Investigation of discordant sibling pairs from hereditary breast cancer families and analysis of a rare PMS1 variant.

BACKGROUND: It is likely that additional genes for hereditary breast cancer can be identified using a discordant sib pair design. Using this design we identified individuals harboring a rare PMS1 c.605G>A variant previously predicted to result in loss of function. OBJECTIVES: A family-based design and predictive algorithms were used to prioritize candidate variants possibly associated with an increased risk of hereditary breast cancer. Functional analyses were performed for one of the candidate variants, PMS1 c.605G>A. METHODS: 1) 14 discordant sister-pairs from hereditary breast cancer families were identified. 2) Whole exome sequencing was performed and candidate risk variants identified. 3) A rare PMS variant was identified in 2 unrelated affected sisters but no unaffected siblings. 4) Functional analysis of this variant was carried out using targeted mRNA sequencing. RESULTS: Genotype-phenotype correlation did not demonstrate tracking of the variant with cancer in the family. Functional analysis revealed no difference in exon 6 incorporation, which was validated by analyzing PMS1 allele specific expression. CONCLUSIONS: The PMS1 c.605G>A variant did not segregate with disease, and there was no variant-dependent impact on PMS1 exon 6 splicing, supporting this variant is likely benign. Functional analyses are imperative to understanding the clinical significance of predictive algorithms.

Human cells contain a factor that facilitates the DNA glycosylase-mediated excision of oxidized bases from occluded sites in nucleosomes.

Reactive oxygen species generate some 20,000 base lesions per human cell per day. The vast majority of these potentially mutagenic or cytotoxic lesions are subject to base excision repair (BER). Although chromatin remodelers have been shown to enhance the excision of oxidized bases from nucleosomes in vitro, it is not clear that they are recruited to and act at sites of BER in vivo. To test the hypothesis that cells possess factors that enhance BER in chromatin, we assessed the capacity of nuclear extracts from human cells to excise thymine glycol (Tg) lesions from exogenously added, model nucleosomes. The DNA glycosylase NTHL1 in these extracts was able to excise Tg from both naked DNA and sites in nucleosomes that earlier studies had shown to be sterically accessible. However, the same extracts were able to excise lesions from sterically-occluded sites in nucleosomes only after the addition of Mg2+/ATP. Gel mobility shift assays indicated that nucleosomes remain largely intact following the Mg2+/ATP -dependent excision reaction. Size exclusion chromatography indicated that the NTHL1-stimulating activity has a relatively low molecular weight, close to that of NTHL1 and other BER glycosylases; column fractions that contained the very large chromatin remodeling complexes did not exhibit this same stimulatory activity. These results indicate that cells possess a factor(s) that promotes the initiation of BER in chromatin, but differs from most known chromatin remodeling complexes.

Periprosthetic fractures of the distal femur after total knee arthroplasty : Plate versus nail fixation.

The incidence of periprosthetic fractures about a TKA is increasing. Traditionally, these fractures are classified by their location and prosthesis integrity. In the setting of a supracondyar fracture about a well-fixed prosthesis, both plate and nail fixation of the fracture present themselves as options, each with unique benefits and pitfalls. Through review and discussion of the literature, we aim to describe some of the patient, fracture, and implant related factors that should be considered when planning fixation of periprosthetic fractures about a TKA. Additionally, we present several technical pearls that may be useful in the successful treatment of these difficult injuries. LEVEL OF EVIDENCE: 4.

Expression of the oxidative base excision repair enzymes is not induced in TK6 human lymphoblastoid cells after low doses of ionizing radiation.

Most of the DNA damage produced by ionizing radiation is repaired by the base excision repair (BER) pathway. To determine whether the BER genes were up-regulated by low doses of ionizing radiation, we investigated their expression in TK6 human lymphoblastoid cells by measuring mRNA levels using real-time quantitative PCR. No induction at the transcriptional level of any of the base excision repair genes, NTH1 (NTHL1), OGG1, NEIL1, NEIL2, NEIL3, APE1, POLB, or accessory protein genes, LIG3, XRCC1 or XPG, was found at gamma-radiation doses ranging from 1 cGy to 2 Gy in a 24-h period. As has been measured in other cell lines, a dose-dependent induction of CDKN1A (WAF1) mRNA levels was observed in TK6 cells in the dose range of 0.5 to 2.0 Gy. We also examined BER enzyme activity on 8-oxoguanine-, dihydrouracil- and furan-containing oligonucleotide substrates and found no increase in extracts of TK6 cells after gamma-ray doses of 0.5-2.0 Gy. These data were corroborated by Western blot analysis of APE1 and NTH1, suggesting that the BER enzymes are also not up-regulated at the post-transcriptional level after ionizing radiation exposure.

Abortive base-excision repair of radiation-induced clustered DNA lesions in Escherichia coli.

It has been postulated that ionizing radiation produces a unique form of cellular DNA damage called "clustered damages" or "multiply damaged sites". Here, we show that clustered DNA damages are indeed formed in Escherichia coli by ionizing radiation and are converted to lethal double-strand breaks during attempted base-excision repair. In wild-type cells possessing the oxidative DNA glycosylases that cleave DNA at repairable single damages, double-strand breaks are formed at radiation-induced clusters during postirradiation incubation and also in a dose-dependent fashion. E. coli mutants lacking these enzymes do not form double-strand breaks postirradiation and are substantially more radioresistant than wild-type cells. Furthermore, overproduction of one of the oxidative DNA glycosylases in mutant cells confers a radiosensitive phenotype and an increase in the number of double-strand breaks. Thus, the effect of the oxidative DNA glycosylases in potentiating DNA damage must be considered when estimating radiation risk.

Inhibition of oxidative DNA repair in cadmium-adapted alveolar epithelial cells and the potential involvement of metallothionein.

This study evaluated the effects of cadmium (Cd) adaptation in cultured alveolar epithelial cells on oxidant-induced DNA damage and its subsequent repair. Using the comet assay, we determined that lower levels of DNA damage occurred in Cd-adapted cells compared with non-adapted cells following treatment of cells with hydrogen peroxide (H(2)O(2)). This may be a consequence of increased thiol-containing antioxidants that were observed in adapted cells, including metallothionein and glutathione. Cd-adapted cells were, however, less efficient at repairing total oxidative DNA damage compared with non-adapted cells. Subsequently, we investigated the effect of Cd adaptation on the repair of particular oxidized DNA lesions by employing lesion-specific enzymes in the comet assay, namely formamidopyrimidine DNA glycosylase (Fpg), an enzyme that predominantly repairs 8-oxoguanine (8-oxoG), and endonuclease III, that is capable of repairing oxidized pyrimidines. The data demonstrated that adaptation to Cd results in significantly impaired repair of both Fpg- and endonuclease III-sensitive lesions. In addition, in situ detection of 8-oxoG using a recombinant monoclonal antibody showed that Cd-adaptation reduces the repair of this oxidative lesion after exposure of cells to H(2)O(2). Activities of 8-oxoG-DNA glycosylase and endonuclease III were determined in whole cell extracts using 32P-labeled synthetic oligonucleotides containing 8-oxoG and dihydrouracil sites, respectively. Cd adaptation was associated with an inhibition of 8-oxoG-DNA glycosylase and endonuclease III enzyme activity compared with non-adapted cells. In summary, this study has shown that Cd adaptation: (1) reduces oxidant-induced DNA damage; (2) increases the levels of key intracellular antioxidants; (3) inhibits the repair of oxidative DNA damage.

Base excision repair processing of radiation-induced clustered DNA lesions.

Energy from low LET ionising radiation, such as X rays and gamma rays, is deposited in the water surrounding the DNA molecule such that between 2 to 5 radical pairs are generated within a radius of I to 4 nm. As a result, multiple single lesions, including oxidised purine or pyrimidine bases, sites of base loss, and single-strand breaks, can be formed in DNA from the same radiation energy deposition event. The single lesions in these so-called multiply damaged sites or clustered lesions are repaired by base excision repair. Here we show that clustered DNA damages are formed in bacterial cells by ionising radiation and are converted to lethal double-strand breaks during attempted repair. In wild type cells possessing the oxidative DNA glycosylases that recognise and cleave DNA at repairable single damages, double-strand breaks are formed at radiation-induced clusters during post-irradiation incubation and in a dose-dependent fashion. Mutant cells lacking these enzymes do not form double-strand breaks post-irradiation and are substantially more radioresistant than wild type cells. These radioresistant mutant cells can be made radiosensitive by overexpressing one of the oxidative DNA glycosylases. Thus the effect of the oxidative DNA glycosylases in potentiating DNA damage must be considered when estimating radiation risk.

Spectroscopic studies of zinc(II)- and cobalt(II)-associated Escherichia coli formamidopyrimidine-DNA glycosylase: extended X-ray absorption fine structure evidence for a metal-binding domain.

Formamidopyrimidine-DNA glycosylase (Fpg) is a 30.2 kDa protein that plays an important role in the base excision repair of oxidatively damaged DNA in Escherichia coli. Sequence analysis and genetic evidence suggest that zinc is associated with a C4-type motif, C(244)-X(2)-C(247)-X(16)-C(264)-X(2)-C(267), located at the C-terminus of the protein. The zinc-associated motif has been shown to be essential for damaged DNA recognition. Extended X-ray absorption fine structure (EXAFS) spectra collected on the zinc-associated protein (ZnFpg) in the lyophilized state and in 10% frozen aqueous glycerol solution show directly that the metal is coordinated to the sulfur atom of four cysteine residues. The average Zn-S bond length is 2.33 +/- 0.01 and 2.34 +/- 0.01 A, respectively, in the lyophilized state and in 10% frozen aqueous glycerol solution. Fpg was also expressed in minimal medium supplemented with cobalt nitrate to yield a blue-colored protein that was primarily cobalt-associated (CoFpg). The profiles of the circular dichroism spectra for CoFpg and ZnFpg are identical, suggesting that the substitution of Co(2+) for Zn(2+) does not alter the structure of Fpg. A similar conclusion is reached upon the analysis of two-dimensional (15)N/(1)H HSQC spectra of uniformly (15)N-labeled samples of ZnFpg and CoFpg; the spectra are similar and display features characteristic of a structured protein. Biochemical assays with a 54 nt DNA oligomer containing 7, 8-dihydro-8-oxoguanine at a specific location show that CoFpg and ZnFpg are equally active at cleaving the DNA at the site of the oxidized guanine. EXAFS spectra of CoFpg indicate that the cobalt is coordinated to the sulfur atom of four cysteine residues with an average Co-S bond length of 2.28 +/- 0.01 and 2.29 +/- 0.01 A, respectively, in the lyophilized state and in 10% frozen aqueous glycerol solution. The structural similarity between CoFpg and ZnFpg suggests that it is biologically relevant to use the paramagnetic properties of Co(2+) as a structural probe.

Multiprobe RNase protection assay analysis of mRNA levels for the Escherichia coli oxidative DNA glycosylase genes under conditions of oxidative stress.

Escherichia coli formamidopyrimidine DNA glycosylase (Fpg), MutY DNA glycosylase, endonuclease VIII, and endonuclease III are oxidative base excision repair DNA glycosylases that remove oxidized bases from DNA, or an incorrect base paired with an oxidized base in the case of MutY. Since genes encoding other base excision repair proteins have been shown to be part of adaptive responses in E. coli, we wanted to determine whether the oxidative DNA glycosylase genes are induced in response to conditions that cause the type of damage their encoded proteins remove. The genes fpg, mutY, nei, and nth encode Fpg, MutY, endonuclease VIII, and endonuclease III, respectively. Multiprobe RNase protection assays were used to examine the transcript levels of these genes under conditions that induce the SoxRS, OxyR, and SOS regulons after a shift from anaerobic to aerobic growth and at different stages along the growth curve. Transcript levels for all four genes decreased as cells progressed from log-phase growth to stationary phase and increased after cells were shifted from anaerobic to aerobic growth. None of the genes were induced by hydrogen peroxide, paraquat, X rays, or conditions that induce the SOS response.

The effect of inter-implant distance on the height of inter-implant bone crest.

BACKGROUND: The biologic width around implants has been well documented in the literature. Once an implant is uncovered, vertical bone loss of 1.5 to 2 mm is evidenced apical to the newly established implant-abutment interface. The purpose of this study was to evaluate the lateral dimension of the bone loss at the implant-abutment interface and to determine if this lateral dimension has an effect on the height of the crest of bone between adjacent implants separated by different distances. METHODS: Radiographic measurements were taken in 36 patients who had 2 adjacent implants present. Lateral bone loss was measured from the crest of bone to the implant surface. In addition, the crestal bone loss was also measured from a line drawn between the tops of the adjacent implants. The data were divided into 2 groups, based on the inter-implant distance at the implant shoulder. RESULTS: The results demonstrated that the lateral bone loss was 1.34 mm from the mesial implant shoulder and 1.40 mm from the distal implant shoulder between the adjacent implants. In addition, the crestal bone loss for implants with a greater than 3 mm distance between them was 0.45 mm, while the implants that had a distance of 3 mm or less between them had a crestal bone loss of 1.04 mm. CONCLUSIONS: This study demonstrates that there is a lateral component to the bone loss around implants in addition to the more commonly discussed vertical component. The clinical significance of this phenomenon is that the increased crestal bone loss would result in an increase in the distance between the base of the contact point of the adjacent crowns and the crest of bone. This could determine whether the papilla was present or absent between 2 implants as has previously been reported between 2 teeth. Selective utilization of implants with a smaller diameter at the implant-abutment interface may be beneficial when multiple implants are to be placed in the esthetic zone so that a minimum of 3 mm of bone can be retained between them at the implant-abutment level.

Fluorescence detection of 8-oxoguanine in nuclear and mitochondrial DNA of cultured cells using a recombinant Fab and confocal scanning laser microscopy.

The presence of 8-oxoguanine (8-oxoG) in DNA is considered a marker of oxidative stress and DNA damage. We describe a multifluorescence technique to detect the localization of 8-oxoG in both nuclear and mitochondrial DNA using a mouse recombinant Fab 166. The Fab was generated by repertoire cloning and combinatorial phage display, and specifically recognized 8-oxoG in DNA, as determined by competitive enzyme-linked immunosorbent assays (ELISAs). In situ detection of 8-oxoG was accomplished using rat lung epithelial (RLE) cells and human B lymphoblastoid (TK6) cells treated with hydrogen peroxide (H(2)O(2)) or ionizing radiation, respectively. Using confocal scanning laser microscopy, we observed nuclear and perinuclear immunoreactivity of 8-oxoG in control cultures. The simultaneous use of a nuclear DNA stain, propidium iodide, or the mitochondrial dye, MitoTracker (Molecular Probes, Eugene, OR, USA), confirmed that 8-oxoG immunofluorescence occurred in nuclear and mitochondrial DNA. Marked increases in the presence of 8-oxoG in nuclear DNA were apparent after treatment with H(2)O(2) or ionizing radiation. In control experiments, Fab 166 was incubated with 200 microM purified 8-oxodG or with formamidopyrimidine DNA-glycosylase (Fpg) to remove 8-oxoG lesions in DNA. These protocols attenuated both nuclear and mitochondrial staining. We conclude that both nuclear and mitochondrial oxidative DNA damages can be simultaneously detected in situ using immunofluorescence labeling with Fab 166 and confocal microscopy.

The genes encoding endonuclease VIII and endonuclease III in Escherichia coli are transcribed as the terminal genes in operons.

Escherichia coli endonuclease VIII and endo-nuclease III are oxidative base excision repair DNA glycosylases that remove oxidized pyrimidines from DNA. The genes encoding these proteins, nei and nth, are both co-transcribed as the terminal genes in operons. nei is the terminal gene in an operon with four open reading frames that encode proteins of unknown function. This operon has two confirmed transcription initiation sites upstream of the first open reading frame and two transcript termination sites downstream of nei. nth is the terminal gene in an operon with seven open reading frames that encode proteins of unknown function. The six open reading frames immediately upstream of nth show homology to the genes rnfA, rnfB, rnfC, rnfD, rnfG and rnfE from Rhodobacter capsulatis. The rnf genes are required for nitrogen fixation in R.capsulatis and have been predicted to make up a membrane complex involved in electron transport to nitrogenase. The nth operon has transcription initiation sites upstream of the first and second open reading frames and a single transcript termination site downstream of nth. The order of genes in these operons has been conserved or partially conserved in other bacteria, although it is not known whether the genes are co-transcribed in these other organisms.

Histologic and clinical comparison of bilateral sinus floor elevations with and without barrier membrane placement in 12 patients: Part 3 of an ongoing prospective study.

In 1993 the Department of Implant Dentistry at New York University College of Dentistry began a long-term clinical, histologic, histomorphometric, and radiographic study of the sinus elevation procedure. One of the parameters under evaluation in this study is the effect of barrier membrane placement on the creation of vital bone in the grafted sinus cavity. This report presents a histologic and histomorphometric evaluation of healing with and without the placement of an expanded polytetrafluoroethylene (e-PTFE) barrier membrane over the lateral window at the time of sinus grafting. The data were collected from 12 patients who underwent bilateral sinus elevation surgery. In each of these 12 patients the same grafting material was used in both sinuses, making the presence or absence of an e-PTFE barrier membrane the only controlled variable. Under the conditions of this study, the results indicate that (1) placement of the barrier membrane tends to increase vital bone formation; (2) placement of a barrier membrane has a positive effect on implant survival; and (3) membrane placement should be considered for all sinus elevation procedures.

Human histologic and histomorphometric analysis comparing OsteoGraf/N with PepGen P-15 in the maxillary sinus elevation procedure: a case report.

The use of the anorganic bovine bone mineral OsteoGraf/N combined with demineralized freeze-dried bone allograft has received widespread use in sinus elevations. This composite graft material has proven to be suitable, predictable, and successful for the placement and integration of endosseous implants in the edentulous, atrophic maxilla. In this case study, the current materials and accepted methodology were compared with the latest tissue-engineered bone replacement graft material, PepGen P-15. PepGen P-15 is a combination of OsteoGraf/N and a synthetic peptide (P-15) that mimics the cell-binding domain of Type-I collagen responsible for cell migration, differentiation, and proliferation. The radiographic, histologic, and histomorphometric evaluations of the sinus grafted with PepGen P-15 showed enhanced bone formation within a shorter time interval compared with the composite graft material of OsteoGraf/N and demineralized freeze-dried bone allograft.

Fabs specific for 8-oxoguanine: control of DNA binding.

Free radicals produce a broad spectrum of DNA base modifications including 7,8-dihydro-8-oxoguanine (8-oxoG). Since free radicals have been implicated in many pathologies and in aging, 8-oxoG has become a benchmark for factors that influence free radical production. Fab g37 is a monoclonal antibody that was isolated by phage display in an effort to create a reagent for detecting 8-oxoG in DNA. Although this antibody exhibited a high degree of specificity for the 8-oxoG base, it did not appear to recognize 8-oxoG when present in DNA. Fab g37 was modified using HCDR1 and HCDR2 segment shuffling and light chain shuffling. Fab 166 and Fab 366 which bound to 8-oxoG in single-stranded DNA were isolated. Fab 166 binds more selectively to single-stranded oligonucleotides containing 8-oxoG versus control oligonucleotides than does Fab 366 which binds DNA with reduced dependency on 8-oxoG. Numerous other clones were also isolated and characterized that contained a spectrum of specificities for 8-oxoG and for DNA. Analysis of the primary sequences of these clones and comparison with their binding properties suggested the importance of different complementarity determining regions and residues in determining the observed binding phenotypes. Subsequent chain shuffling experiments demonstrated that mutation of SerH53 to ArgH53 in the Fab g37 heavy chain slightly decreased the Fab's affinity for 8-oxoG but significantly improved its binding to DNA in an 8-oxoG-dependent manner. The light chain shuffling experiments also demonstrated that numerous promiscuous light chains could enhance DNA binding when paired with either the Fab g37 or Fab 166 heavy chains; however, only the Fab 166 light chain did so in an additive manner when combined with the Fab 166 heavy chain that contains ArgH53. A three-point model for Fab 166 binding to oligonucleotides containing 8-oxoG is proposed. We describe a successful attempt to generate a desired antibody specificity, which was not present in the animal's original immune response.

A novel role for Escherichia coli endonuclease VIII in prevention of spontaneous G-->T transversions.

In the bacterium Escherichia coli, oxidized pyrimidines are removed by two DNA glycosylases, endonuclease III and endonuclease VIII (endo VIII), encoded by the nth and nei genes, respectively. Double mutants lacking both of these activities exhibit a high spontaneous mutation frequency, and here we show that all of the mutations observed in the double mutants were G:C-->A:T transitions; no thymine mutations were found. These findings are in agreement with the preponderance of C-->T transitions in the oxidative and spontaneous mutational databases. The major oxidized purine lesion in DNA, 7,8-dihydro-8-oxoguanine (8-oxoG), is processed by two DNA glycosylases, formamidopyrimidine DNA glycosylase (Fpg), which removes 8-oxoG opposite C, and MutY DNA glycosylase, which removes misincorporated A opposite 8-oxoG. The high spontaneous mutation frequency previously observed in fpg mutY double mutants was significantly enhanced by the addition of the nei mutation, suggesting an overlap in the substrate specificities between endo VIII and Fpg/MutY. When the mutational specificity was examined, all of the mutations observed were G:C-->T:A transversions, indicating that in the absence of Fpg and MutY, endo VIII serves as a backup activity to remove 8-oxoG. This was confirmed by showing that, indeed, endo VIII can recognize 8-oxoG in vitro.

Saccharomyces cerevisiae Ntg1p and Ntg2p: broad specificity N-glycosylases for the repair of oxidative DNA damage in the nucleus and mitochondria.

Saccharomyces cerevisiae possesses two functional homologues (Ntg1p and Ntg2p) of the Escherichia coli endonuclease III protein, a DNA base excision repair N-glycosylase with a broad substrate specificity directed primarily against oxidatively damaged pyrimidines. The substrate specificities of Ntg1p and Ntg2p are similar but not identical, and differences in their amino acid sequences as well as inducibility by DNA damaging agents suggest that the two proteins may have different biological roles and subcellular locations. Experiments performed on oligonucleotides containing a variety of oxidative base damages indicated that dihydrothymine, urea, and uracil glycol are substrates for Ntg1p and Ntg2p, although dihydrothymine was a poor substrate for Ntg2p. Vectors encoding Ntg1p-green fluorescent protein (GFP) and Ntg2p-GFP fusions under the control of their respective endogenous promoters were utilized to observe the subcellular targeting of Ntg1p and Ntg2p in S. cerevisiae. Fluorescence microscopy of pNTG1-GFP and pNTG2-GFP transformants revealed that Ntg1p localizes primarily to the mitochondria with some nuclear localization, whereas Ntg2p localizes exclusively to the nucleus. In addition, the subcellular location of Ntg1p and Ntg2p confers differential sensitivities to the alkylating agent MMS. These results expand the known substrate specificities of Ntg1p and Ntg2p, indicating that their base damage recognition ranges show distinct differences and that these proteins mediate different roles in the repair of DNA base damage in the nucleus and mitochondria of yeast.

In vitro repair of synthetic ionizing radiation-induced multiply damaged DNA sites.

When ionizing radiation traverses a DNA molecule, a combination of two or more base damages, sites of base loss or single strand breaks can be produced within 1-4 nm on opposite DNA strands, forming a multiply damaged site (MDS). In this study, we reconstituted the base excision repair system to examine the processing of a simple MDS containing the base damage, 8-oxoguanine (8-oxoG), or an abasic (AP) site, situated in close opposition to a single strand break, and asked if a double strand break could be formed. The single strand break, a nucleotide gap containing 3' and 5' phosphate groups, was positioned one, three or six nucleotides 5' or 3' to the damage in the complementary DNA strand. Escherichia coli formamidopyrimidine DNA glycosylase (Fpg), which recognizes both 8-oxoG and AP sites, was able to cleave the 8-oxoG or AP site-containing strand when the strand break was positioned three or six nucleotides away 5' or 3' on the opposing strand. When the strand break was positioned one nucleotide away, the target lesion was a poor substrate for Fpg. Binding studies using a reduced AP (rAP) site in the strand opposite the gap, indicated that Fpg binding was greatly inhibited when the gap was one nucleotide 5' or 3' to the rAP site. To complete the repair of the MDS containing 8-oxoG opposite a single strand break, endonuclease IV DNA polymerase I and Escherichia coli DNA ligase are required to remove 3' phosphate termini, insert the "missing" nucleotide, and ligate the nicks, respectively. In the absence of Fpg, repair of the single strand break by endonuclease IV, DNA polymerase I and DNA ligase occurred and was not greatly affected by the 8-oxoG on the opposite strand. However, the DNA strand containing the single strand break was not ligated if Fpg was present and removed the opposing 8-oxoG. Examination of the complete repair reaction products from this reaction following electrophoresis through a non-denaturing gel, indicated that a double strand break was produced. Repair of the single strand break did occur in the presence of Fpg if the gap was one nucleotide away. Hence, in the in vitro reconstituted system, repair of the MDS did not occur prior to cleavage of the 8-oxoG by Fpg if the opposing single strand break was situated three or six nucleotides away, converting these otherwise repairable lesions into a potentially lethal double strand break.

The genes encoding formamidopyrimidine and MutY DNA glycosylases in Escherichia coli are transcribed as part of complex operons.

Escherichia coli formamidopyrimidine (Fpg) DNA glycosylase and MutY DNA glycosylase are base excision repair proteins that work together to protect cells from the mutagenic effects of the commonly oxidized guanine product 7,8-dihydro-8-oxoguanine. The genes encoding these proteins, fpg and mutY, are both cotranscribed as part of complex operons. fpg is the terminal gene in an operon with the gene order radC, rpmB, rpmG, and fpg. This operon has transcription initiation sites upstream of radC, in the radC coding region, and immediately upstream of fpg. There is a strong attenuator in the rpmG-fpg intergenic region and three transcription termination sites downstream of fpg. There is an additional site, in the radC-rpmB intergenic region, that corresponds either to a transcription initiation site or to an RNase E or RNase III cleavage site. mutY is the first gene in an operon with the gene order mutY, yggX, mltC, and nupG. This operon has transcription initiation sites upstream of mutY, in the mutY coding region, and immediately upstream of nupG. There also appear to be attenuators in the yggX-mltC and mltC-nupG intergenic regions. The order of genes in these operons has been conserved or partially conserved only in other closely related gram-negative bacteria, although it is not known whether the genes are cotranscribed in these other organisms.