8-hydroxyguanosine and 5-hydroxycytosine

Several studies have reported enhanced oxidative stress in patients with HIV infection. An important pathophysiologic consequence of increased oxidative stress is endogenous DNA damage, and the base excision repair pathway is the most important mechanism to withstand such deleterious effects. To investigate the role of base excision repair in HIV infection, we examined 7,8-dihydro-8-oxoguanine (8-oxoG) levels as a marker of oxidative DNA damage and DNA glycosylase activities in CD4(+) and CD8(+) T cells of HIV-infected patients and controls. These results showed that the HIV-infected patients, particularly those with advanced disease, had increased levels of 8-oxoG in CD4(+) T cells and marked declines in DNA glycosylase activity for the repair of oxidative base lesions in these cells. In contrast, CD8(+) T cells from HIV-infected patients, with 8-oxoG levels similar to those in healthy controls, showed enhanced capacity to repair oxidative DNA damage. Finally, highly active antiretroviral therapy induced increased glycosylase activity in CD4(+) T cells and normalized 8-oxoG levels. This imbalance between the accumulation of oxidative DNA damage and the capacity to repair such lesions in CD4(+) T cells may represent a previously unrecognized mechanism involved in the numerical and functional impairment of CD4(+) T cells in patients with HIV infection.

Topics: Adult; Anti-Retroviral Agents; Antiretroviral Therapy, Highly Active; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Nucleus; Cytosine; DNA; DNA Damage; DNA Glycosylases; DNA Repair; Female; Guanosine; HIV; HIV Infections; Humans; Leukocytes, Mononuclear; Male; Middle Aged; Oxidative Stress; Oxygen; T-Lymphocytes

Oxidative damage in testicular DNA is associated with poor semen quality, reduced fertility and increased risk of stillbirths and birth defects. These DNA lesions are predominantly removed by base excision repair. Cellular extracts from human and rat testicular cells and three enriched populations of rat male germ cells (primary spermatocytes, round spermatids and elongating/elongated spermatids) all showed proficient excision/incision of 5-hydroxycytosine, thymine glycol and 2,6-diamino-4-hydroxy-5-formamidopyrimidine. DNA containing 8-oxo-7,8-dihydroguanine was excised poorly by human testicular cell extracts, although 8-oxoguanine-DNA glycosylase-1 (hOGG1) was present in human testicular cells, at levels that varied markedly between 13 individuals. This excision was as low as with human mononuclear blood cell extracts. The level of endonuclease III homologue-1 (NTH1), which excises oxidised pyrimidines, was higher in testicular than in somatic cells of both species. Cellular repair studies of lesions recognised by formamidopyrimidine-DNA glycosylase (Fpg) or endonuclease III (Nth) were assayed with alkaline elution and the Comet assay. Consistent with the enzymatic activities, human testicular cells showed poor removal of Fpg-sensitive lesions but efficient repair of Nth-sensitive lesions. Rat testicular cells efficiently repaired both Fpg- and Nth-sensitive lesions. In conclusion, human testicular cells have limited capacity to repair important oxidative DNA lesions, which could lead to impaired reproduction and de novo mutations.

Topics: Adolescent; Adult; Aged; Animals; Cell Extracts; Cytosine; Deoxyribonuclease (Pyrimidine Dimer); DNA Repair; DNA-Formamidopyrimidine Glycosylase; Endodeoxyribonucleases; Escherichia coli Proteins; Guanosine; Humans; Leukocytes, Mononuclear; Male; Middle Aged; N-Glycosyl Hydrolases; Rats; Rats, Wistar; Testis

DNA polymerase iota (poliota) is a distributive error-prone enzyme that can incorporate nucleotides opposite a variety of DNA lesions. Further elongation is, however, either substantially inhibited or completely abolished. Here, we provide evidence that poliota can facilitate the efficient bypass of uracil and its derivatives as well as oxidized cytosine and guanine residues. The fidelity of translesion replication depends upon the lesion encountered. Correct nucleotides were inserted preferentially opposite 7,8-dihydro-8-oxoguanine (8-oxoG) and 5-hydroxycytosine (5-OHC). However, when bypassing uracil, 5-hydroxyuracil (5-OHU) or 5,6-dihydrouracil (5,6-DHU), poliota inserted T and G with a 4- to 26-fold preference over the Watson-Crick base, A. While the T:U, T:5-OHU and T:5,6-DHU mispairs were extended poorly, the G:U, G:5-OHU and G:5,6-DHU mispairs were extended with equal or greater efficiency than the correctly paired primer termini. Thus, poliota-dependent misinsertion of G opposite uracil and its derivatives may actually provide a mechanism whereby mammalian cells can decrease the mutagenic potential of lesions formed via the deamination of cytosine.

Topics: Animals; Base Pair Mismatch; Base Pairing; Cytosine; DNA Damage; DNA Polymerase iota; DNA Primers; DNA-Directed DNA Polymerase; Guanine; Guanosine; Humans; Kinetics; Models, Chemical; Mutagenesis; Mutagenesis, Site-Directed; Oxygen; Time Factors; Uracil

We recently showed that abasic sites, uracil mismatches, nicks, and gaps can trap DNA topoisomerase I (top1) when these lesions are introduced in the vicinity of a top1 cleavage site (Pourquier, P., Ueng, L.-M., Kohlhagen, G., Mazumder, A., Gupta, M., Kohn, K. W., and Pommier, Y. (1997) J. Biol. Chem. 272, 7792-7796; Pourquier, P., Pilon, A. A., Kohlhagen, G., Mazumder, A., Sharma, A., and Pommier, Y. (1997) J. Biol. Chem. 26441-26447). In this study, we investigated the effects on top1 of an abundant base damage generated by various oxidative stresses: 7,8-dihydro-8-oxoguanine (8-oxoG). Using purified eukaryotic top1 and oligonucleotides containing the 8-oxoG modification, we found a 3-7-fold increase in top1-mediated DNA cleavage when 8-oxoG was present at the +1 or +2 position relative to the cleavage site. Another oxidative lesion, 5-hydroxycytosine, also enhanced top1 cleavage by 2-fold when incorporated at the +1 position of the scissile strand. 8-oxoG at the +1 position enhanced noncovalent top1 DNA binding and had no detectable effect on DNA religation or on the incision step. top1 trapping by 8-oxoG was markedly enhanced when asparagine adjacent to the catalytic tyrosine was mutated to histidine, suggesting a direct interaction between this residue and the DNA major groove immediately downstream from the top1 cleavage site. Altogether, these results demonstrate that oxidative base lesions can increase top1 binding to DNA and induce top1 cleavage complexes.

Topics: Camptothecin; Cytosine; DNA; DNA Damage; DNA Topoisomerases, Type I; DNA-Binding Proteins; Guanosine; Humans; Kinetics; Molecular Structure; Mutation; Oligonucleotides; Oxidative Stress