Elsevier

DNA Repair

Volume 6, Issue 1, 4 January 2007, Pages 94-99
DNA Repair

Knockdown of ribosomal protein S3 protects human cells from genotoxic stress

https://doi.org/10.1016/j.dnarep.2006.09.004Get rights and content

Abstract

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 24 h 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.

Introduction

Reactive oxygen species (ROS) are produced as by-products of mitochondrial oxidative phosphorylation, inflammatory responses, and as a result of environmental exposure to ionizing radiation and a variety of chemicals. A major consequence of ROS formation is the generation of DNA damage that can take on many forms including strand breaks and a variety of purine and pyrimidine DNA base lesions. If left unrepaired, these altered DNA bases are believed to be a contributing factor to cancer and other pathological states including age related syndromes.

DNA modifications produced by ROS are generally repaired via the base excision repair (BER) pathway, in which oxidized bases are removed by a DNA glycosylase. This pathway was originally viewed simplistically as involving the liberation of the modified base by an N-glycosylase, followed by the cleavage of the remaining abasic site by an apurinic/apyrimidinic (AP) endonuclease. However, recent results show that the repair of oxidized damage to DNA is far more complex and involves a number of different N-glycosylases that possess overlapping specificity. The classification of these N-glycosylases have emerged from studies in Escherichia coli, in which their reaction mechanisms are based upon whether the N-glycosylases carry out a subsequent β-elimination reaction, or instead produce a βδ-elimination reaction. These criteria have led to classifying human N-glycosylases as either belonging to the Nth1 β-elimination catalysts, or the Fpg βδ-elimination catalysts [1]. The human Nth1 family consists of OGG1, even though it is mechanistically similar to Fpg in that it removes 8-oxoG. On the other hand, the Fpg human family is represented by NEIL1, which excises a broad range of oxidatively damaged bases including 8-oxoG.

We have previously shown using surface plasmon resonance (SPR) that human ribosomal protein S3 (hS3) has an extraordinarily high binding affinity for 8-oxoG and abasic sites in DNA [2]. Notably, SPR analysis also showed that hS3 can positively interact with the BER proteins OGG1 and APE/ref-1, but only in cases where these proteins were mixed prior to their exposure to DNA [3]. Subsequent experiments using 5′-end-labeled oligonucleotides containing a single 8-oxoG residue (8-oxoG-37mer) supported our previous observations using SPR technology [4]. We showed that hS3 preincubated with 8-oxoG-37mer completely inhibited the removal of 8-oxoG by OGG1. On the other hand, a specific hS3 site-directed mutant that completely abrogated the binding of hS3 to 8-oxoG in fact stimulated OGG1 mediated removal of 8-xoG by over two-fold [4].

This conundrum between hS3 creating an obstacle to the liberation of 8-oxoG as opposed to its positively influencing BER has led us to question what effect hS3 expression has in vivo on human cells, especially in situations where cells are placed under genotoxic stress. To test this, we have used RNA interference technology to determine the outcome of decreased hS3 expression in cells exposed to DNA damaging agents. We show for the first time that in fact a decrease in ribosomal protein hS3 leads to the increased survival of cells exposed to DNA damaging agents.

Section snippets

Cells and cell culture

Human embryonic kidney cell line HEK293 (ATCC) was used in the studies described here. Cells were cultured in DMEM (Sigma) supplemented with 10% fetal bovine serum (FBS, Sigma) and antibiotics (penicillin, 250 IU/ml and streptomycin, 250 μg/ml, Sigma) at 95% humidity/5% CO2 in air at 37 °C.

Human S3 overexpression

The hS3 gene cloned into the pcDNA3.1 vector was obtained from Dr. Mark Kelley (Indiana University School of Medicine, Indianapolis, Indiana). The plasmid DNA was transiently transfected into the HEK293 cells

Construction of siRNAs to knockdown hS3 expression

Four siRNAs were commercially produced (Qiagen) to knockdown hS3 expression. The generated siRNAs were designed towards two different regions proximal to the N-terminus, and two that were directed towards the middle portion of the hS3 gene. These siRNAs were subsequently pooled to see whether they collectively could decrease hS3 mRNA expression in HEK293 cells, and in turn result in a diminished expression of hS3 protein. As seen in Fig. 1A, the pooled siRNAs resulted in a 70% reduction in mRNA

Discussion

The S3 protein has been shown to be remarkably versatile in its ability to influence both ribosomal function and DNA repair transactions. For example, recent studies show that S3 is an integral part of the organization of the pre-40S subunit in yeast [9]. Briefly, S3 was shown to be susceptible to Hrr25 phosphorylation that caused its dissociation from the pre-ribosome, but its dephosphorylation induced integration into the 40S subunit. Depletion of Hrr25 showed the accumulation of immature 40S

Acknowledgements

This work was supported by National Institutes of Health/NCI grant to W.A.D. (CA 109798).

Conflict of interest: None.

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