work was to study the effects of gamma and proton irradiation and single administration of methylnitrosourea (MNU) on mice retina. We assessed morphological changes, DNA damage and repair, as well as expression of proteins (p53, ATM, PARP, FasR, and caspase 3) participating in apoptosis in retina. 14 Gy was the equitoxic dose for induction of DNA single-strand breaks by both gamma- and proton irradiation. However, protons were twice as effective as γ rays in induction of DNA double-strand breaks. All breaks have been repaired for ≤10 h. Irradiation resulted in increased expression of p53 and ATM. Seven days after irradiation, no signs of cell death and retinal degeneration were observed. Proton irradiation with 25 Gy resulted in destructive changes in retina localized mainly in the photoreceptor layer. These changes were accompanied by enhanced expression of proapoptotic proteins. A single systemic administration of MNU (70 mg/kg) increased intracellular levels of p53, PARP, FasR, and Caspase 3 followed by destructive changes in retina with sings of apoptosis in photoreceptors. Similarly to irradiation, a halved MNU dose did not exhibit a cytotoxic effect on retina. A high level of spontaneous DNA damage at apurine and apyrimidine sites were observed in mouse retina. The results show that there is a genotoxic threshold in initiation of retinal cell death in vivo. It is suggested that topoisomerase 2 translates primary DNA damage into a cytotoxic effect in retina.     

A new binary system for photosensitized labeling of DNA polymerases in nuclear extract

A binary system of reagents was used for photosensitized labeling of proteins of bovine testis nuclear extract. A dUTP analog containing 4-azido-2,5-difluoro-3-chloropyridyl group (FAP-dUTP) was used for the first time as a component of the binary system, and a dUTP analog containing the pyrenyl group (Pyr-dUTP) was used as a photosensitizer. Photoaffinity labeling of proteins of nuclear extract was performed using the radioactively labeled DNA duplex with the photoreactive FAP group at the 3"-end of elongating DNA strand and analog of the deoxyribose phosphate residue (3-hydroxy-2-hydroxymethyltetrahydrofuran (F) 5"-phosphate) at the 5"-end of the nick. Such structure is formed by the action of nuclear extract enzymes from the initial DNA duplex containing a synthetic apurine/apyrimidine site and is a photoreactive analog of a long-patch base excision repair intermediate. UV-irradiation modified a limited number of proteins of the nuclear extract. As shown using specific antibodies, the new binary system of photoreagents increases the efficiency of DNA polymerase labeling.     

Purification and partial characterization of a DNA 3'-phosphatase from Schizosaccharomyces pombe

Cells that depend on oxygen for survival constantly produce reactive oxygen species that attack DNA to produce a variety of lesions, including single-strand breaks with 3'-blocking groups such as 3'-phosphate and 3'-phosphoglycolate. These 3'-blocking ends prevent the activity of DNA polymerase and are generally removed by DNA repair proteins with 3'-diesterase activity. We report here the purification and partial characterization of a 45 kDa protein from Schizosaccharomyces pombe total extract based on the ability of this protein to process bleomycin- or H(2)O(2)-damaged DNA in vitro to allow DNA repair synthesis by DNA polymerase I. Further analysis revealed that the 45 kDa protein removes 3'-phosphate ends created by the Escherichia coli fpg AP lyase following the incision of AP site but is unable to process the 3'-alpha,beta unsaturated aldehyde generated by E. coli endonuclease III. The protein cannot cleave DNA bearing AP sites, suggesting that it is not an AP endonuclease or AP lyase. We conclude that the 45 kDa protein purified from S. pombe is a DNA 3'-phosphatase.     

Cross-linking of 2-deoxyribonolactone and its beta-elimination product by base excision repair enzymes

2-Deoxyribonolactone (3) is produced in DNA as a result of reaction with a variety of DNA damaging agents. The lesion undergoes beta-elimination to form a second metastable electrophilic product (4). In this study, DNA containing 2-deoxyribonolactone (3) and its beta-elimination product (4) are generated at specific sites using a photolabile nucleotide precursor. 2-Deoxyribonolactone is not incised by any of the 8 AP lyases tested. One enzyme, Escherichia coli endonuclease III, cross-links to 3, and the lesion strongly inhibits excision of typical abasic sites by this enzyme. Two of the enzymes, FPG and NEIL1 known to cleave normal abasic sites (1) by effecting beta,delta-elimination form cross-links to the butenolide lesion (4). The observed results are ascribable to characteristics of the enzymes and the lesions. These enzymes are also important for the removal of oxidative base lesions. These results suggest that high concentrations of 3 and 4 may exert significant effects on the repair of normal AP site and oxidative base lesions in cells by reducing the cellular activity of these BER enzymes either via cross-linking or competing with binding to the BER enzymes.     

Functional identification of an 8-oxoguanine specific endonuclease from Thermotoga maritima

To date, no 8-oxoguanine-specific endonuclease-coding gene has been identified in Thermotoga maritima of the order Thermotogales, although its entire genome has been deciphered. However, the hypothetical protein Tm1821 from T. maritima, has a helix-hairpin-helix motif that is considered to be important for DNA binding and catalytic activity. Here, Tm1821 was overexpressed in Escherichia coli and purified using Ni-NTA affinity chromatography, protease digestion, and gel filtration. Tm1821 protein was found to efficiently cleave an oligonucleotide duplex containing 8-oxoguanine, but Tm1821 had little effect on other substrates containing modified bases. Moreover, Tm1821 strongly preferred DNA duplexes containing an 8-oxoguanine:C pair among oligonucleotide duplexes containing 8-oxoguanine paired with four different bases (A, C, G, or T). Furthermore, Tm1821 showed AP lyase activity and Schiff base formation with 8-oxoguanine in the presence of NaBH4, which suggests that it is a bifunctional DNA glycosylase. Tm1821 protein shares unique conserved amino acids and substrate specificity with an 8-oxoguanine DNA glycosylase from the hyperthermophilic archaeon. Thus, the DNA recognition and catalytic mechanisms of Tm1821 protein are likely to be similar to archaeal repair protein, although T. maritima is an eubacterium.     

Yeast ribosomal protein S3 possesses a β-lyase activity on damaged DNA

Yeast ribosomal protein S3 has multifunctional activities that are involved in both protein translation and DNA repair. Here, we report that yeast Rps3p cleaves variously damaged DNA that contains not only AP sites and pyrimidine dimers but also 7,8-hydro-8-oxoguanine. This study also revealed that Rps3p has a β-lyase activity with a broad range of substrate specificity which cleaves phosphodiester bonds of UV or oxidatively damaged DNA substrates. Mutation analysis of the yeast Rps3 protein including introduction of domain deletions and residue replacements identified the residues Asp154 and Lys200 are important for the catalytic activity. In addition, the repair enzyme activity of yeast Rps3p was confirmed by complementation in xth, nfo Escherichia coli cells in which the DNA repair process is defective.     

A general role of the DNA glycosylase Nth1 in the abasic sites cleavage step of base excision repair in Schizosaccharomyces pombe

One of the most frequent lesions formed in cellular DNA are abasic (apurinic/apyrimidinic, AP) sites that are both cytotoxic and mutagenic, and must be removed efficiently to maintain genetic stability. It is generally believed that the repair of AP sites is initiated by the AP endonucleases; however, an alternative pathway seems to prevail in Schizosaccharomyces pombe. A mutant lacking the DNA glycosylase/AP lyase Nth1 is very sensitive to the alkylating agent methyl methanesulfonate (MMS), suggesting a role for Nth1 in base excision repair (BER) of alkylation damage. Here, we have further evaluated the role of Nth1 and the second putative S.pombe AP endonuclease Apn2, in abasic site repair. The deletion of the apn2 open reading frame dramatically increased the sensitivity of the yeast cells to MMS, also demonstrating that the Apn2 has an important function in the BER pathway. The deletion of nth1 in the apn2 mutant strain partially relieves the MMS sensitivity of the apn2 single mutant, indicating that the Apn2 and Nth1 act in the same pathway for the repair of abasic sites. Analysis of the AP site cleavage in whole cell extracts of wild-type and mutant strains showed that the AP lyase activity of Nth1 represents the major AP site incision activity in vitro. Assays with DNA substrates containing base lesions removed by monofunctional DNA glycosylases Udg and MutY showed that Nth1 will also cleave the abasic sites formed by these enzymes and thus act downstream of these enzymes in the BER pathway. We suggest that the main function of Apn2 in BER is to remove the resulting 3′-blocking termini following AP lyase cleavage by Nth1.     

Activation of human MutS homologs by 8-oxo-guanine DNA damage.

The DNA lesion 8-oxo-guanine (8-oxo-G) is a highly mutagenic product of the interaction between reactive oxygen species and DNA. To maintain genomic integrity, cells have evolved mechanisms capable of removing this frequently arising oxidative lesion. Mismatch repair (MMR) appears to be one pathway associated with the repair of 8-oxo-G lesions (DeWeese, T. L., Shipman, J. M., Larrier, N. A., Buckley, N. M., Kidd, L. R., Groopman, J. D., Cutler, R. G., te Riele, H., and Nelson, W. G. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 11915-11920; Ni, T. T., Marsischky, G. T., and Kolodner, R. D. (1999) Mol. Cell 4, 439-444). Here we report the effect of double-stranded DNA oligonucleotides containing a single 8-oxo-G on the DNA binding affinity, ATPase, and ADP right arrow ATP exchange activities of hMSH2-hMSH6 and hMSH2-hMSH3. We found that hMSH2-hMSH6 binds the oligonucleotide DNA substrates with the following affinities: 8-oxo-G/T > 8-oxo-G/G > 8-oxo-G/A > 8-oxo-G/C approximately G/C. A similar trend was observed for DNA-stimulated ATPase and ADP --> ATP exchange activities of hMSH2-hMSH6. In contrast, hMSH2-hMSH3 did not appear to bind any of the 8-oxo-G containing DNA substrates nor was there enhanced ATPase or ADP --> ATP exchange activities. These results suggest that only hMSH2-hMSH6 is activated by recognition of 8-oxo-G lesions. Our data are consistent with the notion that post-replication MMR only participates in the repair of mismatched 8-oxo-G lesions.