The DNA Glycosylases Ogg1 and Nth1 Do Not Contribute to Ig Class Switching in Activated Mouse Splenic B Cells

During activation of B cells to undergo class switching, B cell metabolism is increased, and levels of reactive oxygen species (ROS) are increased. ROS can oxidize DNA bases resulting in substrates for the DNA glycosylases Ogg1 and Nth1. Ogg1 and Nth1 excise oxidized bases, and nick the resulting abasic sites, forming single-strand DNA breaks (SSBs) as intermediates during the repair process. In this study, we asked whether splenic B cells from mice deficient in these two enzymes would show altered class switching and decreased DNA breaks in comparison with wild-type mice. As the c-myc gene frequently recombines with the IgH S region in B cells induced to undergo class switching, we also analyzed the effect of deletion of these two glycosylases on DSBs in the c-myc gene. We did not detect a reduction in S region or c-myc DSBs or in class switching in splenic B cells from Ogg1- or Nth1-deficient mice or from mice deficient in both enzymes

Expression of the human DNA glycosylase hSMUG1 in Trypanosoma brucei causes DNA damage and interferes with J biosynthesis

In kinetoplastid flagellates such as Trypanosoma brucei, a small percentage of the thymine residues in the nuclear DNA is replaced by the modified base β-d-glucosyl-hydroxymethyluracil (J), mostly in repetitive sequences like the telomeric GGGTTA repeats. In addition, traces of 5-hydroxymethyluracil (HOMeUra) are present. Previous work has suggested that J is synthesised in two steps via HOMedU as an intermediate, but as J synthesising enzymes have not yet been identified, the biosynthetic pathway remains unclear. To test a model in which HOMeUra functions as a precursor of J, we introduced an inducible gene for the human DNA glycosylase hSMUG1 into bloodstream form T.brucei. In higher eukaryotes SMUG1 excises HOMeUra as part of the base excision repair system. We show that expression of the gene in T.brucei leads to massive DNA damage in J-modified sequences and results in cell cycle arrest and, eventually, death. hSMUG1 also reduces the J content of the trypanosome DNA. This work supports the idea that HOMeUra is a precursor of J, freely accessible to a DNA glycosylase

Ig class switching and proliferation of splenic B cells are not reduced in glycosylase-deficient mice.

<p>(A) Splenic B cell proliferation as assayed by dilution of CFSE assayed ∼44 hrs after treatment to switch to the indicated isotypes. (B) Flow cytometry results showing surface isotype expression (Y-axis) vs CFSE fluorescence (X axis) in cells induced to switch to the indicated isotypes for ∼44 hr. The % of the cells expressing the switched isotype are indicated within the gates. (C) Summaries of the flow cytometry data normalized to WT CSR in each experiment. The numbers of mice used are WT, 5 mice; <i>aid</i>^−/−, 1; <i>ogg1</i>^−/−, 5; <i>nth1</i>^−/− 1, DKO, 2.</p

RT-PCR for Ogg1 and Nth1 mRNA demonstrates that these genes are expressed in activated WT splenic B cells.

<p>Splenic B cells were treated for 2 days as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036061#s4" target="_blank">Methods</a> in order to induce CSR to IgG3, IgG2a, or IgA. The lanes are labeled as to the isotype to which the cells were induced to switch. Lanes labeled “water" have no template; “genomic DNA" is from mouse B cells. The <i>hprt</i> cDNA segment is ∼250 bp, the nth1 segment is 391 bp, and the <i>ogg1</i> segment is 355 bp.</p

LM-PCR assay demonstrates that DSBs in the IgH Sμ region and <i>c-myc</i> gene are not detectably affected by deletion of <i>nth1</i> and/or or <i>ogg1</i> genes in splenic B cells induced to undergo CSR.

<p>The upper panel shows Sμ DSBs and the middle panel shows DSBs in the first intron of the <i>c-myc</i> gene in splenic B cells induced to switch to IgG1 for 2 days. Three-fold dose titrations of DNA were assayed (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036061#s4" target="_blank">Methods</a>). The <i>gapdh</i> gene is amplified (lowest panel) as an internal control for template loading. The amounts used for the <i>gapdh</i> assay correspond to the 3 lowest doses of input DNA that were used for the LM-PCR assays. Similar results were obtained in two other experiments for Sμ and one other for <i>c-myc</i> DSBs.</p

Targeted deletion of the genes encoding NTH1 and NEIL1 DNA N-glycosylases reveals the existence of novel carcinogenic oxidative damage to DNA

We have generated a strain of mice lacking two DNA N-glycosylases of base excision repair (BER), NTH1 and NEIL1, homologs of bacterial Nth (endonuclease three) and Nei (endonuclease eight). Although these enzymes remove several oxidized bases from DNA, they do not remove the well-known carcinogenic oxidation product of guanine: 7,8-dihydro-8-oxoguanine (8-OH-Gua), which is removed by another DNA N-glycosylase, OGG1. The Nth1(-/-)Neil1(-/-) mice developed pulmonary and hepatocellular tumors in much higher incidence than either of the single knockouts, Nth1(-/-) and Neil1(-/-). The pulmonary tumors contained, exclusively, activating GGT -> GAT transitions in codon 12 of K-ras of their DNA. Such transitions contrast sharply with the activating GGT -> GTT transversions in codon 12 of K-ras of the pathologically similar pulmonary tumors, which arose in mice lacking OGG1 and a second DNA N-glycosylase, MUTY. To characterize the biochemical phenotype of the knockout mice, the content of oxidative DNA base damage was analyzed from three tissues isolated from control, single and double knockout mice. The content of 8-OH-Gua was indistinguishable among all genotypes. In contrast, the content of 4,6-diamino-5-formamidopyrimidine (FapyAde) and 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyGua) derived from adenine and guanine, respectively, were increased in some but not all tissues of Neil1(-/-) and Neill-1-Nth1(-/-) mice. The high incidence of tumors in our Nth1(-/-)Neil1(-/-) mice together with the nature of the activating mutation in the K-ras gene of their pulmonary tumors, reveal for the first time, the existence of mutagenic and carcinogenic oxidative damage to DNA which is not 8-OH-Gua. (C) 2009 Elsevier B.V. All rights reserved

Targeted Deletion of mNth1 Reveals a Novel DNA Repair Enzyme Activity

DNA N-glycosylase/AP (apurinic/apyrimidinic) lyase enzymes of the endonuclease III family (nth in Escherichia coli and Nth1 in mammalian organisms) initiate DNA base excision repair of oxidized ring saturated pyrimidine residues. We generated a null mouse (mNth1(−/−)) by gene targeting. After almost 2 years, such mice exhibited no overt abnormalities. Tissues of mNth1(−/−) mice contained an enzymatic activity which cleaved DNA at sites of oxidized thymine residues (thymine glycol [Tg]). The activity was greater when Tg was paired with G than with A. This is in contrast to Nth1, which is more active against Tg:A pairs than Tg:G pairs. We suggest that there is a back-up mammalian repair activity which attacks Tg:G pairs with much greater efficiency than Tg:A pairs. The significance of this activity may relate to repair of oxidized 5-methyl cytosine residues (5meCyt). It was shown previously (S. Zuo, R. J. Boorstein, and G. W. Teebor, Nucleic Acids Res. 23:3239-3243, 1995) that both ionizing radiation and chemical oxidation yielded Tg from 5meCyt residues in DNA. Thus, this previously undescribed, and hence novel, back-up enzyme activity may function to repair oxidized 5meCyt residues in DNA while also being sufficient to compensate for the loss of Nth1 in the mutant mice, thereby explaining the noninformative phenotype