Browsing by Author "Xu, Yi"
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Item Conversion of the Bifunctional 8-oxoguanine/β-δ AP DNA Repair Activities of Drosophila Ribosomal Protein S3 into the Human S3 Monofunctional β- elimination Catalyst Through a Single Amino Acid Change(2001-07) Hegde, Vijay; Kelley, Mark R.; Xu, Yi; Mian, Saira; Deutsch, Walter A.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 β-elimination reaction that cleaves phosphodiester bonds 3′ and adjacent to an AP lesion in DNA. In certain situations, this is followed by a δ-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 β-elimination reaction, thus further distinguishing itself from the Drosophila S3 protein in lacking a δ-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 δ-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 Drosophilawild-type S3 and its ability to protect a bacterial mutant from the harmful effects of DNA-damaging agents.Item Enhanced mtDNA repair and cellular survival following oxidative stress by targeting the hOGG repair enzyme to mitochondria.(2000-12) Dobson, Allison W.; Xu, Yi; Kelley, Mark R.; LeDoux, Susan P.; Wilson, Glenn L.Oxidative damage to mtDNA has been implicated as a causative factor in many disease processes and in aging. We have recently discovered that different cell types vary in their capacity to repair this damage, and this variability correlates with their ability to withstand oxidative stress. To explore strategies to enhance repair of oxidative lesions in mtDNA, we have constructed a vector containing a mitochondrial transport sequence upstream of the sequence for human 8-oxoguanine glycosylase. This enzyme is the glycosylase/AP lyase that participates in repair of purine lesions, such as 8-oxoguanine. Western blot analysis confirmed this recombinant protein was targeted to mitochondria. Enzyme activity assays showed that mitochondrial extracts from cells transfected with the construct had increased enzyme activity compared to cells transfected with vector only, while nuclear enzyme activity was not changed. Repair assays showed that there was enhanced repair of oxidative lesions in mtDNA. Additional studies revealed that this augmented repair led to enhanced cellular viability as determined by reduction of tetrazolium compound to formazan, Trypan blue dye exclusion, and clonogenic assays. Therefore, targeting of DNA repair enzymes to mitochondria may be a viable approach for the protection of cells against some of the deleterious effects of oxidative stress.Item A novel fluorometric oligonucleotide assay to measure O 6-methylguanine DNA methyltransferase, methylpurine DNA glycosylase, 8-oxoguanine DNA glycosylase and abasic endonuclease activities: DNA repair status in human breast carcinoma cells overexpressing methylpurine DNA glycosylase(2001-04) Kreklau, Emiko L.; Limp-Foster, Melissa; Liu, Naili; Xu, Yi; Kelley, Mark R.; Erickson, Leonard C.DNA repair status plays a major role in mutagenesis, carcinogenesis and resistance to genotoxic agents. Because DNA repair processes involve multiple enzymatic steps, understanding cellular DNA repair status has required several assay procedures. We have developed a novel in vitro assay that allows quantitative measurement of alkylation repair via O6‐methylguanine DNA methyltransferase (MGMT) and base excision repair (BER) involving methylpurine DNA glycosylase (MPG), human 8-oxoguanine DNA glycosylase (hOGG1) and yeast and human abasic endonuclease (APN1 and APE/ref-1, respectively) from a single cell extract. This approach involves preparation of cell extracts in a common buffer in which all of the DNA repair proteins are active and the use of fluorometrically labeled oligonucleotide substrates containing DNA lesions specific to each repair protein. This method enables methylation and BER capacities to be determined rapidly from a small amount of starting sample. In addition, the stability of the fluorometric oligonucleotides precludes the substrate variability caused by continual radiolabeling. In this report this technique was applied to human breast carcinoma MDA-MB231 cells overexpressing human MPG in order to assess whether up-regulation of the initial step in BER alters the activity of selected other BER (hOGG1 and APE/ref-1) or direct reversal (MGMT) repair activities.Item Protection of Human Lung Cells against Hyperoxia Using the DNA Base Excision Repair Genes hOgg1 and Fpg(2002-07) Wu, Min; He, Ying-Hui; Kobune, Masayoshi; Xu, Yi; Kelley, Mark R.; Martin II, William J.Hyperoxia causes pulmonary toxicity in part by injuring alveolar epithelial cells. Previous studies have shown that toxic oxygen-derived species damage DNA and this damage is recognized and repaired by either human enzyme 8-oxoguanine DNA glycosylase (hOgg1) or Escherichia coli enzyme formamidopyrimidine DNA glycosylase (Fpg). To determine whether these DNA repair proteins can reduce O2-mediated DNA damage in lung cells, A549 lung epithelial cells were transduced with either hOgg1 or Fpg using a retroviral vector containing enhanced green fluorescent protein. Expression of each gene in the transduced cells was confirmed by fluorescent microscopy, Northern blotting, Western blotting, and an enzymatic oligonucleotide cleavage assay. A549 cells expressing either hOgg1 or Fpg were protected from hyperoxia as evidenced by a decrease in DNA damage and a corresponding increase in cell survival. Further, we determined that overexpression of hOgg1 or Fpg partially mitigated the toxic effects of hydrogen peroxide in lung cells. Our data suggest that increased expression of DNA base excision repair genes might represent a new approach for protecting critical lung cells from the toxic effects of hyperoxia.Item Protection of pulmonary epithelial cells from oxidative stress by hMYH adenine glycosylase(2004-09) Kremer, Ted M.; Rinne, Mikael L.; Xu, Yi; Chen, Xian Ming; Kelley, Mark R.Background: Oxygen toxicity is a major cause of lung injury. The base excision repair pathway is one of the most important cellular protection mechanisms that responds to oxidative DNA damage. Lesion-specific DNA repair enzymes include hOgg1, hMYH, hNTH and hMTH. Methods: The above lesion-specific DNA repair enzymes were expressed in human alveolar epithelial cells (A549) using the pSF91.1 retroviral vector. Cells were exposed to a 95% oxygen environment, ionizing radiation (IR), or H2O2. Cell growth analysis was performed under non-toxic conditions. Western blot analysis was performed to verify over-expression and assess endogenous expression under toxic and non-toxic conditions. Statistical analysis was performed using the paired Student's t test with significance being accepted for p < 0.05. Results: Cell killing assays demonstrated cells over-expressing hMYH had improved survival to both increased oxygen and IR. Cell growth analysis of A549 cells under non-toxic conditions revealed cells over-expressing hMYH also grow at a slower rate. Western blot analysis demonstrated over-expression of each individual gene and did not result in altered endogenous expression of the others. However, it was observed that O2 toxicity did lead to a reduced endogenous expression of hNTH in A549 cells. Conclusion: Increased expression of the DNA glycosylase repair enzyme hMYH in A549 cells exposed to O2 and IR leads to improvements in cell survival. DNA repair through the base excision repair pathway may provide an alternative way to offset the damaging effects of O2 and its metabolites.Item Testing for Replication Competent Lentivirus Associated with HIV1 Lentiviral Vectors(Elsevier, 2003-05-01) Sastry, Lakshmi; Xu, Yi; Johnson, Terry; Cornetta, Ken; Medical and Molecular Genetics, School of MedicineItem The yeast 8-oxoguanine DNA glycosylase (Ogg1) contains a DNA deoxyribophosphodiesterase (dRpase) activity(1997-10) Sandigursky, Margarita; Yacoub, Adly; Kelley, Mark R.; Xu, Yi; Franklin, William A.; Deutsch, Walter A.The yeast OGG1 gene was recently cloned and shown to encode a protein that possesses N-glycosylase/AP lyase activities for the repair of oxidatively damaged DNA at sites of 7,8-dihydro-8-oxoguanine (8-oxoguanine). Similar activities have been identified for Escherichia coli formamidopyrimidine-DNA glycosylase (Fpg) and Drosophila ribosomal protein S3. Both Fpg and S3 also contain a deoxyribophosphodiesterase (dRpase) activity that removes 2-deoxyribose-5-phosphate at an incised 5′ apurinic/apyrimidinic (AP) sites via a β-elimination reaction. Drosophila S3 also has an additional activity that removes trans-4-hydroxy-2-pentenal-5-phosphate at a 3′ incised AP site by a Mg2+-dependent hydrolytic mechanism. In view of the substrate similarities between Ogg1, Fpg and S3 at the level of base excision repair, we examined whether Ogg1 also contains dRpase activities. A glutathione S-transferase fusion protein of Ogg1 was purified and subsequently found to efficiently remove sugar-phosphate residues at incised 5′ AP sites. Activity was also detected for the Mg2+-dependent removal of trans-4-hydroxy-2-pentenal-5-phosphate at 3′ incised AP sites and from intact AP sites. Previous studies have shown that DNA repair proteins that possess AP lyase activity leave an inefficient DNA terminus for subsequent DNA synthesis steps associated with base excision repair. However, the results presented here suggest that in the presence of MgCl2, Ogg1 can efficiently process 8-oxoguanine so as to leave a one nucleotide gap that can be readily filled in by a DNA polymerase, and importantly, does not therefore require additional enzymes to process trans-4-hydroxy-2-pentenal-5-phosphate left at a 3′ terminus created by a β-elimination catalyst.Item Yeast apurinic/apyrimidinic endonuclease Apn1 protects mammalian neuronal cell line from oxidative stress(2007-07) Ho, Renee; Rachek, Lyudmila I.; Xu, Yi; Kelley, Mark R.; LeDoux, Susan P.; Wilson, Glenn L.Reactive oxygen species (ROS) have been implicated as one of the agents responsible for many neurodegenerative diseases. A critical target for ROS is DNA. Most oxidative stress-induced DNA damage in the nucleus and mitochondria is removed by the base excision repair pathway. Apn1 is a yeast enzyme in this pathway which possesses a wider substrate specificity and greater enzyme activity than its mammalian counterpart for removing DNA damage, making it a good therapeutic candidate. For this study we targeted Apn1 to mitochondria in a neuronal cell line derived from the substantia nigra by using a mitochondrial targeting signal (MTS) in an effort to hasten the removal of DNA damage and thereby protect these cells. We found that following oxidative stress, mitochondrial DNA (mtDNA) was repaired more efficiently in cells containing Apn1 with the MTS than controls. There was no difference in nuclear repair. However, cells that expressed Apn1 without the MTS showed enhanced repair of both nuclear and mtDNA. Both Apn1-expressing cells were more resistant to cell death following oxidative stress compared with controls. Therefore, these results reveal that the expression of Apn1 in neurons may be of potential therapeutic benefit for treating patients with specific neurodegenerative diseases.