Faculty Bibliography

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.

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 Neil1(-/-)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

Oxidation of thymine yields 5,6-dihydroxy-5,6-dihydrothymine (thymine glycol. Tg) which, as cis 5S,6R and 5R,6S 2'-deoxyribonucleoside diastereoisomers (dTg1, dTg2), are in equilibrium with their trans 5S,6S and 5R,6R epimers. The stereoselective excision of Tg from DNA by the mammalian orthologs of E. coli DNA N-glycosylase/AP lyases Nth and Nei was reported using substrates in which Tg opposed adenine. Since we showed that Tg is the major product of oxidation of 5-methylcytosine, we asked if the opposing purine influenced stereospecific enzymatic excision. The human ortholog hNth1 released Tg2 much more rapidly than Tg1 regardless of the opposing purine. In contrast, hNeil1 released Tg non-stereoselectively, but the rate of excision was much greater when Tg opposed guanine. Remarkably, the kinetics of excision of Tg by hNth1 and hNeil1 were biphasic, describing a double exponential curve which yielded two rate constants. We suggest that the greater rate constant describes the rate of enzymatic excision of Tg. The smaller rate constant represents the equilibrium constant for the cis and trans epimerization of dTg1 and dTg2 in high molecular weight DNA. Thus, only one of the epimers of dTg1 and dTg2 are enzymatically processed but it is not yet known whether it is cis or trans. Thus, base excision repair of Tg in mammals is mediated by at least two DNA N-glycosylase/AP lyases which are affected by the nature of the diastereoisomer of dTg, the rate of cis-trans epimerization of each diastereoisomer, and the nature of the opposing purine

DNA polymerase lambda (pol lambda) is a member of the X family of DNA polymerases that has been implicated in both base excision repair and non-homologous end joining through in vitro studies. However, to date, no phenotype has been associated with cells deficient in this DNA polymerase. Here we show that pol lambda null mouse fibroblasts are hypersensitive to oxidative DNA damaging agents, suggesting a role of pol lambda in protection of cells against the cytotoxic effects of oxidized DNA. Additionally, pol lambda co-immunoprecipitates with an oxidized base DNA glycosylase, single-strand-selective monofunctional uracil-DNA glycosylase (SMUG1), and localizes to oxidative DNA lesions in situ. From these data, we conclude that pol lambda protects cells against oxidative stress and suggest that it participates in oxidative DNA damage base excision repair

Human apurinic/apyrimidinic endonuclease (APE1) is an enzyme of DNA base excision repair (BER) which catalyzes endonucleolytic cleavage immediately 5' to abasic (AP) sites. APE1 has long been thought to act on AP sites only in double stranded (ds) DNA, in order to generate the appropriate site for insertion of the correct nucleotide of DNA repair synthesis effected by DNA polymerase beta. We now present evidence that APE1 also acts on AP sites in single-stranded (ss) DNA. The catalytic efficiency of this activity (defined within as k(cat)/Km) is approximately 20-fold less than the activity against AP sites in ds DNA, with the disparity stemming largely from a difference in Km. Similar to its action on AP sites in ds DNA, catalysis of endonucleolytic cleavage of ss DNA by APE1 is Mg(2+) dependent, DNA N-glycosylase independent, and requires an active site aspartate. In contrast to its activity against AP sites in ds DNA, APE1 does not display product inhibition when acting on an AP site in ss DNA. We suggest that this novel activity is related to the processing of DNA N-glycosylase initiated BER in ss DNA perhaps during replication and/or transcription

Base excision repair of oxidized pyrimidines in human DNA is initiated by the DNA N-glycosylase/apurinic/apyrimidinic (AP) lyase, human NTH1 (hNTH1), the homolog of Escherichia coli endonuclease III (Nth). In contrast to Nth, the DNA N-glycosylase activity of hNTH1 is 7-fold greater than its AP lyase activity when the DNA substrate contains a thymine glycol (Tg) opposite adenine (Tg:A) (Marenstein, D. R., Ocampo, M. T. A., Chan, M. K., Altamirano, A., Basu, A. K., Boorstein, R. J., Cunningham, R. P., and Teebor, G. W. (2001) J. Biol. Chem. 276, 21242-21249). When Tg is opposite guanine (Tg:G), the two activities are of the same specific activity as the AP lyase activity of hNTH1 against Tg:A (Ocampo, M. T. A., Chaung, W., Marenstein, D. R., Chan, M. K., Altamirano, A., Basu, A. K., Boorstein, R. J., Cunningham, R. P., and Teebor, G. W. (2002) Mol. Cell. Biol. 22, 6111-6121). We demonstrate here that hNTH1 was inhibited by the product of its DNA N-glycosylase activity directed against Tg:G, the AP:G site. In contrast, hNTH1 was not as inhibited by the AP:A site arising from release of Tg from Tg:A. Addition of human APE1 (AP endonuclease-1) increased dissociation of hNTH1 from the DNA N-glycosylase-generated AP:A site, resulting in abrogation of AP lyase activity and an increase in turnover of the DNA N-glycosylase activity of hNTH1. Addition of APE1 did not abrogate hNTH1 AP lyase activity against Tg:G. The stimulatory protein YB-1 (Marenstein et al.), added to APE1, resulted in an additive increase in both activities of hNTH1 regardless of base pairing. Tg:A is formed by oxidative attack on thymine opposite adenine. Tg:G is formed by oxidative attack on 5-methylcytosine opposite guanine (Zuo, S., Boorstein, R. J., and Teebor, G. W. (1995) Nucleic Acids Res. 23, 3239-3243). It is possible that the in vitro substrate selectivity of mammalian NTH1 and the concomitant selective stimulation of activity by APE1 are indicative of selective repair of oxidative damage in different regions of the genome

In kinetoplastid flagellates such as Trypanosoma brucei, a small percentage of the thymine residues in the nuclear DNA is replaced by the modified base beta-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

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

Purification from calf thymus of a DNA N-glycosylase activity (HMUDG) that released 5-hydroxymethyluracil (5hmUra) from the DNA of Bacillus subtilis phage SPO1 was undertaken. Analysis of the most purified fraction by SDS-polyacrylamide gel electrophoresis revealed a multiplicity of protein species making it impossible to identify HMUDG by inspection. Therefore, we renatured the enzyme after SDS-polyacrylamide gel electrophoresis and assayed slices of the gel for DNA N-glycosylase activity directed against 5hmUra. Maximum enzymatic activity was identified between molecular mass markers 30 and 34 kDa. Protein was extracted from gel slices and subjected to tryptic digestion and analysis by mass spectrometry. Analysis revealed the presence of 11 peptides that were homologous or identical to the sequence of the recently characterized human single-stranded monofunctional uracil DNA N-glycosylase (hSMUG1). The cDNA of hSMUG1 was isolated and expressed as a recombinant glutathione S-transferase fusion protein that was shown to release 5hmUra with 20x the specific activity of the most purified bovine fraction. We conclude that hSMUG1 and HMUDG are the same protein

Human endonuclease III (hNth1) is a DNA glycosylase/apurinic/apyrimidinic (AP) lyase that initiates base excision repair of pyrimidines modified by reactive oxygen species, ionizing, and ultraviolet radiation. Using duplex 2'-deoxyribose oligonucleotides containing an abasic (AP) site, a thymine glycol, or a 5-hydroxyuracil residue as substrates, we found the AP lyase activity of hNth1 was 7 times slower than its DNA glycosylase activity, similar to results reported for murine and human 8-oxoguanine-DNA glycosylase, which are also members of the endonuclease III family. This difference in rates contrasts with the equality of rates found in Escherichia coli and Saccharomyces cerevisiae endonuclease III homologs. A yeast two-hybrid screen for potential modulators of hNth1 activity revealed interaction with the damage-inducible transcription factor Y box-binding protein 1 (YB-1), also identified as DNA-binding protein B (DbpB). The in vitro addition of His(6)YB-1 to hNth1 increased the rate of DNA glycosylase and AP lyase activity. Analysis revealed that YB-1 affects the steady state equilibrium between the covalent hNth1-AP site Schiff base ES intermediate and the noncovalent ES intermediate containing the AP aldehydic sugar and the epsilon-amino group of the hNth1 active site lysine. This equilibrium may be a checkpoint in modulating hNth1 activity